Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Table 2. Detection of Teicoplanin in Human Blood Using the Current Assay or a Commercial FPIA Kit spiked (µg/mL) this method recovery (%) FPIA recovery (%) 9.5 9.2 ± 0.2 97 9.8 ± 0.6 103 19 19.6 ± 0.4 103 18.5 ± 0.6 97 38 39.0 ± 1.0 103 37.8 ± 4.4 99 obtain the original drug concentration. Meanwhile, each plasma sample was also analyzed using a commercial FPIA kit. The spiked and determined concentrations were summarized in Table 2. When human blood samples from different donors were used as the background matrix for spiking, the variation between the determined drug concentrations was similar to the deviation among multiple tests performed using blood samples from the same donor. The accuracy and precision of the current assay is comparable to the commercial kit. In conclusion, we developed a simple analytical assay for the detection and quantification of selected glycopeptides antibiotics, including vancomycin, teicoplanin, and telavancin, in various samples. The method is highly selective toward glycopeptide 7048 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 antibiotics and does not need complicated pretreatment for serum and blood samples. In addition, no antibody is used in our method; therefore, we expect the shelf life and storage stability of our assay to greatly exceed those of the commercial FPIA kits. We have stored the AF680-peptide probe at room temperature in the dark for 9 months. No significant loss of signal intensity or binding affinity to antibiotics could be detected. ACKNOWLEDGMENT This work was supported by a RCTF fellowship and the faculty startup fund from University of Kentucky. The authors thank Mr. Raymond Miracle from LABfx LLC for suggestions on the teicoplanin analysis using the FPIA kit. 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 February 28, 2010. Accepted July 7, 2010. AC100543E
Anal. Chem. 2010, 82, 7049–7052 Development of a High Sensitivity Rapid Sandwich ELISA Procedure and Its Comparison with the Conventional Approach Chandra Kumar Dixit, †,‡ Sandeep Kumar Vashist, †,|,# Feidhlim T. O’Neill, † Brian O’Reilly, † Brian D. MacCraith, †,§ and Richard O’Kennedy* ,†,‡,§ Centre for Bioanalytical Sciences (CBAS), National Centre for Sensor Research, Applied Biochemistry Group, School of Biotechnology, and Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin 9, Ireland, and Bristol-Myers Squibb (BMS), Swords Laboratories, Watery Lane, Swords, Co. Dublin, Ireland A highly sensitive and rapid sandwich enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of human fetuin A/AHSG (r2-HSglycoprotein), a specific biomarker for hepatocellular carcinoma and atherosclerosis. Anti-human fetuin A antibody was immobilized on aminopropyltriethoxysilanemediated amine-functionalized microtiter plates using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide-based heterobifunctional cross-linking. The analytical sensitivity of the developed assay was 39 pg/mL, compared to 625 pg/ mL for the conventional assay. The generic nature of the developed procedure was demonstrated by performing human fetuin A assays on different polymeric matrixes, i.e., polystyrene, poly(methyl methacrylate), and polycyclo-olefin (Zeonex), in a modified microtiter plate format. Thus, the newly developed procedure has considerable advantages over the existing method. Conventional enzyme-linked immunosorbent assay (ELISA) procedures have been followed for decades for the detection of analytes of importance in industrial, healthcare, and academic research. However, improvement on existing ELISA technologies are continuously attempted by many groups. 1,2 We report the development of a high-sensitivity ELISA-based assay for human fetuin A (HFA), with lower detection limits and a higher sensitivity than commercially available assays. The biological importance of HFA, a member of the cystatin superfamily, which is commonly present in the cortical plate of the immature cerebral cortex and hemopoietic matrix of bone marrow, is discussed elsewhere. 