Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 7044–7048 Direct Fluorescence Polarization Assay for the Detection of Glycopeptide Antibiotics Linliang Yu, Meng Zhong, and Yinan Wei* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506 Glycopeptide antibiotics are widely used in the treatment of infections caused by Gram-positive bacteria. They inhibit the biosynthesis of the bacterial cell wall through binding to the D-alanyl-D-alanine (D-Ala-D-Ala) terminal peptide of the peptidoglycan precursor. Taking advantage of this highly specific interaction, we developed a direct fluorescence polarization based method for the detection of glycopeptide antibiotics. Briefly, we labeled the acetylated tripeptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore to create a peptide probe. Using three glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin, as model compounds, we demonstrated that the fluorescence polarization of the peptide probe increased upon binding to antibiotics in a concentration dependent manner. The dissociation constants (Kd) between the peptide probes and the antibiotics were consistent with those reported between free D-Ala-D-Ala and the antibiotics in the literature. The assay is highly reproducible and selective toward glycopeptide antibiotics. Its detection limit and work concentration range are 0.5 µM and 0.5-4 µM for vancomycin, 0.25 µM and 0.25-2 µM for teicoplanin, and 1 µM and 1-8 µM for telavancin. Furthermore, we compared our assay in parallel with a commercial fluorescence polarization immunoassay (FPIA) kit in detecting teicoplanin spiked in human blood samples. The accuracy and precision of the two methods are comparable. We expect our assay to be useful in both research and clinical laboratories. Glycopeptide antibiotics are cyclic peptides that bind to the peptidoglycan precursor D-alanyl-D-alanine to inhibit the biosynthesis of bacterial cell walls. They are widely used to treat infections caused by Gram-positive bacteria, including Methicillin Resistant Staphylococcus aureus (MRSA), and are regarded as the drug of “last resort”. 1 Vancomycin, teicoplanin, and telavancin are three representative glycopeptide antibiotics that are currently in clinical use. 2-4 Monitoring of drug levels in patients’ serum during * To whom correspondence should be addressed. Address: 305 Chemistry- Physics Building, University of Kentucky, Lexington, KY 40506-0055. Telephone: (859) 257-7085. Fax: (859) 323-1069. E-mail: yinan.wei@uky.edu. (1) Perkins, H. R. Pharmacol. Ther. 1982, 16, 181–197. (2) Levine, D. P. Clin. Infect. Dis. 2006, 42, S5–S12. (3) Delalla, F.; Nicolin, R.; Rinaldi, E.; Scarpellini, P.; Rigoli, R.; Manfrin, V.; Tramarin, A. Antimicrob. Agents Chemother. 1992, 36, 2192–2196. (4) Higgins, D. L.; Chang, R.; Debabov, D. V.; Leung, J.; Wu, T.; Krause, K. M.; Sandvik, E.; Hubbard, J. M.; Kaniga, K.; Schmidt, D. E.; Gao, Q.; Cass, R. T.; Karr, D. E.; Benton, B. M.; Humphrey, P. P. Antimicrob. Agents Chemother. 2005, 49, 1127–1134. 7044 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 the treatment is critical in maximizing the effectiveness of the treatment while minimizing drug toxicities and side effects. 5-7 Such data also provide important pharmacokinetic and pharmacodynamic parameters for clinical studies 8-12 and, thus, guide the optimization of therapies. 7,13,14 In addition, vancomycin has been extensively used as a model antibiotic in tissue engineering and controlled drug release studies. 15-20 Various assays have been developed to measure the concentration of glycopeptide antibiotics,includingmethodsbasedonUVabsorbance, 17 immunoassay, 21-24 high performance liquid chromatography (HPLC), 25-28 and agar diffusion bioassay. 29,30 Each of these existing methods suffer from (5) James, C. W.; Gurk-Turner, C. Proc. (Bayl. Univ. Med. Cent.) 2001, 14, 189–190. (6) Williams, A. H.; Gruneberg, R. N. J. Antimicrob. Chemother. 1984, 14, 441– 445. (7) Brink, A. J.; Richards, G. A.; Cummins, R. R.; Lambson, J.; Teicoplanin, G. U. Int. J. Antimicrob. Agents 2008, 32, 455–458. (8) Svetitsky, S.; Leibovici, L.; Paul, M. Antimicrob. Agents Chemother. 2009, 53, 4069–4079. (9) Wood, M. J. J. Antimicrob. Chemother. 1997, 40, 147–147. (10) Cobo, J.; Fortun, J. J. Antimicrob. Chemother. 1996, 38, 1113–1114. (11) Wood, M. J. J. Antimicrob. Chemother. 1996, 38, 919–919. (12) Wood, M. J. J. Antimicrob. Chemother. 1996, 37, 209–222. (13) Pea, F.; Brollo, L.; Viale, P.; Pavan, F.; Furlanut, M. J. Antimicrob. Chemother. 