Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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FL, USA) with three-dimensional adjustment capabilities. A cylindrical cathodic/detection reservoir (2 cm diameter ×1 cm height) contained Pt wires (1 mm in diameter, 99.9% purity), serving as the counter electrode for amperometric detection and the cathode for electrophoresis. An Ag/AgCl (3 M NaCl) reference electrode was placed vertically into the reservoir, whereas the BDD electrode was inserted upward from the reservoir’s bottom and sealed with epoxy (the working reservoir volume was ∼3 mL). The micromanipulator and a laboratory jack (to which the reservoir was solidly mounted) were attached to a solid breadboard to prevent movement during alignment. The capillary outlet was aligned to the detecting electrode using the micromanipulator with the aid of a surgical microscope (World Precision Instruments). The capillary outlet was adjusted until it touched the electrode surface (evident by a slight bend in the capillary observed by microscopic inspection) and it was then backed off 25-30 µm using the micromanipulator’s z-control. The BDD electrode was connected to an electrochemical workstation (CHI660C, CH Instruments, Austin, TX, USA) consisting also of a platinum wire (1 mm in diameter) as counter electrode and an Ag/AgCl (3 M NaCl) electrode as reference electrode. The BDD electrode (3 mm in diameter, 0.1% doped diamond) was purchased from Windsor Scientific (Slough, Berkshire, U. K.). The analytes are detected by a boron doped diamond (BDD) electrode which is positioned close to the capillary outlet. The BDD electrode is advocated in this work due to its stable low background current and a wide applied potential window. BDD is also resistant to fouling due to the hydrogen surface termination and sp 3 carbon bonding (no extended pi-electron system). 14a,b BDD can be considered as general-purpose working electrodes with a broad array of applications for use with HPLC and CE. Pioneering work in the early 1990s was conducted by Swain and co-workers 14c,d and the Fujishima group. 14e The electrophoretic separation was conducted at -10 kV (reversed polarity) unless otherwise stated. A plastic cap with a central hole of ∼1 mm was firmly attached to the surface of the BDD electrode to reduce the active sensing area. The analyte sample was injected electrokinetically for 5sat-10 kV. Peak identification was based on the migration time of a single standard with that of unknown peaks. However, if the resolution between any peak pair was low, then peak identification was performed by spiking both solutes individually. The pKa values and aqueous solubility of the analytes were obtained using the ACD/ Structure Designer software (Advanced Chemistry Development, Toronto, ON, Canada). The degree of ionization was estimated as pH ) pKa+ log [A - /AH] for acid and pH ) pKb+log [BH + /B] for base (the Henderson-Hasselbalch equation). RESULTS AND DISCUSSION Performance of the PDDA Coated Capillary. PDDA was firmly adsorbed on the inner walls of the capillary via ionic interactions between the negatively charged SiO - of fused silica and the quaternary ammonium groups of the polymer. Indeed, immersion of a substrate (glass, quartz, silica wafer, gold, silver, and even Teflon) into an aqueous 1% solution of this positively charged polymer results in the strong adsorption of a mono- 6898 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 1. Electropherograms obtained using a PDDA coating capillary (50 µm id and 45 cm effective length) for the separation of 20 µM IXS, 25 µM VMA, 25 µM HVA, 25 µM TRP, 100 µM isoproterenol (ISP), 100 µM normetanephrine (NMN), 100 µM epinephrine (EP), 50 µM 5-hydroxytryptamine (5-HT), 125 µM 4-hydroxy-3-methoxybenzylamine (HMBA), and 75 µM tryptamine (TA). The running buffer consisted of 50 mM H3PO4-Tris, (a) pH 3, (b) pH 4, and (c) pH 5. The separation voltage was applied at -10 kV with an injection time of 5s at -10 kV. BDD at +1.0 V vs Ag/AgCl, 3 M NaCl. layer (1.6 nm) of PDDA on the substrate. 15,16 The adsorption of a PDDA thin film on a glass substrate was also reported elsewhere. 