Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6854–6861 Improved Sensitivity of DNA Microarrays Using Photonic Crystal Enhanced Fluorescence Patrick C. Mathias, †,‡ Sarah I. Jones, § Hsin-Yu Wu, ‡,| Fuchyi Yang, ‡,| Nikhil Ganesh, ‡,⊥ Delkin O. Gonzalez, § German Bollero, § Lila O. Vodkin, § and Brian T. Cunningham* ,†,‡,| Department of Bioengineering, 1304 W. Springfield Ave., Micro and Nanotechnology Laboratory, 208 N. Wright St., Department of Crop Sciences, 1102 S. Goodwin Ave., Department of Electrical and Computer Engineering, 1406 W. Green St., and Department of Materials Science and Engineering, 1304 W. Green St., University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 DNA microarrays are used to profile changes in gene expression between samples in a high-throughput manner, but measurements of genes with low expression levels can be problematic with standard microarray substrates. In this work, we expand the detection capabilities of a standard microarray experiment using a photonic crystal (PC) surface that enhances fluorescence observed from microarray spots. This PC is inexpensively and uniformly fabricated using a nanoreplica molding technique, with very little variation in its optical properties within- and between-devices. By using standard protocols to process glass microarray substrates in parallel with PCs, we evaluated the impact of this substrate on a onecolor microarray experiment comparing gene expression in two developmental stages of Glycine max. The PCs enhanced the signal-to-noise ratio observed from microarray spots by 1 order of magnitude, significantly increasing the number of genes detected above substrate fluorescence noise. PC substrates more than double the number of genes classified as differentially expressed, detecting changes in expression even for low expression genes. This approach increases the dynamic range of a surface-bound fluorescence-based assay to reliably quantify small quantities of DNA that would be impossible with standard substrates. The DNA microarray is a valuable tool for high-throughput quantification of gene expression, allowing a large number of candidate genes to be examined for differential expression simultaneously without extensive prior knowledge of gene functions. Eukaryotic gene expression is typically characterized by a large number of genes expressed at very low levels and a decreasing number of genes expressed at high levels. 1,2 Often the noise present in DNA microarray experiments is high enough that only a small fraction of genes assayed can be * To whom correspondence should be addressed. Phone: 217-265-6291. E-mail: bcunning@illinois.edu. † Department of Bioengineering. ‡ Micro and Nanotechnology Laboratory. § Department of Crop Sciences. | Department of Electrical and Computer Engineering. ⊥ Department of Materials Science and Engineering. (1) Kuznetsov, V. A.; Knott, G. D.; Bonner, R. F. Genetics 2002, 161, 1321– 1332. 6854 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 detected by fluorescence measurements. While sample variation and nonspecific binding play an important role in this experimental noise, microarray substrate fluorescence can contribute to noise as well. This may explain the poor performance of microarrays in detecting genes with low expression levels relative to other methods. 3,4 To overcome the difficulties of quantifying the abundance of low expression genes, substrates that enhance the fluorescence observed from microarray spots can be used to achieve better assay performance. Researchers have utilized various nanostructured metal substratres to achieve increased intensity from common microarray dyes, with signal enhancements ranging from 1 to 2 orders of magnitude. These methods include the growth of metal island films on a substrate 5,6 or the deposition of nanoparticles fabricated by spray pyrolysis 7 to produce optical resonances to which microarray dye excitation and/or emission can couple. However, the practical impact of these enhancement methods is unclear, because previous work has not characterized the signal-to-noise ratio (SNR) enhancement for a large number of spots over many substrates in a conventional gene expression microarray experiment. One potential obstacle in achieving this result is the need for an inexpensive nanoscale fabrication method achieving highthroughput and good uniformity over large areas; previous reports of microarray dye enhancement have not utilized photolithography to generate consistent patterns and thus are subject to a random arrangement of structures. Another potential obstacle is integration of these substrates with the existing commercial equipment used to fabricate and scan DNA microarrays, as previous literature has not explicitly demonstrated nanostructures over areas as large as conventional microscope slides. We addressed these issues by designing a nanostructured photonic crystal (PC) substrate capable of enhancing Cyanine-5 (Cy-5) fluorescence in a com- (2) Ueda, H. R.; Hayashi, S.; Matsuyama, S.; Yomo, T.; Hashimoto, S.; Kay, S. A.; Hogenesch, J. B.; Iino, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 3765–3769. (3) Kuo, W. P.; Liu, F.; Trimarchi, J.; Punzo, C.; Lombardi, M.; et al. Nat. Biotechnol. 