Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 3. Asymmetric PLA. (A) Amount of background ligations vs dissociation constant (Kd,B) of the variable segments of each connector, Cb,PLA, in the absence of target protein. (B) Titration with human thrombin. Asymmetric connector C16,PLA (filled black circles) and symmetric connector C20,PLA (open gray squares) were used for comparison. Error bars represent the standard error of quadruplicate qPCR measurements. Horizontal bars define the assay dynamic ranges. (C) Linear scale comparisons of dynamic range, sensitivity, and limit of detection (LOD) improvements using asymmetric connector C16,PLA (white bars) compared to the standard connector C20,PLA (gray bars). Error bars depict standard deviations of linear sensitivities. The cross-hatched bar represents the LOD obtained in the original reports of PLA (refs 1 and 6). With significant reductions in background using asymmetric PLA (Figure 3A), we hypothesized that the dynamic range could be improved using higher aptamer concentrations with connector C16,PLA. This hypothesis was confirmed by titration of the system with human thrombin (Figure 3B). Asymmetric (closed black circles) and standard (open gray squares) PLA were carried out under the following optimal conditions in the 5 µL incubation mixture: asymmetric, [C16,PLA] ) 480 nM, [aptA] ) 0.9 nM, and [aptB] ) 1.2 nM; standard, [C20,PLA] ) 45 nM, [aptA] ) 15 pM, and [aptB] ) 20 pM. Using asymmetric PLA, the dynamic range 6982 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 was increased by 2 orders of magnitude, and the sensitivity was improved by a factor 1.57 compared to the standard system with C20,PLA (Figure 3, parts B and C). In addition, the maximum achievable S/BG was larger by a factor of 3.15. These improvements were accomplished with an additional 3-fold improvement in LOD to 5.0 amol of thrombin. When compared to previously reported data, 1,6 our dynamic range was improved by an even larger factor of 323. In Figure 3B, horizontal bars mark the dynamic ranges, with the upper bounds defined as the highest concentration with a statistically higher (p < 0.05) value of signalto-background ratio compared to the adjacent lower concentration. Error bars represent standard errors of quadruplicate runs of qPCR. Since logarithmic scales were used in Figure 3B, linear scale comparisons of dynamic ranges, sensitivities, and limits of detection are shown in Figure 3C. Sensitivities were determined as the slopes of the linear regressions of both data sets in the range from the LOD to 3000 amol. This figure helps to clarify the significant advantages provided by asymmetric PLA, in comparison to standard PLA. Most notably, our approach of using asymmetric connectors has allowed a significant improvement in one of the major limitations of the assay, dynamic range. This improvement should be generally applicable to any of the previously developed PLA methods. 1,6-10 CONCLUSIONS This report describes a method of utilizing asymmetric connectors to significantly improve the dynamic range of PLA, thereby addressing a major limitation of the assay. Sensitivity, LOD, and maximum S/BG were also improved. By enhancing its already successful predecessor, this asymmetric PLA method approaches an ideal assay system, providing a homogeneous and highly specific protein assay with the capability to detect very small quantities of protein in free solution, at high throughput, and with good dynamic range. Furthermore, an experimental model of proximity hybridization was successfully developed and tested. This model system ultimately improved our understanding of the proximity effect and its inherent cooperative assembly in PLA, highlighting the importance of the interplay between connector-to-aptamer affinities and aptamer-to-target affinities in maximizing S/BG and dynamic range. Generally speaking, low connector affinities can be paired with high probe affinities (aptamers or antibodies) to achieve this goal. ACKNOWLEDGMENT Support for this work was provided by the Auburn University Department of Chemistry and Biochemistry. The authors thank Professor Douglas C. Goodwin for use of his PCR and gel preparation equipment, as well as Daniel C. Leslie for helpful discussions about proximity ligation and ligation background. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 14, 2010. Accepted July 8, 2010. AC101762M
Anal. Chem. 2010, 82, 6983–6990 Improving Pressure Robustness, Reliability, and Versatility of Solenoid-Pump Flow Systems Using a Miniature Economic Control Unit Including Two Simple Pressure Pulse Mathematical Models Burkhard Horstkotte, † Erich Ledesma, ‡ Carlos M. Duarte, † and Víctor Cerdà* ,‡ Department of Global Change Research, IMEDEA (CSIC-UIB) Institut Mediterráni d’Estudis Avançats, Miquel Marques 21, 07190 Esporles, Spain, and University of the Balearic Islands, Department of Chemistry, Carreterra de Valldemossa km 7, 5, 07011 Palma de Mallorca, Spain In this work we have systematically studied the behavior of solenoid pumps (SMP) as a function of flow rate and flow resistance. Using a new, economic, and miniature control unit, we achieved improvements of the systems versatility, transportability, and pressure robustness. A further important improvement with respect to pressure resistance was achieved when a flexible pumping tube was inserted between the solenoid pump and the flow resistance acting as a pressure reservoir and pulsation damper. The experimental data were compared with two pressure pulse models for SMP, which were developed during this work and which were well-suited to describe the SMP operation. Solenoid micropumps (SMP) have gained considerable importance as liquid drivers in analytical flow techniques (FT) since their first application in flow systems reported by Weeks and Johnson. 