Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Scheme 1. Top: Formulas of Purine Nucleobases (A, G) and Their 7-Deaza Analogues (A*, G*) a in primer extension (PEX) experiments. 12,13 Nevertheless, many of the nucleobase conjugates have displayed less facile incorporation at adjacent positions 7,9,10 and only some of them have been efficiently used in PCR. 13,14 The 7-deazapurines, 7-deazaguanine (G*), or 7-deazaadenine (A*) (Scheme 1) are substitutes of standard purine nucleobases incorporable into DNA by PCR. Although the rate of G* and A* incorporation by DNA polymerases has been reported 15 to be lower, compared to the “parent” purines, both of these nucleobase analogues allow efficient sequence-specific DNA amplification by PCR and it has been possible to prepare PCR products with a high density of the corresponding modification. The 7-deazapurines are able to form Watson-Crick base pairs, maintaining pairing specificity of the respective natural nucleobases, but cannot form Hoogsteen pairs due to absence of the N7 atom (which is substituted by CH group, Scheme 1). Thus, the 7-deazapurines cannot be involved in triplex and tetraplex DNA structures. 16 Reduced tendency of the “deaza-modified” DNA to adopting multistranded conformations has been utilized to improve PCR amplification of G-rich sequences such as (CGG)n repeat, 17 the length of which is analyzed during molecular diagnostics of fragile X syndrome. Similarly, 7-deaza-8-azaguanine has been used to create G-rich DNA probes with reduced propensity to aggregate and improved specificity. 18 Absence of the nitrogen atom at the 7-position in G* was also utilized in a study of sequence-specificity of DNA modification with cisplatin 19 and in studies of echinomycin-DNA interaction modes. 20 Moreover, DNA substituted with G* or A* has been shown to resist cleavage with certain endonucleases. 15 a Atoms at 7-position are highlighted in red. Bottom: Watson-Crick and Hoogsteen base pairing. N7 is involved in the Hoogsteen pairing of natural purines. (13) Cahova, H.; Pohl, R.; Bednarova, L.; Novakova, K.; Cvacka, J.; Hocek, M. Org. Biomol. Chem. 2008, 6, 3657–3660. (14) Raindlova, V.; Pohl, R.; Sanda, M.; Hocek, M. Angew. Chem., Int. Ed. 2010, 49, 1064–1066. (15) Seela, F.; Roling, A. Nucleic Acids Res. 1992, 20, 55–61. (16) Palecek, E. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 151–226. (17) Cao, J.; Tarleton, J.; Barberio, D.; Davidow, L. S. Mol. Cell. Probes 1994, 8, 177–180. (18) Kutyavin, I. V.; Lokhov, S. G.; Afonina, I. A.; Dempcy, R.; Gall, A. A.; Gorn, V. V.; Lukhtanov, E.; Metcalf, M.; Mills, A.; Reed, M. W.; Sanders, S.; Shishkina, I.; Vermeulen, N. M. J. Nucleic Acids Res. 2002, 30, 4952–4959. (19) Cairns, M. J.; Murray, V. Biochim. Biophys. Acta 1994, 1218, 315–321. (20) Sayers, E. W.; Waring, M. J. Biochemistry 1993, 32, 9094–9107. 6808 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Table 1. Oligonucleotides Used in This Work a prim rnd 5′-CATGGGCGGCATGGG-3′ prim noG 5′-TACTCATCATATCAA-3′ temp rnd16 5′-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3′ temp noG 5′-AATATAAATATATTGATATGATGAGTA-3′ p53-for 5′-GAGGTTGTGAGGCGCTGCCC-3′ p53-rev 5′-TCCTCTGTGCGCCGGTCTCT-3′ a The template strands (temp rnd16 , temp noG ) used in PEX experiments were 5′end-biotinylated. Compared to the standard purines, 7-deazapurines exhibit significantly lower potentials of their oxidation. 9,21,22 G* is thus easily photooxidized by intercalated ethidium and this photooxidation has been interrogated as a function of distance, nucleotide sequence and integrity of π-stacking in studies of DNA-mediated charge transfer. 21 The G* ability of being selectively oxidized by a redox mediator with relatively low redox potential, such as Ru(dmb)3 3+/2+ (dmb )4,4′-dimethyl-2,2′-bipyridine), compared to A* together with natural G requiring stronger oxidants, such as Ru(bpy)3 3+/2+ (bpy )2,2′-bipyridine), has been utilized in a PCR-coupled electrochemical technique proposed for parallel detection of two genes. 22 Mediated electrooxidation of G* (or G) was also applied in an indirect electrochemical real time monitoring of PCR via measuring consumption of the respective dNTPs. 