Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6976–6982 Improvement of Sensitivity and Dynamic Range in Proximity Ligation Assays by Asymmetric Connector Hybridization Joonyul Kim, Jiaming Hu, Rebecca S. Sollie, and Christopher J. Easley* Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University, Auburn, Alabama 36849 The proximity ligation assay (PLA) is one of the most sensitive and simple protein assays developed to date, yet a major limitation is the relatively narrow dynamic range compared to other assays such as enzyme-linked immunosorbent assays. In this work, the dynamic range of PLA was improved by 2 orders of magnitude and the sensitivity was improved by a factor of 1.57. To accomplish this, asymmetric DNA hybridization was used to reduce the probability of target-independent, background ligation. An experimental model of the aptamer-target-connector complex (apt A-T-aptB-C20,PLA) in PLA was developed to study the effects of asymmetry in aptamer-connector hybridization. Connector base pairing was varied from the PLA standard of 20 total bases (C 20) to an asymmetric combination with 15 total bases (C15). The results of this model suggested that weakening the affinity of one side of the connector to one aptamer would significantly reduce target-independent ligation (background) without greatly affecting target-dependent ligation (signal). These predictions were confirmed using PLA with asymmetric connectors for detection of human thrombin. This novel, asymmetric PLA approach should impact any previously developed PLA method (using aptamers or antibodies) by reducing target-independent ligation events, thus generally improving the sensitivity and dynamic range of the assay. The proximity ligation assay 1 (PLA) is among the most sensitive protein assays developed to date. PLA can be considered a descendent of immuno-PCR methods, 2 in which antibodies are attached to oligonucleotides to allow the coupling of target binding with exponential amplification by polymerase chain reaction (PCR). PLA is a homogeneous assay, requiring no washing steps, with protein detection limits as low as tens of zeptomoles 1 (fM in 1 µL samples). As shown in Figure 1A, the mode of action of PLA results in a four-part complex composed of the targeted protein analyte (T), two proximity probes (DNA-conjugated antibodies or oligonucleotide aptamers; aptA and aptB), and a connector oligonucleotide (C20,PLA). The three separate DNA molecules * To whom correspondence should be addressed. Phone: +1 334-844-6967. Fax: +1 334-844-6959. E-mail: chris.easley@auburn.edu. (1) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gústafsdóttir, S. M.; Ostman, A.; Landegren, U. Nat. Biotechnol. 2002, 20, 473– 477. (2) Adler, M.; Wacker, R.; Niemeyer, C. M. Analyst 2008, 133, 702–718. 6976 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 1. Schematics of PLA and model systems described in the text. (A) Standard PLA, in which a connector length of 20 bases (C20,PLA, orb ) 20) is used. Ligated signal or background complexes are not differentiated by qPCR. (B) Asymmetric PLA, in which connector lengths of 15-19 bases (Cb,PLA, where 15 e b < 20) are tested. (C) Schematic of the experimental model system used to predict signal and background in PLA methods. The “signal” complex is defined as Loop-Cb, whereas the “background” complex is defined as FreeA-Cb-FreeB. Model connectors were varied from C15 to C20 in this study. (or moieties) are designed to hybridize into a structure that promotes a DNA ligase enzyme to covalently couple the two proximity probes. In the presence of target, the four-part complex is preferred over target-independent hybridization (three-part complex) due to a cooperative “proximity effect” that essentially increases the local concentration of the oligo- 10.1021/ac101762m © 2010 American Chemical Society Published on Web 07/23/2010
nucleotide tails. 3-5 Thus, the amount of ligation products is proportional to the amount of protein analyte, and these products can be made recognizable by PCR for exponential amplification and highly sensitive quantitation by qPCR. Due to its simplicity and sensitivity, PLA has found much interest of late and has been successfully utilized in a variety of applications, such as sensitive protein detection, 1,6 in situ analysis of protein localization, 7 DNA-protein interactions, 8 clinical diagnostics, 9 and proteinprotein interaction assays. 10 In the beginning stages of PLA method development, Landegren and co-workers determined the optimal concentrations of each proximity probe and the connector oligonucleotide that maximize the cooperative effect induced by target binding, 1 and they further proved that this cooperative effect was negatively related to the Kd of the proximity probes. 6 Major advantages of PLA are sensitivity, selectivity, and ease of use. Use of at least two proximity probes, with discrete binding sites on the same target, allows unprecedented limits of target detection (LOD) and better specificity compared to other assays with one probe. Furthermore, the assay requires no washing steps due to its homogeneous nature. However, PLA has the limitation of a relatively narrow dynamic range. The reason for this flaw is the necessity to use very low amounts of proximity probes (∼10 -16 mol) to minimize a target-independent hybridization and ligation (background). Such a low amount of proximity probes allows PLA to assay only up to ∼10 -15 mol (10 times more analytes than proximity probes) if the Kd of the proximity probe is less than 0.4 nM. 6 Our goal in this study was to improve the dynamic range of PLA without sacrificing LOD. To address this