Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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slanted or herringbone grooves were fabricated in poly(dimethylsiloxane) (PDMS) chips, and the mixing characteristics of these channels compared to channels without grooves. A thin-film Pt electrode at the end of the fluidic channel served as electrochemical detector of the reaction product, H2O2. 2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. All chemicals were analytical reagent-grade. Fluorescein and potassium iodide (KI) were purchased from Sigma-Aldrich (Germany) and used to prepare 0.002 M fluorescein solution and 0.2 M KI solution, respectively. Potassium ferricyanide (K3Fe(CN)6) and potassium ferrocyanide (K4Fe(CN) 6) were supplied by Sigma-Aldrich (Germany) and used to prepare a1mMsolution in a 20 mM phosphate buffer (pH 7.2). pH was measured using a pH meter (inoLab pH 720, WTW, Germany). The phosphate buffer was prepared by mixing solutions of NaH2PO4 · H2O (63 mL) and Na2HPO4 · 2H2O (100 mL) containing 0.1 M potassium nitrate (KNO3) asa supporting electrolyte. H2O2 (30%) was supplied by VWR (The Netherlands) and prepared freshly every day at various concentrations in phosphate buffer. D-glucose and GOx were supplied from Merck (Germany) and used to prepare 100 mM and 5000 U/mL stock solutions, respectively. D-glucose was stored in a refrigerator at 2 °C and used for 1 month. GOx was stored at -20 °C and used for 3 months. All solutions were prepared with 18 MΩ ultrapure water purified in an Arium 611 (Sartorius Stedim Biotech, Germany). 2.2. Microfluidic Chip Fabrication. The first microchannels were constructed by standard microfabrication and replicated in the silicone rubber, PDMS (Sylgard 184, Dow Corning, U.S.). The chip layout and design were drawn using the program, Clewin (Wieweb software, Hengelo, The Netherlands). The structure on the silicon master, which served as a mold, was processed with two steps of standard photolithography (Figure 1, right column). 28 A 4 in. p-type (100) silicon wafer (Si-Mat, Germany) (525 µm thickness) was employed as a substrate. The silicon wafer was first cleaned sequentially with acetone, isopropyl alcohol, and deionized water, and dried with N2 gas. The wafer was then treated with hexamethyldisilazane (HMDS) (Sigma-Aldrich, Germany) in a vacuum desiccator for 30 min to improve adhesion of the photoresist (PR). A thick positive PR, AZ4562 (Microchemicals GmbH, Germany), was coated on the silicon wafer using a spin-coater at 1000 rpm for 3 s and baked at 100 °C for 30 min. The resulting PR layer was 35 µm thick. After rehydration at ambient temperature for 3 h, the coated wafer was exposed to ultraviolet (UV) light (365 nm, 10 mW/cm 2 ) using a photomask printed on a high-resolution transparency to pattern the microchannels (resolution 3,810 dpi; Pro-Art BV, Groningen, The Netherlands). The exposed PR was then removed by dipping in a developer solution (AZ351B: deionized water (DI) ) 1:3, Microchemicals GmbH, Germany) and agitating the beaker for 20 min, leaving behind PR ridges for microchannel replication. The wafer was then rinsed in DI water and dried with N 2 gas. Contrary to usual protocol, no postbake step was undertaken for this layer. Rather, the substrate was again exposed to UV light for 10 s using a second transparency photomask to pattern the groove arrays. The (28) Kim, J.; Heo, J.; Crooks, R. M. Langmuir 2006, 22, 10130–10134. 6758 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 1. Fabrication process of microfluidic chip. See text for details. patterned PR was again immersed in a developer (AZ351B: DI ) 1:4) solution for 6 min to form groove structures on top of the microchannel structures. PDMS resin and a curing agent were used for microchannel replication in a PDMS slab. The two liquids were mixed at a weight ratio of 10:1 PDMS: curing agent, and the solution left to stand at ambient conditions for 20 min to remove air bubbles. Afterward, the mixture was poured over the prepared silicon master. A polycarbonate alignment piece was used together with fused-silica capillaries to form access holes to the microchannels during the replication process. Holes (300 µm in diameter) were drilled in the polycarbonate using a CNC machine (Sherline, U.S.) at points corresponding to the locations of the required inlets/ outlets when this piece was aligned with the silicon master. Fusedsilica capillaries were then inserted through the holes and aligned on the microchannel structures on the silicon master. The prepolymer mixture was allowed to cure against the master at 50 °C for4h. 2.3. Thin-Film Electrode Formation. Platinum (Pt) sensing electrodes were formed on a glass wafer by a standard photolithography and lift-off process (Figure 1, left column). A 4 in., 525- µm-thick borofloat glass wafer (Telic, U.S.) was used as a substrate for the electrode patterning. The glass wafer was first cleaned using a piranha solution (96% H 2SO4: 30% H2O2 ratio of 3:1), then thoroughly rinsed in DI water and dried with N2 gas. (WARN- ING: Piranha solution must be handled with extreme caution, as it is highly oxidizing and reacts explosively when it comes into contact with easily oxidized organic solvents. This solution must be used in a well-ventilated fumehood with absolutely no organic solvents in the vicinity. Explosive reactions also occur in the presence of trace amounts of metals such as Pt, Ag, and Mn. Metal tweezers must not be used when cleaning wafers in piranha.) The wafer was then treated with HMDS in a vacuum desiccator for 30 min. An image reversal PR 5214E (Microchemicals GmbH, Germany) was coated using a spin-
