Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Table 2. Linearity and Detection Limit of the Analytes a,b
analytes linear range (µM) calibration equation R2 detection limit (µM)
indoxyl sulfate (IXS) 1.00-40 I ) 0.4839X+0.1490 0.9998 0.3
vanillylmandelic acid (VMA) 1.25-50 I ) 0.3635X+0.3036 0.9998 0.4
homovanillic acid (HVA) 1.25-50 I ) 0.3034X+0.5667 0.9998 0.4
tryptophan (TRP) 1.25-50 I ) 0.3025X+1.0870 0.9998 0.4
isoproterenol (ISP) 5.00-200 I ) 0.0871X+0.7481 0.9995 1.7
normetanephrine (NMN) 5.00-200 I ) 0.0865X+0.6429 0.9995 1.7
epinephrine (EP) 5.00-200 I ) 0.0945X+0.3850 0.9995 1.7
5-hydroxytryptamine (5-HT) 2.50-100 I ) 0.1686X+0.9364 0.9995 0.8
4-hydroxy-3-methoxybenzylamine (HMBA) 6.25-250 I ) 0.0822X+0.0446 0.9991 2.1
3,4- dihydroxybenzylamine (DHBA) 6.25-250 I ) 0.0866X+0.3842 0.9991 2.1
tryptamine (TA) 3.75-150 I ) 0.1476X+0.5307 0.9995 1.3
a I ) peak current (nA), X ) analyte concentration (µM). b Electrophoretic separation was carried out using a PDDA-AuNP coated capillary (50
µm id, 365 µm od) with an effective length of 45 cm, running buffer, 50 mM H3PO4-Tris, pH 3.0, separation voltage, -10 kV, injection for 5s at -10
kV. Detection: BDD electrode at +1.0 V vs. Ag/AgCl, 3M NaCl.
Figure 4. The comparison electropherograms for (a) nonsample
stacking using a running buffer of 50 mM H3PO4-Tris, pH 4 with an
injection time of 5 s (b) sample stacking, injection buffer: 10 mM
H3PO4-Tris, pH 2, and the running buffer of 50 mM H3PO4-Tris, pH 4
with an injection time of 10 s. (c) sample stacking, injection buffer:
10 mM H3PO4-Tris, pH 2, and the running buffer was similar to the
injection buffer with an injection time of 10 s. Concentration of
analytes: 50 µM (each) IXS, VMA, AA, HVA, UA, TRP. Separation
voltage: -10 kV. BDD at +1.0 V vs Ag/AgCl, 3 M NaCl.
becomes almost constant (∼-45 to -50 mV). The negative
charge of the gold surface may be explained by the fact that
the Au-OH present on the gold surface can lose protons as
the pH increases to form Au-O-groups on the nanoparticle
surface. 29 AuNPs have a partially hydroxylated surface with a
pKa value of 3.2 (Au-OH) in the presence of water. 29 At pH 3,
around the pKa value of AuNPs, -OH groups are predominant
(-OH groups/O - groups ) 1.6). The rest of the gold surface
should be metallic, i.e., essentially hydrophobic. Therefore,
AuNPs would display ionic and hydrophobic interaction, as well
as hydrogen bonding with the PDDA network. The synthesis
and incorporation of AuNPs in the polymer network would
affect the PDDA structural change, which in turn increased
the charge density and coverage efficiency of the coating. At
pH4or5,Au-O - groups were predominant, resulting in a
(29) Sylvestre, J.-P.; Kabashin, A. V.; Sacher, E.; Meunier, M.; Luong, J. H. T.
J. Am. Chem. Soc. 2004, 126, 7176–7177.
decrease of the �-potential of the PDDA-AuNPs composite, i.e.,
the EOF is reduced. Notice that TRP also becomes more
neutral at pH 5, whereas the charges of the catechoamines and
indolamines were highly negative and such analytes were
expected to interact strongly with PDDA. Indeed, all catecholamines
and indoleamines were not eluted after 24 min into the
experiment. In contrast, Zhang et al. 28 reported that the
separation using the PDDA coated capillary is significantly
longer than the one modified with PDDA-AuNPs for analysis
of heroin and impurities. In such a study, the separation is
performed at pH 5.2 with addition of 3% methanol into the
running buffer consisting of 120 mM ammonium acetate.
Therefore, it was somewhat difficult to compare the result
obtained in this work with that of Zhang et al. 28
Other Optimal Conditions and Sample Stacking. The 50
mM phosphoric-Tris buffer pH 3 appeared to provide the highest
detection sensitivity with good separation resolution. Indeed,
4-hydroxy-3-methoxybenzylamine comigrated with 3,4-dihydroxybenzylamine
when the buffer strength increased to 100 mM and
the run was longer with lower detection sensitivity (Figure 3A).
This pair was coeluted as one single peak if the separation was
carried out with the PDDA coated capillary. Thus, a running buffer
consisting of 50 mM phosphoric-Tris, pH 3 was used to optimize
the separation potential and the detection potential of the BDD
electrode. With respect also to detection sensitivity and separation
efficiency (N/cm), the electrokinetic injection of the sample at
-10 kV for 5 s was optimal compared to the results obtained at
shorter (3 s) or longer (7 and 10 s) injection times (Figure 3B).
Beyond 5 s, the resulting peaks were very broad with compromised
detection sensitivity and separation efficiency. In contrast,
3 s was not sufficient, as reflected by considerably lower peaks.
The BDD detection potential, poised at +1 V vs 3 M Ag/AgCl
provided highest detection sensitivity compared to the detection
performed at lower or higher applied potentials (Figure 3C). Both
resolution and detection sensitivity were severely deteriorated
when the separation was conducted at -15 kV and -20 kV,
compared to the run at -10 kV (Figure 3D). Linearity and LOD
obtained for 11 analytes are summarized in Table 2. The four fast
migrating analytes, IXS, VMA, HVA, and tryptophan exhibited a
LOD of 0.3 µM, whereas a higher LOD, ∼1 µM was obtained for
the slower migrating group.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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