Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6770–6774 Surface-Enhanced Raman Spectroscopy as a Tool for Detecting Ca 2+ Mobilizing Second Messengers in Cell Extracts Elina A. Vitol, † Eugen Brailoiu, ‡ Zulfiya Orynbayeva, § Nae J. Dun, ‡ Gary Friedman, † and Yury Gogotsi* ,| Department of Electrical and Computer Engineering, and Department of Materials Science and Engineering, Drexel University, Philadelphia, Pennsylvania 19104, and Department of Pharmacology, Temple University, Philadelphia, Pennsylvania 19140 Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca 2+ release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surfaceenhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications. Calcium signaling is one of the fundamental cellular processes involved in any cell metabolic and physiologic activity. 1 Calcium signals convey information from the cell plasma membrane to intracellular targets. The mechanism of calcium concentration modulations is a complex problem associated with calcium influx from the extracellular matrix and release from intracellular stores mobilized by calcium messengers. Calcium signaling pathways of two calcium messengers, inositol trisphosphate (IP3) 2,3 and cyclic adenine dinucleotide ribose (cADPR), 4 have been studied extensively in different types of cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) has a unique physiological role in cells 5 in the release of Ca 2+ from acid-filled calcium stores * To whom correspondence should be addressed. E-mail: gogotsi@drexel.edu. † Department of Electrical and Computer Engineering, Drexel University. ‡ Temple University. § School of Biomedical Engineering, Science and Health System, Drexel University. | Department of Materials Science and Engineering, Drexel University. (1) Patel, S.; Churchill, G. C.; Galione, A. Biochem. J. 2000, 352, 725–729. (2) Taylor, C. W.; Thorn, P. Curr. Biol. 2001, 11, R352–R355. (3) Cancela, J. M.; Gerasimenko, O. V.; Gerasimenko, J. V.; Tepikin, A. V.; Petersen, O. H. EMBO J. 2000, 19, 2549–2557. (4) Guse, A. H.; da Silva, C. P.; Berg, I.; Skapenko, A. L.; Weber, K.; Heyer, P.; Hohenegger, M.; Ashamu, G. A.; Schulze-Koops, H.; Potter, B. V. L.; Mayr, G. W. Nature 1999, 398, 70–73. (5) Lee, H. C.; Aarhus, R. J. Biol. Chem. 1995, 270, 2152–2157. 6770 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 through two-pore channels 1, 2, and 3. 6,7 NAADP is the least investigated Ca 2+ mobilizing second messenger, because of the lack of widely accessible and efficient techniques for detecting and quantifying its concentration in cells. Enzymatic bioassays and radioreceptor binding assays are the primary methods which have been used for detecting NAADP in cell extracts. 8,9 The enzymatic assay 9 requires NAADP to be first converted to nicotinamide adenine dinucleotide phosphate (NADP) using ADPribosyl cyclase, which is followed by two enzymatic cycling reactions of oxidation/reoxidation of NADP. 10 Diaphorase, the enzyme for reoxidation of NADP to nicotinamide adenine dinucleotide phosphate (NADPH), also serves as a catalyst for conversion of the reaction indicator resazurin to a highly fluorescent resorufin. The latter is then used for fluorimetric assessment of the NAADP concentration. Importantly, the described assay requires a very high purity of all of the components and takes more than 12 h. 10 The radioreceptor binding assay is less time-consuming and can be conducted without extensive sample purification, 8 but due to the need for unique specialized equipment, the availability of this method is extremely limited. Here we present an alternative, label-free technique for the detection of NAADP enabled by surface-enhanced Raman spectroscopy (SERS). 11-13 SERS enhances Raman scattering due to the amplification of the electric field around metal nanostructures. 14,15 Solutions of metal colloids have been used for SERS, 16,17 but in some cases they show relatively poor data reproducibility resulting from uncontrollable aggregation of colloidal particles. 