Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Table 3. LODs in nM for Measured Elements in Different Operating Conditions (NM: Not Measurable) flow injection flow injection precolumn precolumn measured masses STD mode DRC mode SEC STD mode SEC DRC mode 63Cu 4.8 5.8 13.0 20.0 65Cu 6.8 7.6 17.0 27.0 48SO 27 077 264 55 760 747 50SO NM 1816 NM 5241 Figure 5. (A) chromatogram of diluted (1:3) healthy subject plasma and (B) Wilson disease untreated patient plasma by using Biosep 3000 and Biosep 2000 connected in series. Cu peak (blue line): (1) TC-Cu; (2) Cp-Cu; (3) Alb-Cu; (4) LMWM-Cu. 50 SO peak (red line): (5) unknown protein(s); (6) albumin. type 1 copper from ceruloplasmin reported to be as labile copper. 37 (37) Musci, G.; Fraterrigo, T. Z.; Calabrese, L.; McMillin, D. R. J. Biol. Inorg. Chem. 1999, 4, 441–446. 6910 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Even if SEC columns are known to be neutral toward the analytes/solid phase interactions, our results show that weakly bound Cu by unspecific coordination with proteins such as albumin and the type 1 Cu of ceruloplasmin can be partially released. SEC columns are neutral toward proteins but not toward metals of the proteins if it is not structurally protected. The copper atoms in our case only coordinate to amino acids of the protein and can readily be captured by remaining silanol groups in this silica based column. Upon the profile obtained from the WD we better understand the deposits of Cu in target organs which is unlikely to happen in healthy subjects because their loosely bound Cu is much lower. We believe that this fraction represents the copper that proteins can deliver and that its quantification is useful in WD. CONCLUSION This method is designated to be applied to a larger number of WD patients in order to use it for diagnostic purpose. Moreover, a possible correlation between the loosely bound Cu and the clinical state should be explored in WD. The same approach could be undertaken for other metalloproteins where metal ions are bound by coordination such as zinc proteins. At last, the sulfur monitoring improved tremendously our proteins analysis method. It allowed the SEC column calibration and offered a wide dynamic range for S determination, and hence proteins quantification, by offering the possibility to use both S isotopes. ACKNOWLEDGMENT We thank the Association Bernard PEPIN pour la Maladie de Wilson for financial support. Received for review April 29, 2010. Accepted July 7, 2010. AC101128X
Anal. Chem. 2010, 82, 6911–6918 Direct Quantification of Single-Molecules of MicroRNA by Total Internal Reflection Fluorescence Microscopy Ho-Man Chan, † Lai-Sheung Chan, ‡ Ricky Ngok-Shun Wong, ‡ and Hung-Wing Li* ,† Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. China, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. China MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT- PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis. MicroRNAs (miRNAs) are categorized as a class of small noncoding RNAs with approximately 19-23 nucleotides. It serves as the gene expression and cell development regulator in animals, plants, and viruses by interfering protein synthesis. 1,2 Evidences indicated that miRNAs play important roles in cell proliferation, differentiation, and apoptosis. 2-4 Recent researches have also explored the differential expression levels of miRNAs in cancerous * Corresponding author: (phone) +852-3411-7065; (fax) +852-3411-7348; (e-mail) hwli@hkbu.edu.hk. † Department of Chemistry, Hong Kong Baptist University. ‡ Department of Biology, Hong Kong Baptist University. (1) Ambros, V. Cell 2003, 113, 673–676. (2) Ambros, V. Nature 2004, 431, 350–355. (3) Carthew, R. W. Curr. Opin. Genet. Dev. 2006, 16, 203–208. (4) Nakahara, K.; Carthew, R. W. Curr. Opin. Cell Biol. 2004, 16, 127–133. and noncancerous tissues and displayed its roles as tumor suppressors or oncogenes. 5-7 Tumor formation caused by miR- NAs is notable since it has been proven that a single sequence of miRNA can regulate multiple gene targets. In the meanwhile, a single gene target can also be regulated by multiple miRNAs. Therefore, on account of the connections between miRNA and cancer development, profiling of miRNA expression levels is proposed as a vital tool for the preliminary diagnosis and prognosis of cancer. 8 A majority of the miRNA detection methods are based on the approach of hybridization, in which target molecules of interest arecapturedorhybridizedwiththecomplementaryoligonucleotides. 8,9 Hereafter, the frequency of hybridization events are presented in form of measurable signals for the quantification of miRNA. However, the detection of the small size and trace amount of miRNAs is always challenging. Northern blotting is the most prevalent and accredited method for the quantification of miR- NAs. 10 The technique allows multiplex detection, but it is laborious and sample intensive. Another convincing technique for the determination of miRNA is the real-time polymerase chain reaction (qRT-PCR). 11-13 With PCR, copies of specific miRNAs and their corresponding signals are amplified and intensified, respectively. Nonetheless, the short length of target miRNA and intricate design of primer limited the reliability of amplification and labeling. 8 Other amplification strategies have also been proposed in recent years aiming at higher detection sensitivity, specificity, and lower consumption of starting materials. For instance, nanoparticles such as OsO2, 14 quantum dots, 15 and gold 15,16 are chemically labeled on detecting probes as signal transducers and amplifiers. Besides, (5) Calin, G. A.; Croce, C. M. Nat. Rev. Cancer 2006, 6, 857–866. (6) Esquela-Kerscher, A.; Slack, F. J. Nat. Rev. Cancer 2006, 6, 259–269. (7) Volinia, S.; Calin, G. A.; Liu, C. G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2257–2261. (8) Wark, A. W.; Lee, H. J.; Corn, R. M. Angew. Chem., Int. Ed. 2008, 47, 644–652. (9) Cissell, K. A.; Deo, S. K. Anal. Bioanal. Chem. 2009, 394, 1109–1116. (10) Lagos-Quintana, M.; Rauhut, R.; Lendeckel, W.; Tuschl, T. Science 2001, 294, 853–858. (11) Chen, C. F.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z. H.; Lee, D. H.; Nguyen, J. T.; Barbisin, M.; Xu, N. L.; Mahuvakar, V. R.; Andersen, M. R.; Lao, K. Q.; Livak, K. J.; Guegler, K. J. Nucleic Acids Res. 2005, 33, 9. (12) Raymond, C. K.; Roberts, B. S.; Garrett-Engele, P.; Lim, L. P.; Johnson, J. M. RNA 2005, 11, 1737–1744. (13) Schmittgen, T. D.; Jiang, J. M.; Liu, Q.; Yang, L. Q. Nucleic Acids Res. 2004, 32, e43. (14) Gao, Z. Q.; Yang, Z. C. Anal. Chem. 2006, 78, 1470–1477. 10.1021/ac101133x © 2010 American Chemical Society 6911 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Published on Web 07/23/2010
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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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Page 117 and 118: data as well. The 41 genes in Table
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Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
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Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165: Table 2. Molecular Properties and C
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Page 171 and 172: solution under appropriate incubati
Page 173 and 174: Figure 4. Standardization curve for
Page 175 and 176: Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
Page 181 and 182: the NB signal in a much better foot
Page 183 and 184: Scheme 1. Reactions of Selenium Rea
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206: Figure 1. Schematic flowchart showi
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Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: 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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