Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6756–6763 An Enzymatic Microreactor Based on Chaotic Micromixing for Enhanced Amperometric Detection in a Continuous Glucose Monitoring Application Byeong-Ui Moon, †,‡ Sander Koster, § Klaas J. C. Wientjes, † Radosław M. Kwapiszewski, | Adelbert J. M. Schoonen, † Ben H. C. Westerink, † and Elisabeth Verpoorte* ,‡ Biomonitoring and Sensoring, Pharmaceutical Analysis, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, P.O. Box 196, 9700 AD Groningen, The Netherlands, TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands, and Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, Warsaw, 00-664, Poland The development of continuous glucose monitoring systems is a major trend in diabetes-related research. Small, easy-to-wear systems which are robust enough to function over many days without maintenance are the goal. We present a new sensing system for continuous glucose monitoring based on a microreactor incorporating chaotic mixing channels. Two different types of chaotic mixing channels with arrays of either slanted or herringbone grooves were fabricated in poly(dimethylsiloxane) (PDMS) and compared to channels containing no grooves. Mixing in channels with slanted grooves was characterized using a fluorescence method as a function of distance and at different flow rates, and compared to the mixing behavior observed in channels with no grooves. For electrochemical detection, a thin-film Pt electrode was positioned at the end of the fluidic channel as an on-chip detector of the reaction product, H 2O2. Glucose determination was performed by rapidly mixing glucose and glucose oxidase (GOx) in solution at a flow rate of 0.5 µL/min and 1.5 µL/min, respectively. A 150 U/mL GOx solution was selected as the optimum concentration of enzyme. In order to investigate the dependence of device response on flow rate, experiments with a premixed solution of glucose and GOx were compared to experiments in which glucose and GOx were reacted on-chip. Calibration curves for glucose (0-20 mM, in the clinical range of interest) were obtained in channels with and without grooves, using amperometric detection and a 150 U/mL GOx solution for in-chip reaction. 1. INTRODUCTION Diabetes mellitus is a widespread disease causing heart disease, weight loss, blurry vision, neurological disorders, and * Corresponding author. E-mail: e.m.j.verpoorte@rug.nl, Telephone: +31-50- 363-3337; fax: +31-50-363-7582. † Biomonitoring and Sensoring, University of Groningen. ‡ Department of Pharmacy, University of Groningen. § TNO Quality of Life. | Warsaw University of Technology. 6756 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 even death. 1 Proper management of blood glucose is thus of crucial importance for diabetic patients. The conventional way blood glucose determinations are carried out involves the finger-prick method. Usually, diabetic patients measure their own blood glucose several times per day by applying a drop of blood to a portable device. However, this intermittent monitoring does not yield the full range of information necessary to effectively control glucose levels 24 h a day. The current research trend is thus toward real-time in vivo monitoring over longer periods (days). 2-5 Type 1 diabetic patients in particular could benefit from a portable glucose sensor to keep their glucose values within a reasonable range. Theoretically, a portable glucose sensor could include an insulin pump in a feedback loop to serve as an artificial pancreas. Electrochemical detection is commonly used for glucose analysis. 6 The advantages of electrochemical detection include sensitivity and ease of interfacing with detection electronics. Several reports incorporating electrochemical detection on a microchip have been published for the detection of glucose. 7-9 The principle of the enzymatic reaction involved is shown in eqs 1 and 2. Glucose, O2 and glucose oxidase (GOx) react to generate hydrogen peroxide (H2O2) at concentrations proportional to the original glucose concentrations. The H2O2 is then oxidized under applied potential into O2 and H + , with the resulting electrons being recorded as an electrical current. (1) Patient Education Association. http://www.nlm.nih.gov/medlineplus/ tutorials/diabetesintroduction/htm/index.htm. (Accessed July 12, 2010). (2) Klonoff, D. C. Diabetes Care 2005, 28, 1231–1239. (3) Feldman, B.; Brazg, R.; Schwartz, S.; Weinstein, R. Diabetes Technol. Ther. 2003, 5, 769–779. (4) Lutgers, H. L.; Hullegie, L. M.; Hoogenberg, K.; Sluiter, W. J.; Dullaart, R. P. F.; Wientjes, K. J.; Schoonen, A. J. M. Neth. J. Med. 2000, 57, 7–12. (5) Tamada, J. A.; Garg, S.; Jovanovic, L.; Pitzer, K. R.; Fermi, S.; Potts, R. O. JAMA, J. Am. Med. Assoc. 