17–19 For this reason, SERS sensors with metal nanostructures fixed (6) Brailoiu, E.; Churamani, D.; Cai, X. J.; Schrlau, M. G.; Brailoiu, G. C.; Gao, X.; Hooper, R.; Boulware, M. J.; Dun, N. J.; Marchant, J. S.; Patel, S. J. Cell Biol. 2009, 186, 201–209. (7) Calcraft, P. J.; Ruas, M.; Pan, Z.; Cheng, X. T.; Arredouani, A.; Hao, X. M.; Tang, J. S.; Rietdorf, K.; Teboul, L.; Chuang, K. T.; Lin, P. H.; Xiao, R.; Wang, C. B.; Zhu, Y. M.; Lin, Y. K.; Wyatt, C. N.; Parrington, J.; Ma, J. J.; Evans, A. M.; Galione, A.; Zhu, M. X. Nature 2009, 459, 596–U130. (8) Lewis, A. M.; Masgrau, R.; Vasudevan, S. R.; Yarnasaki, M.; O’Neill, J. S.; Garnham, C.; James, K.; Macdonald, A.; Ziegler, M.; Galione, A.; Churchill, G. C. Anal. Biochem. 2007, 371, 26–36. (9) Graeff, R.; Lee, H. C. Biochem. J. 2002, 367, 163–168. (10) Gasser, A.; Bruhn, S.; Guse, A. H. J. Biol. Chem. 2006, 281, 16906–16913. (11) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667–1670. (12) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783–826. (13) Nabiev, I. R.; Morjani, H.; Manfait, M. Eur. Biophys. J. 1991, 19, 311–316. (14) Vitol, E. A.; Orynbayeva, Z.; Bouchard, M. J.; Azizkhan-Clifford, J.; Friedman, G.; Gogotsi, Y. ACS Nano 2009, 3, 3529–3536. 10.1021/ac100563t © 2010 American Chemical Society Published on Web 07/26/2010
on a substrate are preferred. 20-24 The SERS sensor employed in this work is comprised of a glass substrate coated with gold nanoparticles. Similar sensors fabricated in the form of glass nanopipets have been recently demonstrated for minimally invasive in situ intracellular SERS measurements. 14 In the future, the results of this study could be potentially extended to NAADP detection inside cells using the SERS-based approach enabled by SERS-active nanopipets. EXPERIMENTAL SECTION Cell Culture. Breast cancer SkBr3 cells were grown in McCoy’s 5A modified medium, supplemented with 10% fetal serum, streptomycin, and penicillin. Cells were purchased from ATCC. Acid Extraction of NAADP. For the extraction of NAADP we used the protocol reported by Lewis et al. 8 All chemicals were purchased from Sigma-Aldrich. Briefly, SkBr3 cells were treated with trypsin and suspended in the cell medium. Before treatment with the agonists, cells were preincubated for 30 min with BAPTA- AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). Then in the presence of BAPTA-AM cells were stimulated for 20 min with the agonists, according to the technique described in ref 8. This technique has been shown to trigger the prolonged NAADP synthesis for at least 20 min during the agonist stimulation. This results in highly amplified NAADP production. Here we used histamine, adenosine triphosphate (ATP), and acetylcholine, all at a 5 µM concentration. The reaction was stopped by adding 0.75 M ice-cold HClO4. Next the cells were disrupted by sonication and then kept on ice for 10 min. The disrupted cells were centrifuged at 9000g for 10 min. Supernatant was neutralized with 1 M KHCO3 and vortexed. The resulting KClO4 precipitate was removed by centrifugation at 9000g for 10 min. Samples were stored at -80 °C for later analysis. Fabrication of the SERS Sensor. Microscope glass slides were cut into 1 cm × 1 cm pieces and sonicated in a mixture of NaOH and ethanol. After being washed with plenty of 15 MΩ deionized water, the slides were dried at room temperature. Next the slides were dip coated with 0.001% poly-L-lysine, dried at room temperature for 24 h, and then coated with gold nanoparticles by dipping them in the gold colloid for 3 h. Poly-L-lysine promotes the adhesion of the gold nanoparticles to the glass surface. The mechanism of nanoparticle attachment is based on the electrostatic interaction between the negatively charged particles and (15) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. J. Raman Spectrosc. 