Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 4. (A) Dependence of the intensities of peak G ox (measured for unmodified 347-bp amplicon, black) and peak G* ox (measured for the same DNA fragment amplified in the presence of dG*TP instead of dGTP, red) on DNA concentration. Other conditions as in Figure 2. (B) Staining of the 347-bp amplicon in polyacryalmide gels with (from top to bottom) ethidium bromide, SYBR Green I and Stains- All. The PCR reaction contained either four standard dNTPs, G/G* ) 1, or dGTP fully replaced with dG*TP as indicated on the top. Loading of the PCR products: 1 µL of the undiluted reaction mixture (about 30 ng of the amplicon), followed by binary dilutions as indicated. product, compared to PCR with standard dNTP mix. To support this conclusion, we performed a real-time PCR experiment using standard and dG*TP-containing dNTP mixes. Figure 5B shows increase of the fluorescence of SYBR Green I (which was used here as fluorescent dye indicating progression of DNA amplification) as a function of the number of PCR cycles for dNTP mixes containing 0, 10, 20, 30, 50, and 100% of dG*TP (of dGTP+dG*TP total). The steepest increase of the signal was observed for the unmodified amplicon and the amplification rate decreased with the G* content, showing clear difference even for 10% G*. For 100% of G* the apparent amplification rate was remarkably depressed. However, it should be taken into consideration that SYBR Green I fluorescence is quenched by G* incorporated (see above), and thus the weak signal detected for the G*-substituted amplicon can have reflected the dense DNA modification rather than the amount of the PCR product. We therefore focused on the evaluation of the shapes of the amplification curves rather than the absolute signal magnitudes. RotorGene- 3000 software enables to compare reactions parameters in the 6812 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 5. (A) Effects of the number of PCR cycles on intensities of peak G ox (measured for unmodified 347-bp amplicon, gray columns) and peak G* ox (measured for the same DNA fragment amplified in the presence of dG*TP instead of dGTP, red columns). Other conditions as in Figure 2. (B) Quantitative (real time) PCR amplification of the 347-bp DNA fragment: standard dNTP mix (no G*) (1); 10% G* (2); 20% G*(3); 30% G* (4); 50% G (5); G fully replaced with G* (6); negative “no template” control for standard dNTP mix (7). The graph shows fluorescence of SYBR Green I complexes with the DNA amplicons as a function of the number of PCR cycles. Inset, takeoff graphs derived from the real time PCR data (colors correspond to the raw data plot). All samples are plotted in duplicate. comparative quantification mode. The takeoff points for each reaction (sample) are calculated from the second derivatives of raw data (Figure 5B, inset). Generally, the takeoff value is not possible to determine exactly and it is taken as 80% below the peak of second derivative (i.e., below the point where the amplification curve is increasing most steeply). In the same mode, reaction efficiencies of particular reactions are reporting the amplificability of the template under the given conditions. For example, amplification factors of 1.72, 1.65, and 1.61 obtained for reactions with 100% G, G/G* ) 1 and 100% G*, respectively, clearly show less efficient amplification in the dG*TP-substituted reactions. Similar effects of G* on the PCR kinetics were obtained using Taqman hydrolytic probes which release fluorescent indicators into solution as the PCR progresses, and thus the fluorescence intensity was not affected by the incorporated G* as it was in the case of the intercalative dyes (not shown). Hence, results of the real time PCR experiment were in a qualitative accordance with the above electrochemical data indicating lower amplification rate for the reaction with dG*TP. CONCLUSIONS 7-deazapurines G* and A* enzymatically incorporated into DNA are electrochemically oxidizable at carbon electrodes, producing
analytically useful signals at less positive potentials, compared to the corresponding natural purine nucleobases. Peak A* ox due to electrooxidation of A* occurs at the same potential as the peak G ox due to oxidation of natural G, preventing qualitative discrimination of A* incorporated into DNA containing guanine. On the other hand, total or partial substitution of G with G* results in appearance of a new anodic signal, peak G* ox ,at potential less positive than potentials of oxidation of any natural component of DNA, allowing independent determination of G* incorporated. G* can thus be utilized as an inexpensive, commercially available electroactive label for easy monitoring of primer extension or polymerase chain reactions via simple direct electrochemistry using cheap, widely accessible carbon electrodes. In turn, considering applications of 7-deazaguanine as DNA modifications preventing formation of multistranded alternative structures in PCR analysis of G-rich sequences 17 and problems with quenching of fluorescence of ethidium 26 or “SYBR” (to our knowledge, for the first time reported in this paper) dyes, electrochemical analysis appears an attractive complementary approach providing modification-specific signal suitable for quantitation of the fully modified amplified DNA as well as for the determination of the DNA modification extent. Specifically for the G*-modified DNA, the voltammetric analysis represents a simple and direct way to differentiate between the (27) Fojta, M.; Brazdilova, P.; Cahova, K.; Pecinka, P. Electroanalysis 2006, 18, 141–151. (28) Fojta, M.; Havran, L.; Vojtísˇková, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532–6533. natural G and G* residues and to determine relative content of both. In contrast to using peak G ox or peak A ox , produced by natural purines, determination of DNA amplicons based on the measurement of peak G* ox is not affected by signals produced by residual ODN primers and/or the primary template. It has to be naturally taken into consideration that peak G* ox intensity must depend on the G + C content within the amplified region; alternatively, this feature may potentially be useful for the determination of the G + C content or estimation of the length of the amplified DNA fragment (e.g., triplet repeat expansion 27,28 ) provided that a proper normalization of the signal intensity (such as fragment ends “counting” through the intensity of natural G in primers) is used. Besides the PCR applications, the G* electroactive is also potentially useful for taillabeling of DNA probes for electrochemical hybridization assays and other bioanalytical applications which are metter of our ongoing research and will be reported elsewhere. ACKNOWLEDGMENT This work was supported by Grant Agency of the ASCR (grant IAA400040901), by the ASCR (AV0Z50040507 and AV0Z50040702) and by the MEYS CR (LC06035, MSM0021622415). H.P. and P.H. contributed equally to this work. Received for review March 24, 2010. Accepted July 10, 2010. AC100757V Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6813
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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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Page 29 and 30: Figure 4. SERS analysis of NAADP co
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Page 41 and 42: Figure 1. TEM images of the prepare
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Page 49 and 50: Table 1. Extraction Yields, Liquid
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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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