3-10 There are many commercially available ELISA kits for fetuin A, * To whom correspondence should be addressed. Email: richard.okennedy@dcu.ie. Tel.: +353 1 700 7810. Fax: +353 1 700 5412. † Centre for Bioanalytical Sciences (CBAS), National Centre for Sensor Research, Dublin City University. ‡ Applied Biochemistry Group, School of Biotechnology, Dublin City University. § Biomedical Diagnostics Institute (BDI), Dublin City University. | Bristol-Myers Squibb (BMS). # Current Address: Nanoscience and Nanotechnology Initiative (NUSNNI), National University of Singapore, Engineering Drive 1, Singapore 117576. (1) Kaur, J.; Boro, R. C.; Wangoo, N.; Singh, R. K.; Suri, C. R. Anal. Chim. Acta 2008, 607 (1), 92–99. (2) Jia, C. P.; Zhong, H. Q.; Liu, M. Y.; Jing, F. X.; Yao, S. H.; Xiang, J. Q.; Jin, Q. H.; Zhao, J. L. Biosens. Bioelectron. 2009, 24 (9), 2836–2841. which are listed in Supplementary Table 1, Supporting Information. The assay was demonstrated on different commercially relevant solid supports. The developed ELISA is better than the conventional procedure in terms of greatly reduced overall assay duration, higher sensitivity, and greater reproducibility. HFA was taken as the model assay system to demonstrate the utility of our developed ELISA procedure since all the assay components were commercially available in kit form. This enabled us to do robust and highly precise comparison of the developed ELISA with the commercially existing conventional ELISA procedures, as the same assay components were used under the same conditions. The developed procedure can be employed on any commercially relevant substrate. Therefore, this approach of immobilizing antibody on chemically modified solid supports (Figure 1) has potential applications in many other assays and formats. EXPERIMENTAL SECTION Plate Preparation, Amine-Functionalization with Silane, and Cross-Linking Carboxyl Groups of Anti-HFA Antibody to the Amino Groups of the Surface of Microtiter Plate Wells. Each well of the 96-well plate was treated with 100 µL of absolute ethanol for 5 min at 37 °C and washed five times with 300 µL of deionized water (DIW). Subsequently, each well was treated with (3) Kalabay, L.; Gráf, L.; Vörös, K.; Jakab, L.; Benk|Ado˜, Z.; Telegdy, L.; Fekete, B.; Rohászka, Z.; Füst, G. BMC Gastroenterol. 2007, 7, 15. (4) Srinivas, P. R.; Wagner, A. S.; Reddy, L. V.; Deutsch, D. D.; Leon, M. A.; Goustin, A. S.; Grunberger, G. Mol. Endocrinol. 1993, 7, 1445–1455. (5) Mathews, S. T.; Srinivas, P. R.; Leon, M. A.; Grunberger, G. Life Sci. 1997, 61 (16), l383–1392. (6) Kalabay, L.; Prohászka, Z.; Füst, G.; Benkõ, Z.; Telegdy, L.; Szalay, F.; Tóth, K.; Gráf, L.; Jakab, L.; Pozsonyi, T.; Arnaud, P.; Fekete, B.; Karádi, I. In Liver Cirrhosis: New Research; Chen, T. M., Ed.; Nova Science: New York, 2005; pp 63-75. (7) Reynolds, J. L.; Skepper, J. N.; McNair, R.; Kasama, T.; Gupta, K.; Weissberg, P. L.; Dechent, W. J.; Shanahan, C. M. J. Am. Soc. Nephrol. 2005, 16, 2920–2930. (8) Jethwaney, D.; Lepore, T.; Hassan, S.; Mello, K.; Rangarajan, R.; Dechent, W. J.; Wirth, D.; Sultan, A. A. Infec. Immun. 2005, 73 (9), 5883–5891. (9) Ix, J. H.; Shlipak, M. J.; Brandenburg, V. M.; Ali, S.; Ketteler, M.; Whooley, M. A. Circulation 2006, 113, 1760–1767. (10) Hermans, M. M. H.; Brandenburg, V.; Ketteler, M.; Kooman, J. P.; Sande, F. M. V. D.; Boeschoten, E. W.; Leunissen, K. M. L.; Krediet, R. T.; Dekker, R. T. Kidney Int. 2007, 72, 202–207. (11) Park, S. J.; Jung, W. Y. J. Colloid Interface Sci. 2002, 250, 93–98. (12) Cass, T.; Ligler, F. S. Immobilized biomolecules in analysis: a practical approach; Oxford University Press Inc.: New York, 1998. 10.1021/ac101339q © 2010 American Chemical Society 7049 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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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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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
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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 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
Page 287 and 288: Figure 2. SEM images of Au substrat
Page 289 and 290: Table 3. Film Thickness Measurement
Page 291 and 292: Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294: Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296: Figure 4. (A) Variation in the FL i
Page 297 and 298: Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300: Scheme 1. Proposed Mechanism for Fl
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Page 303: Figure 2. Free fluorophores do not
Page 307 and 308: Figure 2. Comparative analysis of d
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