2006, 24, 832–840. (4) t Hoen, P. A. C.; Ariyurek, Y.; Thygesen, H. H.; Vreugdenhil, E.; Vossen, R. H. A. M.; de Menezes, R. X.; Boer, J. M.; van Ommen, G.-J. B.; den Dunnen, J. T. Nucleic Acids Res. 2008, 36, e141. (5) Sabanyagam, C. R.; Lakowicz, J. R. Nucleic Acids Res. 2007, 35, e13. (6) Moal, E. L.; Leveque-Fort, S.; Potier, M.-C.; Fort, E. Nanotechnology 2009, 20, 225502. (7) Guo, S.-H.; Tsai, S.-J.; Kan, H.-C.; Tsai, D.-H.; Zachariah, M. R.; Phaneuf, R. J. Adv. Mater. 2008, 20, 1424–1428. 10.1021/ac100841d © 2010 American Chemical Society Published on Web 07/16/2010
mercial microarray scanner and fabricating it by nanoreplica molding to fit standard microscope slides. The PC substrates are composed of a subwavelength, periodic SiO2 surface structure coated with a high refractive index dielectric layer of TiO2, creating a periodic modulation in refractive index along the device surface. The periodic modulation gives rise to optical resonances 8 that can be used to achieve fluorescence enhancement. These resonances can be used to generate strong optical near-fields at the device surface when spectrally aligned to the excitation wavelength 9 and to spatially alter the fluorescence emission pattern to maximize light collection. 10 The overall effect of these phenomena is to amplify the fluorescent signal from molecules within approximately 100 nm of the PC surface. While the first demonstrations of PC enhanced fluorescence for microarrays required expensive lithographic procedures for each device and yielded modest enhancement factors of approximately 6× signal enhancement in a commercial scanner, 11,12 inexpensive and uniform fabrication over large areas in a nanoreplica molding process currently used to make commercial label-free biosensors 13 has since been employed to make these structures. Recently, PCs have been engineered by our group to enhance the common microarray dye Cyanine-5 (Cy-5) by more than 1 order of magnitude when scanned in a commercial microarray scanner. 14 This work details the application of this PC design to a microarray experiment assessing differential expression between Glycine max cotyledons and trifoliates, which represent tissues from two distinct developmental stages in the soybean plant. Multiple PCs exhibiting highly uniform optical characteristics over the area of entire microscope slides were fabricated by nanoreplica molding. These PCs were processed using published protocols in parallel with commercial microarray substrates. By enhancing fluorescence, a larger number of genes can be detected above noise on the PC compared with glass substrates. This effect more than doubles the number of genes identified as differentially expressed between the trifoliate and cotyledon tissues, demonstrating that enhanced fluorescence offers practical benefit to a DNA microarray experiment. MATERIALS AND METHODS Photonic Crystal Fabrication and Characterization. The PCs used for this work were designed by simulation software employing Rigorous Coupled-Wave Analysis (DiffractMOD, RSoft Design Group, Inc.) to align optical resonances to the excitation (632.8 nm) and emission wavelengths (670-710 nm) of Cy-5. As described in previous work, 14 the period of the structure, grating depth, and thickness of the high refractive index TiO2 layer were (8) Rosenblatt, D.; Sharon, A.; Friesem, A. A. IEEE J. Quantum Electron. 1997, 33, 2038–2059. (9) Ganesh, N.; Mathias, P. C.; Zhang, W.; Cunningham, B. T. J. Appl. Phys. 2008, 103, 083104. (10) Ganesh, N.; Block, I. D.; Mathias, P. C.; Zhang, W.; Chow, E.; Malyarchuk, V.; Cunningham, B. T. Opt. Express 2008, 16, 21626–21640. (11) Neuschafer, D.; Budach, W.; Wanke, C.; Chibout, S.-D. Biosensors Bioelectron. 2003, 18, 489–497. (12) Budach, W.; Neuschafer, D.; Wanke, C.; Chibout, S.-D. Anal. Chem. 2003, 75, 2571–2577. (13) Cunningham, B.; Lin, B.; Qiu, J.; Li, P.; Pepper, J.; Hugh, B. Sensors Actuators B 2002, 85, 219–226. (14) Mathias, P. C.; Wu, H.