issue, we made two hypotheses: (1) target-independent hybridization (background; Figure 1A, bottom) is inversely proportional to the Kd of the connector oligonucleotide at a given concentration of proximity probes, and (2) the width of the dynamic range is proportional to the amount of a proximity probe to be used. If confirmed, these hypotheses suggest that dynamic range and signal to background could be improved by simply reducing the number of bases in the connector. To test these hypotheses, first we developed and tested an experimental model of the proximity effect. This model suggested that asymmetric connectors with higher K d values could significantly decrease target-independent hybridization (background; Figure 1A, bottom). In turn, this would lead to higher signal-to-background ratios and allow the use of more proximity probes, which should increase the dynamic range of PLA. As described below, we successfully (3) Zhou, H. X. Biochemistry 2001, 40, 15069–15073. (4) Zhou, H. X. J. Mol. Biol. 2003, 329, 1–8. (5) Tian, L.; Heyduk, T. Biochemistry 2009, 48, 264–275. (6) Gullberg, M.; Gústafsdóttir, S. M.; Schallmeiner, E.; Jarvius, J.; Bjarnegård, M.; Betsholtz, C.; Landegren, U.; Fredriksson, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8420–8424. (7) Söderberg, O.; Leuchowius, K. J.; Gullberg, M.; Jarvius, M.; Weibrecht, I.; Larsson, L. G.; Landegren, U. Methods 2008, 45, 227–232. (8) Gustafsdottir, S. M.; Schlingemann, J.; Rada-Iglesias, A.; Schallmeiner, E.; Kamali-Moghaddam, M.; Wadelius, C.; Landegren, U. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3067–3072. (9) Nordengrahn, A.; Gustafsdottir, S. M.; Ebert, K.; Reid, S. M.; King, D. P.; Ferris, N. P.; Brocchi, E.; Grazioli, S.; Landegren, U.; Merza, M. Vet. Microbiol. 2008, 127, 227–236. (10) Gustafsdottir, S. M.; Wennström, S.; Fredriksson, S.; Schallmeiner, E.; Hamilton, A. D.; Sebti, S. M.; Landegren, U. Clin. Chem. 2008, 54, 1218– 1225. Table 1. Oligonucleotide Sequences Used in the Experimental Model of Proximity Hybridization a name sequence b C20 5′-TAATACTTGCTGAGGAATAA-3′ (similar to that used in standard PLA) C19 5′-TAATACTTGCTGAGGAATA-3′ C18 5′-TAATACTTGCTGAGGAAT-3′ C17 5′-TAATACTTGCTGAGGAA-3′ C16 5′-TAATACTTGCTGAGGA-3′ C15 5′-TAATACTTGCTGAGG-3′ FreeA 5′-/IABkFQ/GCA AGT ATT ATT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTC TCT TTT TC-3′ FreeB 5′-/Phos/TCT TCT CTC TCT CTC TTT TTT TTT TTT TTT TTT TTT TTT TTT ATT CCT CA/6-FAM/-3′ linker 5′-GAG AGA GAG AGA AGA GAA AAA GAG AAA AAA-3′ Loop 5′-/IAbFQ/GCA AGT ATT ATT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTC TCT TTT TC TCT TCT CTC TCT CTC TTT TTT TTT TTT TTT TTT TTT TTT TTT ATT CCT CA/6-FAM/-3′ a Bases in bold depict the variable segments of the connectors used to alter connector affinity (see Table 2 for affinities). b Notes: IABkFQ, Iowa black FQ; Phos, phosphorylated end; 6-FAM, 5(6)-carboxyfluorescein (mixed isomer). improved the sensitivity and substantially widened the dynamic range for thrombin detection compared to standard PLA. This novel, asymmetric PLA approach should be generally applicable to any of the various PLA methods developed to date, using either aptamer- or antibody-based probes. 1,6-10 EXPERIMENTAL SECTION Reagents and Materials. All solutions were prepared with deionized, ultrafiltered water (Fisher Scientific). T4 DNA ligase was purchased from New England BioLabs. Human thrombin was obtained from Sigma-Aldrich. All oligonucleotides except the Taqman probe were obtained from Integrated DNA Technologies (IDT; Coralville, Iowa), with purity and yield confirmed by mass spectrometry and HPLC, respectively. Sequences of ssDNA strands used in the experimental model are given in Table 1. Sequence Free A was labeled at its 5′-end with Iowa black FQ quencher (IABkFQ; absorbance maximum at 531 nm). Sequence FreeB was labeled at its 5′-end with a phosphate group (Phos) and at its 3′-end with 5(6)-carboxyfluorescein (6-FAM, mixed isomer; emission maximum at 520 nm). Loop strands (Table 1) were synthesized by ligation of FreeA and FreeB using T4 DNA ligase; further details on Loop synthesis are included in the Supporting Information text and Figure S-2. Sequences (listed 5′ to 3′) for PLA or asymmetric PLA were as follows. Thrombin aptamer A, aptA, CAG TCC GTG GTA GGG CAG GTT GGG GTG ACT TCG TGG AAC TAT CTA GCG GTG TAC GTG AGT GGG CAT GTA GCA AGA GG; thrombin aptamer B, aptB, /5Phos/GT CAT CAT TCG AAT CGT ACT GCA ATC GGG TAT TAG GCT AGT GAC TAC TGG TTG GTG AGG TTG GGT AGT CAC AAA; symmetric connector, C20,PLA, AAG AAT GAT GAC CCT CTT GCT AAA A; asymmetric connector, C18,PLA, TTA TGA TGA CCC TCT TGC TAA AA; asymmetric connector, C16,PLA, TAG ATG ACC CTC TTG CTA AAA; forward PCR primer, GTG ACT TCG TGG AAC TAT CTA GCG; reverse PCR primer, AAT ACC CGA TTG CAG TAC GAT TC; Taqman probe (Applied Biosystems, ABI), 4-chlorofluorescein-TGT ACG TGA GTG GGC ATG TAG CAA GAG G-carboxytetramethylrhodamine. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6977
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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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Page 183 and 184: Scheme 1. Reactions of Selenium Rea
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Page 199 and 200: Figure 2. Normalized product ion ab
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Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
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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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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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