2003, 51, 971–975. (14) Mohammedi, I.; Descloux, E.; Argaud, L.; Le Scanff, J.; Robert, D. Int. J. Antimicrob. Agents 2006, 27, 259–262. (15) Adams, C. S.; Antoci, V., Jr.; Harrison, G.; Patal, P.; Freeman, T. A.; Shapiro, I. M.; Parvizi, J.; Hickok, N. J.; Radin, S.; Ducheyne, P. J. Orthop. Res. 2009, 27, 701–709. (16) Antoci, V., Jr.; King, S. B.; Jose, B.; Parvizi, J.; Zeiger, A. R.; Wickstrom, E.; Freeman, T. A.; Composto, R. J.; Ducheyne, P.; Shapiro, I. M.; Hickok, N. J.; Adams, C. S. J. Orthop. Res. 2007, 25, 858–866. (17) Radin, S.; Chen, T.; Ducheyne, P. Biomaterials 2009, 30, 850–858. (18) Radin, S.; Ducheyne, P.; Kamplain, T.; Tan, B. H. J. Biomed. Mater. Res. 2001, 57, 313–320. (19) Perelman, L. A.; Pacholski, C.; Li, Y. Y.; VanNieuwenhz, M. S.; Sailor, M. J. Nanomedicine 2008, 3, 31–43. (20) Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S. Y. J. Am. Chem. Soc. 2003, 125, 4451–4459. (21) Kitzis, M. D.; Goldstein, F. W. Clin. Microbiol. Infect. 2006, 12, 92–95. (22) Wilson, J. F.; Davis, A. C.; Tobin, C. M. J. Antimicrob. Chemother. 2003, 52, 78–82. (23) Lee, H. B.; Kwak, B. Y.; Lee, J. C.; Kim, C. J.; Shon, D. H. J. Microbiol. Biotechnol. 2004, 14, 612–619. (24) Lam, M. T.; Le, X. C. Analyst 2002, 127, 1633–1637. (25) Valle, M. J. D.; Lopez, F. G.; Navarro, A. S. J. Pharm. Biomed. Anal. 2008, 48, 835–839. (26) Abu-Shandi, K. H. Anal. Bioanal. Chem. 2009, 395, 527–532. (27) Saito, M.; Santa, T.; Tsunoda, M.; Hamamoto, H.; Usui, N. Biomed. Chromatogr. 2004, 18, 735–738. (28) Shen, J.; Jiao, Z.; Zhou, Y. N.; Zhu, H. L.; Song, Z. J. Chromatographia 2007, 65, 9–12. (29) Kureishi, A.; Jewesson, P. J.; Bartlett, K. H.; Cole, C. D.; Chow, A. W. Antimicrob. Agents Chemother. 1990, 34, 1642–1647. (30) Bantar, C.; Durlach, R.; Nicola, F.; Freuler, C.; Bonvehi, P.; Vazquez, R.; Smayevsky, J. J. Antimicrob. Chemother. 1999, 43, 737–740. 10.1021/ac100543e © 2010 American Chemical Society Published on Web 07/20/2010
one or more drawbacks including poor selectivity, requirement of large amount of samples, and involvement of cumbersome pretreatment processes. Fluorescence polarization immunoassay (FPIA) is the most popular method currently used in the quantification of glycopeptide antibiotics in clinical samples. 23,31 For example, in the FPIA assay for vancomycin, fluorescentlabeled vancomycin forms a complex with antivancomycin antibody. Free vancomycin in test samples competitively binds to the antibody to displace the labeled one, decreases the fluorescence polarization signal, and thus allows the quantification of the analyte. 32 In this study, we developed a direct fluorescence polarization based assay, taking advantage of the specific interaction between glycopeptide antibiotics and their therapeutic target, the dipeptide D-alanyl-D-alanine (D-Ala-D-Ala). Briefly, we labeled acetylated peptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore and monitored the increase of fluorescence polarization of the labeled peptide in the presence of drugs. Our method is a class-specific assay, which can be used to detect glycopeptide antibiotics in general. In addition, since no protein (such as the antivancomycin antibody) is used in the assay, we expect the shelf life and storage stability of our assay to greatly exceed those of the immunoassay. In this study, we have characterized the interaction between the fluorescent peptide probes and three representative glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin. We were able to measure the concentration of these drugs in serum and blood samples without complicated pretreatment. In addition, we compared the performance of our assay with a commercial FPIA kit in determining the concentration of teicoplanin in human blood samples. We expect our assay to be useful in both research and clinical applications. EXPERIMENTAL SECTION Chemicals. The acetylated Ac-L-Lys-D-Ala-D-Ala-OH peptide, teicoplanin, fluorescein isothiocyanate (FITC), HPLC grade acetonitrile, and heat inactivated, sterile-filtered fetal bovine serum (FBS) were from Sigma-Aldrich (St. Louis, MO). Vancomycin hydrochloride was from Duchefa (Haarlem, The Netherlands). Telavancin was from Astellas (Deerfield, IL). Alexa Fluor 680maleimide (AF680) was from Invitrogen (Eugene, OR). Teicoplanin FPIA kit was from LABfx (Portland, OR). Human whole blood was from Bioreclamation (Westbury, NY). All other reagents were of analytical grade and purchased from Sigma-Aldrich. Fluorescent Labeling of the Peptide. The acetylated peptide Ac-L-Lys-D-Ala-D-Ala-OH (3 mM) was incubated with fluorescein isothiocyanate (FITC, 4.5 mM) in a phosphate buffer (PBS) (10 mM