Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 2. Results of the experimental model. Fluorescence response curves for the signal (Loop, black circles) and background (Free, gray open squares) complexes when titrated with (A) C20 or (B) C16. NLLS fits to the Hill equation (eq 2) are shown as solid curves. Other curves (titration with C15,C17,C18, and C19) are given in Supporting Information Figure S-1. Also shown are (C) relative amounts of signal and background using each connector and (D) signal-to-background ratios as functions of the dissociation constants (Kd,B) of the variable segments of each connector, Cb. would have significantly increased cooperativity (higher n) compared to the FreeA-Cb-FreeB complexes (background). Example data sets from these titrations are shown in Figure 2, with error bars representing standard deviations of the triplicate measurements. Figure 2A shows that the Loop-C20 complex (filled black circles) is more thermodynamically favored compared to the FreeA-C20-FreeB complex (open gray squares). This result was expected, owing to the success of various PLAs 1,6-10 using 20-base connectors such as C20. This data, and all titrations to follow, were fit to eq 2 via NLLS fitting to determine Kd,eff values and S/BG (via eq 1) for each asymmetric connector (Cb); measured and calculated values are shown in Table 2. With the use of the symmetric connector, C20, the S/BG value was determined to be 5.72. Figure 2B shows the fluorescence measurements from a similar experiment using C16. This figure clearly shows that the relative stability of the signal complex (Loop-C16, filled black circles) over the background complex (FreeA-C16-FreeB, open gray squares) is much greater than that measured with C20, without a large decrease in stability of the signal complex. In fact, the S/BG value using C16 was determined to be 838. Thus, a 146-fold increase in S/BG was achieved by simply decreasing the connector length in an asymmetric manner. Titration curves for C15, C17, C18, and C19 (Supporting Information Figure S-1) follow this trend of increasing S/BG with decreasing connector length (Figure 2, parts 6980 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 C and D). As shown in Figure 2C, the background levels with C20 were 382-fold larger than the background with C16, whereas the signal differed by only 3.16-fold. These large decreases in background and only modest decreases in signal gave the expected increase in S/BG with decreasing connector affinity BG (Figure 2D). The agreement between measured Kd,eff and calculated Kd,B values was also encouraging (Figures S-2 and S-3 in the Supporting Information). It must be noted that the background complex titration experiment for C15 was impractical, due to the expense involved in working with millimolar concentrations of DNA. Although, using nearest-neighbor calculations, 12 it is predicted that the S/BG could be as high as 1.7 × 104 for C15. These results provided a first step toward confirmation of our hypothesis. Also shown in Figure 2D is a curve (dashed line) calculated from data presented by Tian and Heyduk, 5 who developed a similar experimental model using bivalent ligands of various affinities. It is clear that the general trend observed in our data matches with that seen by Tian and Heyduk with an obvious offset in magnitude. This offset is likely due to the model differences, in which Tian and Heyduk used a6nmspacer between their binding sites, whereas our model has essentially no spacer between the binding sites. Inclusion of an equivalent spacer in our system would be unreasonable, since PLA requires subse-
quent ligation of the aptamers after hybridization of the signal complex. Nonetheless, the similarities were encouraging. Figure 2D presents the relationship between S/BG and the affinities of the variable segments of the connectors, which are represented by the calculated Kd,B values 12 (Table 2). This analysis is useful, since Kd,B will ultimately become the independent variable in the asymmetric PLA approach. As shown in the figure, S/BG increases proportionally with Kd,B. In theory, one could increase Kd,B even further. In the extreme example, one could use C11 (only one base pair in the variable segment). However, the S/BG parameter is not useful if there is insufficient signal present with respect to the noise of measurement, and it is likely that the amount of either signal or background (or both) complexes using C11 would be negligible. The connector length, b, could be considered an alternative for the independent variable (shown as upper axis in Figure 2, parts C and D), but care must be taken, since b remains dependent upon base composition, making it less precise than Kd,B. Hill coefficients (n) were also determined by NLLS fitting of the data to eq 2. The mean n values for formation of all of the background (FreeA-Cn-FreeB) and signal (Loop-Cn) complexes were 1.42 ± 0.19 and 1.76 ± 0.11, respectively. These values were shown to be statistically different (p < 0.01). Cooperativity enhancement in the signal complex was expected, since this effect gives PLA the ability to discriminate signal from background. Furthermore, as reported by Weiss, 16 these Hill coefficients suggest that the signal complex forms in a sequential manner, whereas the background complex forms in an independent manner, as reflected in Figure 1C. In summary, the improvements in S/BG in the experimental model is derived from the more than 2 orders-of-magnitude BG increase in Kd,eff (reduced affinity) that is achieved using S asymmetric connectors, whereas Kd,eff is increased by less than 1 order of magnitude (Table 2, Figure 2C); thus, by eq 1, S/BG is increased significantly (Figure 2D). However, application of asymmetric connectors to PLA requires experimental confirmation, as given below, since we began our model with the assumption of infinite aptamer affinities. Design of Asymmetric PLA. A schematic of asymmetric PLA is shown in Figure 1B. Since decreased background was achieved in the experimental model (Figure 2), the next step was to confirm that this approach was useful for PLA. The real assay is inherently more complicated than the experimental model, requiring not only a four-part complex