1999, 282, 1839–1844. (6) Updike, S. J.; Hicks, G. P. Nature 1967, 214, 986–988. (7) Wang, J.; Chatrathi, M. P.; Tian, B.; Polsky, R. Anal. Chem. 2000, 72, 2514– 2518. (8) Wilke, R.; Büttgenbach, S. Biosens. Bioelectron. 2003, 19, 149–153. (9) Yamaguchi, A.; Jin, P.; Tsuchiyama, H.; Masuda, T.; Sun, K.; Matsuo, S.; Misawa, H. Anal. Chim. Acta 2002, 468, 143–152. 10.1021/ac1000509 © 2010 American Chemical Society Published on Web 07/21/2010
GOx glucose + O298 gluconic acid + 0.7 V H2O298 H 2 O 2 (GOx: glucose oxidase) (1) 2H + + O 2 + 2e - The reaction may be carried out by mixing the reagents in solution to produce H2O2 which is then subsequently detected by integrated electrodes, 7,8 or by immobilizing GOx directly onto the detection electrode surface to react with glucose in solution. 9 The electrochemical detectors in these microchips displayed fast response times and low detection limits. Immobilizing GOx certainly reduces its consumption significantly compared to devices in which GOx is added in solution. However, the major disadvantage of this approach is that the enzyme layer is prone to biofouling in in vivo applications, which might cause a decrease in activity over time and poor performance as a result. 10,11 Over the past decade, a number of glucose sensor designs have been developed and become available for continuous monitoring purposes. 12-16 Mastrototaro et al. 14 reported an in vivo needle-type continuous glucose monitoring system (CGMS), which is currently being used by consumers as a kit (MiniMed). The detector in this system is based on the reaction of immobilized glucose oxidase on the electrode with glucose. Schoemaker et al. 15 and Wientjes et al. 17 have both reported a subcutaneous continuous glucose monitoring system (SCGM) based on a microdialysis probe and using the solution-based enzymatic reaction with glucose. They reported reliable measurements in diabetes patients over periods of four days 18 up to two weeks. 19,20 However, due to their bulky size and the need for connecting tubes between system components, these systems exhibited relatively long physical lag times of 30 min or more. Pickup et al. 13 reviewed the clinical use of glucose monitoring systems and introduced new technology for noninvasive glucose sensing, based on near-infrared spectroscopy and fluorescence. Wentholt et al. 21 (10) Gerritsen, M.; Jansen, J. A.; Lutterman, J. A. Neth. J. Med. 1999, 54, 167– 179. (11) Wisniewski, N.; Moussy, F.; Reichert, W. M. Fresenius’ J. Anal. Chem. 2000, 366, 611–621. (12) Maran, A.; Crepaldi, C.; Tiengo, A.; Grassi, G.; Vitali, E.; Pagano, G.; Bistoni, S.; Calabrese, G.; Santeusanio, F.; Leonetti, F.; Ribaudo, M.; Di Mario, U.; Annuzzi, G.; Genovese, S.; Riccardi, G.; Previti, M.; Cucinotta, D.; Giorgino, F.; Bellomo, A.; Giorgino, R.; Poscia, A.; Varalli, M. Diabetes Care 2002, 25, 347–352. (13) Pickup, J. C.; Hussain, F.; Evans, N. D.; Sachedina, N. Biosens. Bioelectron. 2005, 20, 1897–1902. (14) Mastrototaro, J. J. Diabetes Technol. Ther. 2000, 2, 13–18. (15) Schoemaker, M.; Andreis, E.; Roper, J.; Kotulla, R.; Lodwig, V.; Obermaier, K.; Stephan, P.; Reuschling, W.; Rutschmann, M.; Schwaninger, R.; Wittmann, U.; Rinne, H.; Kontschieder, H.; Strohmeier, W. Diabetes Technol. Ther. 2003, 5, 599–608. (16) Suzuki, H.; Tokuda, T.; Miyagishi, T.; Yoshida, H.; Honda, N. Sens. Actuators, B 2004, 97, 90–97. (17) Wientjes, K. J. C.; Grob, U.; Hattemer, A.; Hoogenberg, K.; Jungheim, K.; Kapitza, C.; Schoonen, A. J. M. Diabetes Technol. Ther. 2003, 5, 615–620. (18) Kapitza, C.; Lodwig, V.; Obermaier, K.; Wientjes, K. J. C.; Hoogenberg, K.; Jungheim, K.; Heinemann, L. Diabetes Technol. Ther. 2003, 5, 609–614. (19) Wientjes, K. J.; Vonk, P.; Vonk-van Klei, Y.; Schoonen, A. J.; Kossen, N. W. Diabetes Care 1998, 21, 1481–1488. (20) Schoonen, A. J. M.; Schmidt, F. J.; Hasper, H.; Verbrugge, D. A.; Tiessen, R. G.; Lerk, C. F. Biosens. Bioelectron. 