Na2HPO4,2mMKH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) at room temperature overnight. The reaction was terminated through the addition of a Tris-Cl buffer (5 mM, pH 7.4) followed by an incubated of2hatroom temperature. The FITClabeled peptide was purified using a reverse-phase HPLC (Waters, Milford, MA) on a C18 column (Waters, Milford, MA). The purified FITC-labeled peptide was dried under vacuum and then resuspended in water. The concentration of the peptide was determined based on the absorption at 515 nm. Labeling with AF680 was performed similarly. The peptide probe concentration was determined by absorption at 680 nm. The molecule weight of the labeled peptides was confirmed by mass spectroscopy analysis. Fluorescence Polarization Measurements. Fluorescence polarization measurements were performed using a Perkin-Elmer LS-55 fluorescence spectrometer (Perkin-Elmer, Waltham, MA). The temperature was kept at 20 °C. Fluorophore-conjugated peptide (FITC-peptide or AF680-peptide) was added into 400 µL of PBS or FBS to a final concentration of 1 µM. The excitation and emission wavelengths were 479 and 515 nm for FITC-peptide and 679 and 702 nm for AF680-peptide. Titration and Data Fitting. For titration experiments, FITCpeptide or AF680-peptide was added into 400 µL PBS or FBS to a final concentration of 1 µM, and then, small aliquots of glycopeptide antibiotics were titrated into the samples. The increases of fluorescence polarization upon the formation of drug-peptide complexes were recorded. The titration curve was fitted using a one-to-one binding model according to the following equation. 33 ∆P ) ∆Pmax( ([F] + x + Kd ) - √([F] + x + Kd ) 2 - 4[F]x) 2[F] (1) Where ∆P is the monitored change of fluorescence polarization at each drug concentration, ∆Pmax is the maximum change of the fluorescence polarization upon saturation, [F] is the peptide probe concentration, Kd is the dissociation constant between the peptide probe and the drug, and x is the drug concentration. The titration data were fitted using Origin (Northampton, MA). Binding Selectivity Assay. For each antibiotic, AF680-peptide was added into 400 µL of FBS to a final concentration of 1 µM. Next, antibiotics were added to a final concentration of 8 µM. The change of fluorescent polarization upon the addition of antibiotics was recorded. Determination of Drug Concentration in Test Samples. For tests in serum, AF680-peptide was added to 400 µL of FBS to a final concentration of 1 µM. Then, known quantities of antibiotics were added into the sample. The concentrations of the drugs were determined based on the calibration curves derived in FBS. For tests in blood, three human blood samples were spiked with teicoplanin in the therapeutic range by a person not involved in this project. The exact values were kept blind to the authors during the analysis. The blood samples were centrifuged at 2000g for 10 min. The supernatant (plasma) was collected and analyzed together with a series of teicoplanin standards in FBS as described above. For each plasma sample, we prepared two sets of dilutions so the final concentration of teicoplanin would be in the working concentration range of the assay. The first set of three test samples contain 10 µL of plasma and 390 µL of FBS with 1 µM of AF680peptide. The second set of three test samples contain 20 µL of plasma and 380 µL of FBS with 1 µM of AF680-peptide. The teicoplanin concentration in each set of samples was determined. The original teicoplanin concentration calculated from the set of samples falls in the working concentration range. The same (31) Urakami, T.; Maiguma, T.; Kaji, H.; Kondo, S.; Teshima, D. J. Clin. Pharm. Ther. 2008, 33, 357–363. (32) Adamczyk, M.; Grote, J.; Moore, J. A.; Rege, S. D.; Yu, Z. G. Bioconjugate Chem. 1999, 10, 176–185. (33) Yu, L. L.; Fang, J.; Wei, Y. N. Biochemistry 2009, 48, 2099–2108. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7045
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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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Anal. Chem. 2010, 82, 6991-6999 Int
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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 279 and 280: luciferase (T7 control vector) was
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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
Page 297 and 298: Figure 6. (A) FL spectra of BSPOTPE
Page 299: Scheme 1. Proposed Mechanism for Fl
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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