coater at 2000 rpm for 20 s, and the substrate was placed on a hot plate at 105 °C for 50 s to soft-bake the resist and remove solvents. Subsequently, the substrate was exposed to UV light (365 nm, 10 mW/cm 2 ) for 2 s using a photomask with electrode pattern. After reversal baking of the substrate at 115 °C for 2 min, a flood exposure was performed for 20 s without mask to solubilize unexposed areas, resulting in a negative image of the mask. Finally, the substrate was immersed in a developer solution (AZ351B: DI water, ratio 1:4) to remove soluble PR and expose the glass surface where the electrodes were to be formed. A lift-off process was performed to obtain Pt sensing electrodes on the patterned glass wafer. A Ti thin-film layer (20 nm) was deposited over the entire wafer by E-beam evaporation (Temescal, U.S.) under a high vacuum of 9 × 10 -6 Torr. Ti was deposited first, as it adheres better to glass than Pt, and in turn, Pt adheres well to Ti. Subsequently, a Pt thin-film layer (150 nm) was grown by E-beam evaporation at 2 × 10 -5 Torr. The glass substrate was then immersed in acetone to dissolve the remaining PR layer, thereby simultaneously lifting off the thin Pt/Ti film on top of it. The metal remained behind in the predefined electrode regions. Groups of three Pt electrodes were patterned; the patterned electrode dimensions were 100 × 1500 µm, and the distance between two electrodes was 30 µm. 2.4. Chip Integration. The dimensions of the channel and grooves on the structured silicon master were determined using a stylus profilometer (Veeco Instrument BV, The Netherlands) before casting PDMS. There are two inlets and one outlet (waste) to the microreactor channel. The two inlet channels are half as wide as the microreactor channel after the Y-junction, to ensure a constant linear flow velocity throughout the device upon introduction of two solutions at the same flow rate. A 1:1 dilution of solutions will also result under these conditions of operation. The inlet channels are both 100 µm wide, 35 µm deep, and 5 mm in length. A ruler is located along the channel to show the distance from the Y-junction. The total length of channel from the Y-junction is 22 mm, while the width and depth of the channel are 200 and 35 µm, respectively, and groove widths are 50 µm. The 122 grooves in the mixing channel were 6-8 µm deep and were patterned at an angle of 45° with respect to the central axis of the channel. The total volume of the mixing/reaction channel is about 150 nL. In order to bond the PDMS slab to the glass chip with the electrodes, the PDMS was smoothly peeled off from the master and cut. The two devices were first treated with UV-generated ozone for 15 min to oxidize the PDMS surface, which creates hydrophilic properties and improves bonding strength. 29 Subsequently, the PDMS slab and glass were immediately aligned under a microscope and brought into contact with each other. The assembled chip was then placed on a hot plate at 140 °C and allowed to cool down to room temperature, after which the chip was irreversibly bonded. The whole bonding procedure was carried out in the cleanroom. Figure 2 shows a fabricated microfluidic chip and examples of the three different types of PDMS channels with integrated electrodes investigated in this (29) Berdichevsky, Y.; Khandurina, J.; Guttman, A.; Lo, Y. H. Sens. Actuators, B 2004, 97, 402–408. Figure 2. (a) Photograph of the detection region of a fabricated microfluidic chip. (b) Three different types of PDMS channels with integrated electrodes: from top to bottom, channels with no grooves, slanted grooves, and herringbone grooves. (c) Schematic diagram of grooves in a channel structure. Note that the grooves were 6-8 µm deep. study, containing either no grooves, slanted grooves, or herringbone grooves. 2.5. Experimental Setup for Fluorescence. In order to characterize mixing, fluorescence detection was used to gain quantitative information about the degree of mixing along the length of the channel. A fluorescence microscope, model “DMIL” (Leica Microsystems, The Netherlands), was equipped with a 10× objective, a mercury arc lamp, and a CCD camera. The mixing process was visualized using a fluorescein filter, which is made for 488 nm excitation and 518 nm emission. Fluorescence quenching was carried out for quantitative analysis using 2 mM fluorescein and 200 mM potassium iodide (KI). KI quenches the fluorescence produced by fluorescein when it is excited at 488 nm. Fluorescein and KI were introduced separately through the inlets into the Y-junction at various flow rates, and the image was captured with a CCD camera using a 20.1 ms exposure time, a gamma setting of 1.0, and a gain of 1.0. The images were acquired at different distances from the Y-junction and analyzed by determining the standard deviation (SD) of the intensity distribution across the entire channel using Lx95P image analysis software. Flow rates were controlled by syringe pumps (ProSense, The Netherlands) connected with silica capillaries to the chip inlets using 350-µm-o.d. and 250-µm-i.d. silica capillaries. 2.6. Experimental Setup for Electrochemical Detection. Cyclic voltammetry and chronoamperometry were carried out using a potentiostat, “Electrochemical Analyzer” (CH Instrument, U.S.), interfaced to a computer. Two thin-film Pt electrodes were used, one as a working electrode (WE), the other as a counter electrode (CE). The Pt electrodes were located 21 mm downstream from the Y-junction. The active area of the Pt WE was 0.02 mm 2 (200-µm-wide channel ×100-µm-wide electrode). A Ag/AgCl wire was used as a reference electrode (RE) and positioned in the reservoir at the end of the device. The RE was prepared by dipping a short piece of Ag wire (250-µmdiameter) for 5 s into a crucible containing melted silver chloride (Sigma-Aldrich, Germany). For the glucose analysis experiments, a 1:3 flow rate ratio of glucose sample to GOx solution was introduced through the two inlets, and measurements were carried out under continuous flow conditions controlled by the syringe pumps. (For microdialysis experi- Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6759
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 and 12: Figure 4. Ion mobility mass spectra
Page 13: GOx glucose + O298 gluconic acid +
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
Page 63 and 64: 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),
Page 161 and 162:
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
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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
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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