1990, 5, 37–46. (21) Wentholt, I. M. E.; Hoekstra, J. B. L.; DeVries, J. H. Diabetes Technol. Ther. 2007, 9, 399–409. (2) reported an overview of the current applications and clinically relevant aspects of continuous glucose monitors (CGMs), with emphasis on the calibration procedure, interpretation of continuous glucose data, and some important limitations. Overall, these authors concluded that improved accuracy, reliability for longer periods of time, miniaturization, and cost-effectiveness are the main issues which need to be considered in the further development of continuous glucose monitoring systems. 21 Chip-based microfluidic technologies are a good alternative to improve on conventional monitoring approaches such as the SCGM described above. Microfluidic systems, also termed “miniaturized total analysis systems (µTAS)” 22 or “lab-on-a-chip”, are now widely used in analytical chemistry and biological applications. Advantages of these systems include dramatically reduced consumption of chemical reagents, faster reaction times and cost-effectiveness. Micro SCGM systems can thus be envisaged which exploit the solution-based reaction of GOx with glucose in nL volumes in micrometer-sized channels for glucose sensing. The speed of this analysis is determined by the efficiency of mixing reagents in the microfluidic channels. Mixing at the micrometer scale is a challenge, as flows are generally extremely well-defined and laminar. Mixing of two solution streams in a straight microchannel is possible only through means of diffusion, a passive molecular transport process which is very slow. There has therefore been an enormous amount of research done in the past decade or so on how to implement efficient mixing at the nL scale. So-called passive micromixers are generally preferred for many micro analytical flow systems, since these elements do not require the application of an external force to achieve mixing. A large number of passive micromixers have been reported, including a planar laminar flow mixer, 23 a cross-shaped micromixer 24 and a droplet mixer. 25 The approach chosen for our work is one based on chaotic mixing, first described by Stroock et al. 26,27 Mixing is achieved through the incorporation of an array of microgrooves into a microchannel. Flow over the groove array assumes a helical or corkscrew pattern, in which the contact area between two adjacent solutions is increased dramatically to facilitate mixing by diffusion. The long-term goal of the present project is to realize an autonomous, portable sensing system for continuous in vivo glucose monitoring, based on the reaction in solution of GOx with glucose to produce H 2O2. To accomplish this, we have designed a miniaturized glucose sensing system based on microdialysis sampling and lab-on-a-chip technology. In this system, nL amounts of sample and enzyme rapidly mix and react. As highrecovery microdialysis requires flow rates in tissue less than 1 µL/min, 17 we have adapted the microreactor dimensions according to these conditions. In this paper, we describe a new application for chaotic mixing, that is, the efficient and fast mixing of GOx and glucose for reaction. To that end, either (22) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623–2636. (23) Melin, J.; Gimenez, G.; Roxhed, N.; van der Wijngaart, W.; Stemme, G. Lab Chip 2004, 4, 214–219. (24) Wong, S. H.; Bryant, P.; Ward, M.; Wharton, C. Sens. Actuators, B 2003, 95, 414–424. (25) Paik, P.; Pamula, V. K.; Fair, R. B. Lab Chip 2003, 3, 253–259. (26) Stroock, A. D.; Dertinger, S. K. W.; Ajdari, A.; Mezic, I.; Stone, H. A.; Whitesides, G. M. Science 2002, 295, 647–651. (27) Stroock, A. D.; Dertinger, S. K.; Whitesides, G. M.; Ajdari, A. Anal. Chem. 2002, 74, 5306–5312. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6757
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11: Figure 4. Ion mobility mass spectra
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: 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
Page 213 and 214:
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
Page 251 and 252:
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
Page 291 and 292:
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
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
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