Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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pH with a tilted baseline (Figure 1, curve c). It is of interest to<br />
compare the migration order of these peaks with MEKC performed<br />
at normal electrophoretic separation conditions by Caslavska<br />
et al. 11a In such a study, a buffer composed of 75 mM sodium<br />
dodecyl sulfate (SDS), 6 mM Na2B4O7, and 10 mM Na2HPO4<br />
(pH 9.2) is used with the separation potential set at +20 kV.<br />
The migration sequence is TRP, HVA, VMA, and IXS, which<br />
is opposite to the migration order shown in Figure 1. The<br />
remaining 7 catecholamines and indoleamines with a positive<br />
charge from the ammonium ion (NH3 + ) and/or the secondary<br />
amino group (-NH + -) were completely ionized at pH 3 and<br />
migrated to the cathode with high mobilities, i.e., countercurrent<br />
to the EOF. However, they were eventually driven out the<br />
capillary by EOF with higher mobility.<br />
Notice that a buffer composed of 200 mM boric acid, 100 mM<br />
potassium hydroxide, and 0.1% hydroxyethylcellulose, pH 9.2, has<br />
been used to separate VMA from various indoleamines under<br />
normal separation. 19 In this case, VMA trailed far behind other<br />
indoleamines, as expected from its negative charge at this pH.<br />
PDDA can also be added as a buffer additive as described by<br />
Tseng et al., 20 resulting in high and reversed EOF. The mobility<br />
of indolamines and catecholamines decreases as the PDDA<br />
concentration increases. The separation of 14 analytes including<br />
indolamines, catecholamines, and metanephrines is achieved<br />
within 33 min under optimal separation conditions (1.2% PDDA<br />
and 5 mM formic acid at pH 4.0). Indeed, PDDA has been used<br />
to coat fused silica capillary to form a thin film for the subsequent<br />
absorption of carbon nanotubes. 21 Besides adsorption, PDDA can<br />
be chemically bonded onto the interior capillary wall with an<br />
anodal EOF independent of pH ranging from 2.2 to 8.8. 22 The<br />
lifetimes of both the bonded and physically coated capillaries<br />
exceeded 40 h of continuous use at 240 V/cm at pH 4. Our<br />
experimental data confirmed that the PDDA could be reused for<br />
several repeated runs and the capillary was easily reconditioned<br />
as described earlier. The coated capillary also exhibited good<br />
tolerance to methanol and 0.1 HCl.<br />
Separation on Capillary Coated with PDDA-Gold Nanoparticles.<br />
Our next strategy was to form gold nanoparticles<br />
(AuNPs) on the capillary wall, since they have been known to<br />
interact with several compounds with amino, hydroxyl, and<br />
carboxylic groups. 23 In principle, AuNPs can be prepared separately<br />
and adsorbed on the PDDA layer. This concept has been<br />
used to coat the glass microchip channel with citrate-stabilized<br />
AuNPs for the separation of phenols. 24 Another approach is to<br />
form AuNPs by electroless plating via hydroxylamine-mediated<br />
reduction. 17 However, the charge of the PDDA-AuNP layer returns<br />
to negative and the separation must be performed under normal<br />
separation. Indeed, covalent attachment can be used to immobilize<br />
AuNPs onto the capillary wall, for instance, the preparation of<br />
dodecanethiol AuNPs on prederivatized 3-aminopropyl-trimethox-<br />
(19) Stocking, C. J.; Slater, J. M.; Simpson, C. F. Exp. Nephrol. 1998, 6, 415–<br />
420.<br />
(20) Tseng, W.-L.; Chen, S.-M.; Hsu, C.-Y.; Hsieh, M.-M. Anal. Chim. Acta 2008,<br />
613 (1), 108–115.<br />
(21) Luong, J. H.T.; Bouvrette, P.; Liu, Y.; Yang, D.-Q.; Sacher, E. J. Chromatogr.<br />
A 2005, 1 (2), 187–194.<br />
(22) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 35 (3), 126–130.<br />
(23) Zhong, Z. Y.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gendanken,<br />
A. J. Phys. Chem. B 2004, 108 (13), 4046–4052.<br />
(24) Pumera, M.; Wang, J.; Grushka, E.; Polsky, R. Anal. Chem. 2001, 73, 5625–<br />
5628.<br />
Figure 2. Electropherograms obtained using a PDDA-AuNPs coated<br />
capillary at pH 3, 4, and 5. Other conditions were same as Figure 1.<br />
ysilane or 3-mercaptopropyl-trimethoxysilane fused-silica capillaries.<br />
25 Again; several steps are required for the preparation of such<br />
modified capillaries.<br />
In order to maintain the positive charge for the coating layer,<br />
a one step procedure described by Chen et al. 13 was used to<br />
prepare the PDDA-AuNP composite since PDDA acts as both<br />
reducing and stabilizing agents for AuNPs. The PDDA-gold colloid<br />
exhibited a surface plasmon resonance (SPR) around 527-528<br />
nm, which could be related to AuNPs with the mean diameter of<br />
27-28 nm, in agreement with the report on the SPR peak obtained<br />
for AuNPs with an average diameter of 27 nm. 26 Dynamic light<br />
scattering confirmed that AuNPs should have a mean diameter<br />
of 28 nm with narrow particle size distribution. A TEM micrograph<br />
of PDDA-AuNPs indicated that AuNPs exhibited an average size<br />
of 25 nm (figure not shown). Notice that the excellent stability of<br />
AuNPs arises from the electrosteric effect of PDDA, in agreement<br />
with Mayer et al. 27 where PDDA is simply used as a protecting<br />
and stabilizing agent for AuNPs. Under the same electrophoretic<br />
and detection condition for the PDDA coated capillary, the<br />
electropherogram obtained for the 10 analytes using the PDDA-<br />
AuNP coated capillary is shown in Figure 2. At pH 3, EP was<br />
satisfactorily separated from NMN and the peaks were significantly<br />
sharper with low background current, resulting in significantly<br />
improved detection sensitivity. For the microchip channel<br />
with coated PDDA/AuNPs layer-by-layer, improved resolution and<br />
detection sensitivity has been reported for the three aminophenols,<br />
although the rationale behind such behavior is not known. 24 Our<br />
experimental data also confirmed that the run was longer at pH<br />
4 without any improvement in the separation of the NMN-EP pair<br />
with slightly reduced detection sensitivity. At pH 5, only four peaks<br />
(IXS, VMA, HVA and TRP) emerged in the electropherogram with<br />
the TRP peak far behind the HVA peak.<br />
The circumstances determining the effect of the PDDA-AuNP<br />
coated capillary are complex. The viscosity of the PDDA-AuNP<br />
solution is about 90% of the PDDA solution, i.e., it is easier to<br />
pump the PDDA-AuNP solution through the capillary to form a<br />
(25) (a) O’Mahony, T.; Owens, V. P.; Murrihy, J. P.; Guihen, E.; Holmes, J. D.;<br />
Glennon, J. D J. Chromatogr. A 2003, 1004 (1-2), 181–193. (b) Yang, L.;<br />
Guihen, E.; Holmes, J. D.; Loughran, M.; O’Sullivan, G. P.; Glennon, J. D.<br />
Anal. Chem. 2005, 77 (6), 1840–1846.<br />
(26) Nasir, S. M.; Nur, H. J. Fundam. Sci. 2008, 4, 245–252.<br />
(27) Mayer, A. B. R.; Hausner, S. H.; Mark, J. E. Polym. J. 2000, 32, 15–22.<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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