17 Notice also that the charge of PDDA is not pH dependent, as reflected by constant EOF in the range pH 2-8 provided the capillary is coated with high-molecular weight PDDA. 18 Thus, PDDA with MW of 200 000-350 000 was used in this study for coating the capillary. At -10 kV, except for the epinephrine (EP) and normetanephrine (NMN) pair, all analytes were baseline resolved when 50 mM H3PO4-Tris pH 3.0 was used as the running buffer (Figure 1, curve a). On the basis of the calculated pKa values for the analytes (Table 1), fully deprotonated and highly negatively charged IXS exhibited high electrophoretic mobility and migrated concomitantly with EOF as the first peak in the electropherogram. The carboxylate/carboxyl ratio, estimated as 10 (pH-pK a) ,is∼0.16 and 0.04 for VMA and HVA, respectively. Thus, VMA should emerge before HVA and slightly ahead of EOF. At pH 3, the neutral form of TRP should be predominant (neutral TRP/TRP + ) 4.2);therefore, it should migrate very closely to EOF and trail behind both VMA and HMA. The EP-NMN pair was slightly split further by conducting the separation at pH 4 with improved detection sensitivity but the running time was also slightly longer and VMA emerged very close to IXS (Figure 1, curve b). The run was lengthier at pH 5, with only 4 discernible peaks emerging in the electropherogram as HVA comigrated with VMA and TRP became more neutral at this pH and emerged far behind the HVA peak. The remaining analytes acquired more negative charges and interacted strongly with positively charged PDDA and were not eluted after 1200 s into the experiment. The detection sensitivity was also greatly compromised at this running (15) Kotov, N. A.; Harazsti, T.; Turi, L.; Zavala, G.; Geer, R. E.; Dekany, I.; Fendler, J. H. J. Am. Chem. Soc. 1997, 119, 6821–6832. (16) Moriguchi, I.; Teraoka, Y.; Kagawa, S.; Fendler, J. H. Chem. Mater. 1999, 1, 1603–1608. (17) Hrapovic, S.; Liu, Y.; Enright, G.; Bensabaa, F.; Luong, J. H. T. Langmuir 2003, 19, 3958–3965. (18) Wang, Y.; Dubin, P. L. Anal. Chem. 1999, 71, 3463–3468.
pH with a tilted baseline (Figure 1, curve c). It is of interest to compare the migration order of these peaks with MEKC performed at normal electrophoretic separation conditions by Caslavska et al. 11a In such a study, a buffer composed of 75 mM sodium dodecyl sulfate (SDS), 6 mM Na2B4O7, and 10 mM Na2HPO4 (pH 9.2) is used with the separation potential set at +20 kV. The migration sequence is TRP, HVA, VMA, and IXS, which is opposite to the migration order shown in Figure 1. The remaining 7 catecholamines and indoleamines with a positive charge from the ammonium ion (NH3 + ) and/or the secondary amino group (-NH + -) were completely ionized at pH 3 and migrated to the cathode with high mobilities, i.e., countercurrent to the EOF. However, they were eventually driven out the capillary by EOF with higher mobility. Notice that a buffer composed of 200 mM boric acid, 100 mM potassium hydroxide, and 0.1% hydroxyethylcellulose, pH 9.2, has been used to separate VMA from various indoleamines under normal separation. 19 In this case, VMA trailed far behind other indoleamines, as expected from its negative charge at this pH. PDDA can also be added as a buffer additive as described by Tseng et al., 20 resulting in high and reversed EOF. The mobility of indolamines and catecholamines decreases as the PDDA concentration increases. The separation of 14 analytes including indolamines, catecholamines, and metanephrines is achieved within 33 min under optimal separation conditions (1.2% PDDA and 5 mM formic acid at pH 4.0). Indeed, PDDA has been used to coat fused silica capillary to form a thin film for the subsequent absorption of carbon nanotubes. 21 Besides adsorption, PDDA can be chemically bonded onto the interior capillary wall with an anodal EOF independent of pH ranging from 2.2 to 8.8. 22 The lifetimes of both the bonded and physically coated capillaries exceeded 40 h of continuous use at 240 V/cm at pH 4. Our experimental data confirmed that the PDDA could be reused for several repeated runs and the capillary was easily reconditioned as described earlier. The coated capillary also exhibited good tolerance to methanol and 0.1 HCl. Separation on Capillary Coated with PDDA-Gold Nanoparticles. Our next strategy was to form gold nanoparticles (AuNPs) on the capillary wall, since they have been known to interact with several compounds with amino, hydroxyl, and carboxylic groups. 23 In principle, AuNPs can be prepared separately and adsorbed on the PDDA layer. This concept has been used to coat the glass microchip channel with citrate-stabilized AuNPs for the separation of phenols. 24 Another approach is to form AuNPs by electroless plating via hydroxylamine-mediated reduction. 17 However, the charge of the PDDA-AuNP layer returns to negative and the separation must be performed under normal separation. Indeed, covalent attachment can be used to immobilize AuNPs onto the capillary wall, for instance, the preparation of dodecanethiol AuNPs on prederivatized 3-aminopropyl-trimethox- (19) Stocking, C. J.; Slater, J. M.; Simpson, C. F. Exp. Nephrol. 