-Y.; Cunningham, B. T. Appl. Phys. Lett. 2009, 95, 021111. manipulated given the known refractive indices of PC materials to achieve optical resonances overlapping both excitation and emission wavelength ranges. These PCs were then fabricated by nanoreplica molding 13 to create six distinct devices for microarray experiments. The silicon “master” for the molding process consisted of a 360 nm period one-dimensional grating structure with a 60 nm grating depth and 50% duty cycle, patterned on an 8 in. silicon wafer by deep-UV lithography. After immersion in 2% dichlorodimethylsilane (PlusOne Repel- Silane ES, GE Healthcare) to promote clean release, a UVcurable liquid polymer (Gelest, Inc.) with index of refraction npolymer ) 1.46 was dispensed on a sheet of polyethylene terephthalate (PET), and the grating pattern was transferred with a roller. After curing the polymer under a high-intensity ultraviolet lamp (Xenon) for 90 s through the transparent PET sheet, 300 nm of SiO2 (nSiO2 ) 1.46) and 160 nm of TiO2 (nTiO2 ) 2.35) were added to the grating structure by sputter coating. The completed PCs were cut into 1 in. × 3 in. sections and adhered to glass microscope slides with an optically clear adhesive (3M). The PCs were initially profiled for surface characteristics by atomic force microscopy (Dimension 3000, Digital Instruments) to compare the actual dimensions with the PC design. The PCs were then optically characterized by passing broadband light in the visible spectrum from a tungsten halogen lamp (Ocean Optics) through a polarizer and collimator before transmission through the device, which was aligned on a rotational stage to be perpendicular to the direction of incident light. 15 PCs were illuminated with both transverse magnetic (incident electric field perpendicular to grating lines) and transverse electric (incident electric field parallel to grating lines) polarizations to characterize the two distinct resonances. Light transmitted through the PCs was collected by an optical fiber and measured using a UV-visible light spectrometer (Ocean Optics). Slide Preparation. PCs were cleaned by O2 plasma treatment and incubated with 3-glycidoxypropyltrimethoxysilane at 185 mTorr overnight for surface functionalization. The control slides for microarray experiments were commercially available silanized glass slides (Corning GAPS II). Oligonucleotides were printed on the slides using a Genetix QArray 2 robot. A set of previously annotated 192 70-mer oligonucleotides derived from publicly available soybean EST and mRNA sequences was spotted on the slides with 40 repeats per sequence per slide. These 192 oligonucleotides are a subset of a larger 19 200 set of oligonucleotides detailed in previous work. 16 Each of six spotted PCs was matched with a spotted glass control slide to receive identical experimental treatments. Microarray Sample Preparation and Hybridization. Sample RNA was extracted using previously published protocols. 16 Cotyledon RNA was extracted from freeze-dried soybean cultivar Williams seeds with fresh weight between 100-200 mg. Cotyledon RNA was purified using a Qiagen RNeasy kit. Trifoliate RNA was extracted from freeze-dried rolled-up trifoliates of soybean cultivar Williams from leaves between 0.5 and 1.5 in. in length. Sample RNA was labeled with Cy-5 by reverse transcription. Three replicate slides were hybridized for each of the two tissue samples, (15) Yang, F.; Yen, G.; Cunningham, B. T. Opt. Express 2010, 18, 11846–11858. (16) Gonzalez, D. O.; Vodkin, L. O. BMC Genomics 2007, 8, 468. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6855
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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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Page 65 and 66: Polymerase (1 U per sample). Reacti
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Page 75 and 76: Table 2. Concentrations and Ratios
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Page 79 and 80: mg/mL protein, followed by separati
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Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102: Figure 5. Quantitative calibration
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Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
Page 107 and 108: Table 1. Counting Properties and De
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Page 123 and 124: Table 1. Absolute Quantification Re
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Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
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Page 145 and 146: Figure 1. Infrared spectra of (A) u
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Page 153 and 154: Table 1. Chemical Structure, pKa Va
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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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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
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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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