formation but also ligation of aptA to aptB, followed by qPCR. 1,6 Furthermore, aptamer affinities are finite in the real system, compared to the assumption of infinite affinities in the model. On the basis of these factors, we expected that the observations from the model would be less pronounced in the real system. Asymmetric PLA (Figure 1B) is based on the formation of the complex aptA-T-aptB-Cb,PLA, where b < 10 is achieved by decreasing connector lengths on the side complementary to aptB. Shorter connectors, therefore, have higher Kd,B (Table 2), or lower affinity. For the standard PLA system (C20,PLA, b ) 20), the optimal connector concentration was determined to be >40 nM. 1 This correlates well with the model results shown in Figure 2A. At C20,PLA ) 40 nM, the stability of the signal complex is near maximal, whereas the stability of the background complex is minimized as much as possible. Working on this principle, the results from the titrations in the experimental model (Figure 2, parts A and B and Supporting Information Figure S-1) were used to estimate the optimal connector concentrations, [Cn]opt, for each model connector of different length. These values are reported in the rightmost column of Table 2 and were used for all asymmetric PLA experiments to follow. This approach allowed the interrogation of each asymmetric PLA system at its optimal values of signal and background. Background Reduction Using Asymmetric PLA. Starting with the identical reagents used by Landegren and co-workers 1,6 for sensitive detection of human thrombin, connector lengths were systematically decreased from the initial C20,PLA sequence. Background ligation levels were measured by carrying out the assay without the target protein (human thrombin) present. As shown in Figure 3A, the background levels in asymmetric PLA were significantly reduced by increasing Kd,B (or decreasing length, b), matching well with the experimental model (Figure 2C). Background levels in asymmetric PLA with C16,PLA were 46.7fold lower than with standard, symmetric PLA (C20,PLA), thus confirming our hypothesis. It should, therefore, be possible to use higher concentrations of aptamer probes with asymmetric PLA to improve the dynamic range and sensitivity. Dynamic Range and Sensitivity Enhancement with Asymmetric PLA. A major limitation of standard PLA is the relatively narrow dynamic range. This disadvantage is directly related to the generation of background ligations. The original work in PLA for human thrombin detection determined the optimal concentrations of aptamer probes to be 15 and 20 pM for aptA and aptB in the 5 µL incubation mixture, respectively. 1 At higher probe concentrations, increased background ligations were shown to be detrimental to the S/BG of the assay. Since the assay relies on the simultaneous binding of two aptamers to the same target, simple probability theory shows that the population of signal complexes (aptA-T-aptB-Cb,PLA) will first increase then decrease as the concentration of target is increased. The upper limit of the dynamic range is thus positively related to both the concentrations and the Kd’s of the aptamer probes. Gullberg et al. 6 showed that target at 10 times higher concentration than probes could not be assayed with probe Kd < 0.4 nM but could be assayed with a probe Kd ) 2.5 nM, albeit with relatively low S/BG. This implies that in standard PLA, a lower LOD is achievable with higher affinity probes at the expense of narrow dynamic range. Previous publications using standard, aptamer-based PLA for thrombin detection did not report the LOD or the dynamic range. 1,6 However, by analyzing the presented data, the reported LOD was determined to be 16 amol (significantly different from background, p < 0.05), with a dynamic range from 16 to 400 amol. In our laboratory, with the standard PLA system using C20,PLA, we obtained a detection limit about 3-fold higher than 16 amol, but we were able to achieve a significantly wider dynamic range compared to the previous reports. As shown in Figure 3B (open gray squares), our standard PLA detection limit was 50 amol, and our dynamic range was from 50 to 3000 amol (7.68-fold wider than previous reports). Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6981
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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
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
Page 209 and 210: isoforms, its successful use, in th
Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
Page 217 and 218: Scheme 2. Fragmentation Mechanism o
Page 219 and 220: Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222: Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224: Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226: Anal. Chem. 2010, 82, 6969-6975 Ana
Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
Page 231 and 232: for the fill time, and we find that
Page 233 and 234: nucleotide tails. 3-5 Thus, the amo
Page 235: allow the use of higher aptamer con
Page 239 and 240: Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
Page 245 and 246: Since the time for liquid expulsion
Page 247 and 248: Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
Page 253 and 254: Figure 4. (A) Typical raw electroph
Page 255 and 256: field. DNA profiles were delivered
Page 257 and 258: Another approach to handle this lim
Page 259 and 260: (principal components, PCs) in whic
Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: CONCLUSIONS In this paper we have i
Page 265 and 266: electroactive-species loaded liposo
Page 267 and 268: 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
Page 273 and 274: Table 1. Effect of 1 D-LC Condition
Page 275 and 276: Figure 3. Peak-production rate vers
Page 277 and 278: Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280: luciferase (T7 control vector) was
Page 281 and 282: Figure 3. Inhibitory effects of luc
Page 283 and 284: 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
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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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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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