23 To our best knowledge, direct electrochemical analysis of DNA with incorporated G* or A* as electrochemically oxidizable tags has not been reported to date. In this paper we report on adsorptive transfer stripping voltammetric analysis of DNA with enzymatically incorporated G* or A* residues at a carbon electrode. We show that, particularly G* producing a specific signal separated from those yielded by natural DNA components, can be utilized as an excellent electroactive label suitable for monitoring of DNA amplification by PCR. MATERIALS AND METHODS Material. Synthetic ODNs (Table 1) were purchased from VBC genomics (Austria). Templates used in experiments involving the magnetoseparation procedure were biotinylated at their 5′ ends. Plasmid pT77 bearing wild type p53 cDNA insert 24 (used as primary template for PCR amplification of the 347-bp fragment) was isolated from E. coli cells using Qiagen Plasmid Purification Kit and linearized with EcoR I restrictase (Takara). Streptavidincoated magnetic beads (MBstv) were purchased from Novagen (Germany), DyNAzyme II DNA Polymerase from Finnzymes (Finland), Pfu DNA Polymerase from Promega (U.S.), unmodified nucleoside triphosphates (dATP, dTTP, dCTP and dGTP), SYBR Green I, SYBR Gold and Stains-All reagent from Sigma, 7-deaza-dGTP and 7-deaza-dATP from Jena Bioscience. Other chemicals were of analytical grade. Primer Extension (PEX). The primer (0.7 µM) was mixed with corresponding template ODN (0.7 µM), dNTPs (100 µM each; composition of the dNTP is specified in the text and Figure legends for individual experiments) and the DyNAzyme II DNA (21) Kelley, S. O.; Barton, J. K. Chem. Biol. 1998, 5, 413–425. (22) Yang, I. V.; Ropp, P. A.; Thorp, H. H. Anal. Chem. 2002, 74, 347–354. (23) Defever, T.; Druet, M.; Rochelet-Dequaire, M.; Joannes, M.; Grossiord, C.; Limoges, B.; Marchal, D. J. Am. Chem. Soc. 2009, 131, 11433–11441. (24) Hupp, T. R.; Meek, D. W.; Midgley, C. A.; Lane, D. P. Cell 1992, 71, 875– 886.
Polymerase (1 U per sample). Reactions were carried out at 60 °C for 30 min. MBstv Magnetoseparation Procedure. The PEX products were captured at MBstv via biotin tags. Then, 50 µL aliquots of the PEX reaction mixtures were added to the MBstv (25 µL of the stock suspension washed twice with 100 µL of 0.3 M NaCl, 10 mM Tris-HCl, pH 7.4, buffer H). The mixture was incubated on a shaker for 30 min at 20 °C. Then the beads were washed three times with 100 µL of PBS (0.14 M NaCl, 3 mM KCl, 4 mM sodium phosphate, pH 7.4) containing 0.01% Tween20, three times with 100 µL of the buffer H and resuspended in deionized water (50 µL). The extended primers were released by heating at 75 °C for 2 min. Prior to the electrochemical measurements, NaCl in total concentration of 0.2 M was added to the samples. Preparative PCR. Amplification of the DNA fragment: 500 ng of the pT77 template was mixed with p53-for and p53-rev primers (0.5 µM each), Pfu DNA Polymerase (3 U) and mix of dNTPs (125 µM each) in total volume of 100 µL. The PCR involved 30 cycles if not stated otherwise (denaturation 94 °C/90 s, annealing 60 °C/120 s, polymerization 72 °C/180 s) and was run using C1000 Thermal Cycler (BioRad). The PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and their concentrations were determined spectrophotometrically using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, U.S.). Real Time PCR. Reactions were prepared in duplicates in 20 µL handmade PCR mix consisting of 30 ng of pT77 template, p53for and p53-rev primers at concentrations 0.5 µM, 1 × DyNAzyme II buffer, 1.25 mM MgCl 2,20000× diluted SYBR Green I, each dNTP (or sum of dGTP+dG*TP) 125 µM,1UofDyNAzyme II DNA polymerase. PCR involved 20 cycles (denaturation 94 °C/30 s, annealing 56 °C/30 s, polymerization 72 °C/30 s, fluorescence measurement in SYBR Green I channel 15 s/78 °C, wavelength of the source 470 nm, wavelength of the detection filter 585 nm; final extension 72 °C/3 min) and was run and evaluated using the RotorGene-3000 (Qiagen, Germany). Native PAGE. The PCR products (1 µL each) were mixed with loading buffer (0.1% SDS, 5% glycerol, 5 mg mL -1 bromophenol blue) and subjected to electrophoresis in 5% native gel containing 1 × TBE buffer (pH 8) at 150 V, 4 °C for 35 min. Gels stained with ethidium bromide or SYBR dyes were visualized using LAS-3000 (FUJIFILM Corporation), those stained with the Stains-All reagent were scanned. Electrochemical Analysis. The PEX and PCR products were analyzed by using ex situ (adsorptive transfer stripping, AdTS) square-wave voltammetry (SWV). The DNA was accumulated at the basal-plane pyrolytic graphite electrode (PGE; prepared and pretreated as described in ref 25) surface from 5 µL aliquots