1998, 6, 415– 420. (20) Tseng, W.-L.; Chen, S.-M.; Hsu, C.-Y.; Hsieh, M.-M. Anal. Chim. Acta 2008, 613 (1), 108–115. (21) Luong, J. H.T.; Bouvrette, P.; Liu, Y.; Yang, D.-Q.; Sacher, E. J. Chromatogr. A 2005, 1 (2), 187–194. (22) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 35 (3), 126–130. (23) Zhong, Z. Y.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gendanken, A. J. Phys. Chem. B 2004, 108 (13), 4046–4052. (24) Pumera, M.; Wang, J.; Grushka, E.; Polsky, R. Anal. Chem. 2001, 73, 5625– 5628. Figure 2. Electropherograms obtained using a PDDA-AuNPs coated capillary at pH 3, 4, and 5. Other conditions were same as Figure 1. ysilane or 3-mercaptopropyl-trimethoxysilane fused-silica capillaries. 25 Again; several steps are required for the preparation of such modified capillaries. In order to maintain the positive charge for the coating layer, a one step procedure described by Chen et al. 13 was used to prepare the PDDA-AuNP composite since PDDA acts as both reducing and stabilizing agents for AuNPs. The PDDA-gold colloid exhibited a surface plasmon resonance (SPR) around 527-528 nm, which could be related to AuNPs with the mean diameter of 27-28 nm, in agreement with the report on the SPR peak obtained for AuNPs with an average diameter of 27 nm. 26 Dynamic light scattering confirmed that AuNPs should have a mean diameter of 28 nm with narrow particle size distribution. A TEM micrograph of PDDA-AuNPs indicated that AuNPs exhibited an average size of 25 nm (figure not shown). Notice that the excellent stability of AuNPs arises from the electrosteric effect of PDDA, in agreement with Mayer et al. 27 where PDDA is simply used as a protecting and stabilizing agent for AuNPs. Under the same electrophoretic and detection condition for the PDDA coated capillary, the electropherogram obtained for the 10 analytes using the PDDA- AuNP coated capillary is shown in Figure 2. At pH 3, EP was satisfactorily separated from NMN and the peaks were significantly sharper with low background current, resulting in significantly improved detection sensitivity. For the microchip channel with coated PDDA/AuNPs layer-by-layer, improved resolution and detection sensitivity has been reported for the three aminophenols, although the rationale behind such behavior is not known. 24 Our experimental data also confirmed that the run was longer at pH 4 without any improvement in the separation of the NMN-EP pair with slightly reduced detection sensitivity. At pH 5, only four peaks (IXS, VMA, HVA and TRP) emerged in the electropherogram with the TRP peak far behind the HVA peak. The circumstances determining the effect of the PDDA-AuNP coated capillary are complex. The viscosity of the PDDA-AuNP solution is about 90% of the PDDA solution, i.e., it is easier to pump the PDDA-AuNP solution through the capillary to form a (25) (a) O’Mahony, T.; Owens, V. P.; Murrihy, J. P.; Guihen, E.; Holmes, J. D.; Glennon, J. D J. Chromatogr. A 2003, 1004 (1-2), 181–193. (b) Yang, L.; Guihen, E.; Holmes, J. D.; Loughran, M.; O’Sullivan, G. P.; Glennon, J. D. Anal. Chem. 2005, 77 (6), 1840–1846. (26) Nasir, S. M.; Nur, H. J. Fundam. Sci. 2008, 4, 245–252. (27) Mayer, A. B. R.; Hausner, S. H.; Mark, J. E. Polym. J. 2000, 32, 15–22. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6899
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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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Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
Page 107 and 108: Table 1. Counting Properties and De
Page 109 and 110: Table 3. Analysis of 226 Ra in Sedi
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Page 115 and 116: Figure 5. Volcano plots detailing t
Page 117 and 118: data as well. The 41 genes in Table
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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
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153: Table 1. Chemical Structure, pKa Va
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 and 166: Table 2. Molecular Properties and C
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Page 169 and 170: dilution and hybridization buffer.
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
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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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