2005, 36, 640– 647. (16) Ivleva, N. P.; Wagner, M.; Horn, H.; Niessner, R.; Haisch, C. Anal. Chem. 2008, 80, 8538–8544. (17) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett. 2006, 6, 2225–2231. (18) Chourpa, I.; Lei, F. H.; Dubois, P.; Manfait, M.; Sockalingum, G. D. Chem. Soc. Rev. 2008, 37, 993–1000. (19) Willets, K. A. Anal. Bioanal. Chem. 2009, 394, 85–94. (20) McFarland, A. D.; Van Duyne, R. P. Nano Lett. 2003, 3, 1057–1062. (21) Hartschuh, A.; Qian, H.; Meixner, A. J.; Anderson, N.; Novotny, L. Surf. Interface Anal. 2006, 38, 1472–1480. (22) Haynes, C. L.; Van Duyne, R. P. J. Phys. Chem. B 2003, 107, 7426–7433. (23) Shoute, L. C. T.; Bergren, A. J.; Mahmoud, A. M.; Harris, K. D.; McCreery, R. L. Appl. Spectrosc. 2009, 63, 133–140. (24) Deckert, V.; Zeisel, D.; Zenobi, R.; Vo-Dinh, T. Anal. Chem. 1998, 70, 2646– 2650. Figure 1. (a) Scanning electron micrograph of the SERS sensor, (b) close-up view of the gold nanoparticles on the SERS sensor, (c) extinction spectrum of the SERS sensor, and (d) SERS spectrum of NAADP and a background spectrum from the substrate. positively charged NH2 functional groups of poly-L-lysine. 14,25,26 After fabrication, the substrates were imaged with a scanning electron microscope to confirm the nanoparticle distribution on the surface. SEM images were collected with a field emission Zeiss Supra 50VP scanning electron microscope at a low accelerating voltage (0.7-2 kV) without any conductive coating. In addition, the UV-vis extinction spectra of the substrates were measured using a home-built setup employing a fiber-optic spectrometer, HR-4000, Ocean Optics. SERS Measurements. Raman spectroscopy was performed using a micro-Raman spectrometer (Renishaw, RM 1000) equipped with a 632.8 nm HeNe laser (1800 lines/mm grating) and a diode InGaAs laser operating at 785 nm wavelength (1200 lines/mm grating). The lasers are manufactured by Renishaw Inc., U.K. The laser source was focused on the sample through a long working distance 50× objective to a spot size of approximately 2 µm. The typical sample volume was 1 µL. The acquisition time for all spectra was 10 s. Data analysis was performed using the Renishaw Wire 2.0 software. Experimental data were analyzed using principal component analysis 27 in the Matlab environment. RESULTS AND DISCUSSION Testing the SERS Sensor for Distinguishing between Different Secondary Ca 2+ Mobilizing Messengers: NAADP, cADPR, and IP3. Figure 1a shows the SEM image of the SERS sensor, with the close-up view presented in panel b. The average diameter of the nanoparticles is on the order of 50 nm. Assembly of the SERS sensor is based on the wet chemistry two-step protocol. First, the glass substrates are coated with a positively charged polymer (poly-L-lysine) layer. The functionalized substrates are then coated with a monolayer of negatively charged gold nanoparticles through electrostatic binding from the gold (25) Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.; Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.; Natan, M. J. Science 1995, 267, 1629–1632. (26) Nie, S.; Emory, S. R. Science 1997, 275, 1102–1106. (27) Jackson, J. E. A User’s Guide to Principal Components; John Wiley: New York, 1991. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6771
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 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25: Table 2. Concentrations and Stable
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
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
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: 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),
Page 161 and 162:
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
Page 269 and 270:
Figure 6. Fluorescence imaging of Q
Page 271 and 272:
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
Page 285 and 286:
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
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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