containing 0.3 M NaCl for 60 s. Then the electrode was rinsed by deionized water and was placed into the electrochemical cell. SWV settings: initial potential -1.0 V, final potential +1.5 V, pulse amplitude 25 mV, frequency 200 Hz, potential step 5 mV. The measurements were performed at ambient temperature in 0.2 M acetate buffer pH 5 by using CHI440 Electrochemical Workstation (CH Instruments, Inc., U.S.) in a three-electrode setup (with the PGE as working electrode, Ag/AgCl/3 M KCl as reference, and (25) Fojta, M.; Havran, L.; Kizek, R.; Billova, S. Talanta 2002, 56, 867–874. platinum wire as counter electrode). Baseline correction of the voltammograms was performed by means of a moving average algorithm (GPES 4 software, EcoChemie). RESULTS AND DISCUSSION Primer extension (PEX) has recently been used for the introduction of labeled nucleobases into oligodeoxynucleotides (ODNs). 7-13 Using nucleobase-modified deoxynucleotide triphosphates (dNTPs) and DNA polymerases, we prepared ODNs bearing various tags producing analytically useful electrochemical responses. Since 7-deazapurines are electrochemically oxidized at considerably less positive potentials, compared to the respective natural purine nucleobases, 9,21,22 it is in principle possible to apply these nucleobase analogues themselves (without introducing any extra label groups) as electroactive markers of the in vitro synthesized DNA. We performed PEX with primrnd primer and 5′-end-biotinylated temprnd16 template, the nucleotide sequence of which was designed to accommodate all four DNA bases (four-times each) in the synthesized DNA stretch (Figure 1A). The doublestranded PEX products were pulled-down from the reaction mixture using streptavidin coated magnetic beads (MBstv) and, after magnetic separation, the captured duplex ODN was thermally denatured to release the extended primer strand. The latter was finally analyzed using adsorptive transfer stripping square wave voltammetry (AdTS SWV). As shown in Figure 1B,C, voltammetric responses of the pexrnd16 products reflected the composition of dNTP mix used. For a standard dNTP mix (dATP, dGTP, dCTP, and dTTP), we observed two anodic signals corresponding to electrochemical oxidation of guanine (peak Gox at 1.15 V) and adenine (peak Aox at 1.35 V, Figure 1B,C). When dGTP was replaced by 7-deaza-dGTP (dG*TP), peak Gox was decreased and a new anodic peak appeared at 0.80 V (peak G* ox , Figure 1B,C) which has been ascribed to electrochemical oxidation of G* introduced into the extended ODN stretch. Lower intensity of the peak Gox corresponded to lower number of G residues in the G*-modified PEX product, compared to the PEX product composed of standard nucleotides (note that after PEX in the presence of dG*TP, there are still natural guanines present in the primer stretch, but not in the extended stretch, decreasing the total number of G residues from 12 to 8). When dATP was replaced by 7-deazadATP (dA*TP), we observed a decrease of the peak Aox intensity (due to lowering of the total number of adenines in the entire extended primer strand) and increase of the peak at 1.15 V. The potential of electrochemical oxidation of A* was shown 9 to coincide with the potential of peak Gox , and thus currents due to A* oxidation were responsible for the increase of the apparent signal intensity. To verify this hypothesis, we prepared another PEX product using primnoG primer and tempnoG template (Table 1). Neither of these ODNs contains G residues and no G is incorporated during PEX, which is reflected in the absence of peak Gox at the voltammogram of unmodified pexnoG product, showing only peak Aox (black curve in Figure 1C, inset). After performing PEX with the same primer-template pair but with dATP replaced by dA*TP, the peak Aox was decreased and another signal appeared at 1.15 V, corresponding to the A* oxidation (peak A* ox ; green curve in Figure 1C, inset). Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6809
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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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Page 87 and 88: containing T-T mismatches. 23 Based
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Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
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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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