1 The initial motivation for the use of SMP was miniaturization of field applicable FT systems. SMP present an economic alterative to (multi)syringe and peristaltic pumps, typically used for flow injection analysis (FIA) 2 and sequential injection analysis (SIA) 3 systems, respectively. They provide a semicontinuous flow with highly pronounced pulsation. It has been considered that this pulsation causes intermediate turbulent conditions in the flow manifold improving mixing of the sample and reagents in comparison with other unsegmented FT, where laminar flow conditions are typical. While in the first works, this feature was considered as a disadvantage due to the oscillations of the detector signal, Lapa et al. proved first that mixing of reagent and sample is enhanced and sensitivity improvements of 50% are feasible. 4 The authors further established the denomination “multipumping flow systems” (MPFS). In contrast to syringe pumps, SMP operate continuously, which bears the potential to shorten the time of analysis or to perform * To whom correspondence should be addressed. Phone: +34 971 173 261. Fax: +34 971 173 426. E-mail: Victor.Cerda@uib.es. † IMEDEA (CSIC-UIB) Institut Mediterráni d’Estudis Avançats. ‡ University of the Balearic Islands. (1) Weeks, D. A.; Johnson, K. S. Anal. Chem. 1996, 68, 2717–2719. (2) Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1975, 78, 145–157. (3) Ruzicka, J.; Marshall, G. Anal. Chim. Acta 1990, 237, 329–343. (4) Lapa, R. A. S.; Lima, J. L. F. C.; Reis, B. F.; Santos, J. L. M.; Zagatto, E. A. G. Anal. Chim. Acta 2002, 466, 125–132. analyte from large sample volumes. 5,6 In contrast to and in advantage over peristaltic pumps, in MPFS, each flow channel can be controlled individually. Both FIA and SIA manifold configurations have been successfully applied so far using SMP, i.e., confluent, codirectional flows, and contra-directional (aspiration and dispense), respectively. 4,7 Analytical applications of MPFS have been reviewed in detail elsewhere. 8-10 However, SMP show two considerable shortcomings: they do not work reliable in the presence of gas bubbles and particulate matter affecting both the actuation of the build-in check valves and they are notably liable to flow backpressure leading to a decrease of the effective flow with increasing flow resistance. Although there is an obvious interest in the improvement of the pressure robustness and flow rate reliability, up to date, there have been hardly any efforts to complete this objective. In this work, we studied the pressure robustness of the SMP as a function of backpressure including two strategies to improve of the pressure robustness. These have been the adaptation of the activation time, made possible by the use a versatile and highly economic relay card and software control, and the use of inflatable pumping tubes to decrease the peak backpressure. We further present for the first time simple models of the pressure and flow pulse behavior as a function of pump dimensions and manifold characteristics, which were capable of explaining the observations made from the experiments undertake . MATERIALS AND METHODS Distilled water was used throughout. For the evaluation of flow rates, pumped volumes where quantified by weighing on an analytical balance, including compensation of the water density at ambient temperature. The self-priming SMP from Bio-Chem Fluidics (Boston, NJ) of nominal 8 µL, 40 µL, and two of 25 µL volumes were used (types (5) Pons, C.; Santos, J. L. M.; Lima, J. L. F. C.; Forteza, R.; Cerdà, V. Microchim. Acta 2008, 161, 73–79. (6) Pons, C.; Forteza, R.; Cerdà, V. Anal. Chim. Acta 2005, 550, 33–39. (7) Pinto, P. C. A. G.; Saraiva, M.L.M.F.S.; Santos, J. L. M.; Lima, J. L. F. C. Anal. Chim. Acta 2005, 539, 173–179. (8) Lima, J. L. F. C.; Santos, J. L. M.; Dias, A. C. B.; Ribeiro, M. F. T.; Zagatto, E. A. G. Talanta 2004, 64, 1091–1098. (9) Rocha, F. R. P.; Reis, B. F.; Zagatto, E. A. G.; Lima, J. L. F. C.; Lapa, R. A. S.; Santos, J. L. M. Anal. Chim. Acta 2002, 468, 119–131. (10) Santos, J. L. M.; Ribeiro, M. F. T.; Dias, A. C. B.; Lima, J. L. F. C.; Zagatto, E. E. A. Anal. Chim. Acta 2007, 600, 21–28. 10.1021/ac101250h © 2010 American Chemical Society 6983 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/20/2010
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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
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Figure 2. (a) ESI-MS spectrum showi
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Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
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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
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Page 205 and 206: Figure 1. Schematic flowchart showi
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Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
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Page 217 and 218: Scheme 2. Fragmentation Mechanism o
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Page 223 and 224: Figure 5. (A) MALDI-TOF/TOF product
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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 and 236: allow the use of higher aptamer con
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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
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Page 255 and 256: field. DNA profiles were delivered
Page 257 and 258: Another approach to handle this lim
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Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: CONCLUSIONS In this paper we have i
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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
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Page 279 and 280: luciferase (T7 control vector) was
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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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