Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Anal. Chem. 2010, 82, 6895–6903<br />
Electrophoretic Analysis of Biomarkers using<br />
Capillary Modification with Gold Nanoparticles<br />
Embedded in a Polycation and Boron Doped<br />
Diamond Electrode<br />
Lin Zhou, † Jeremy D. Glennon, † and John H. T. Luong* ,†,‡<br />
Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of <strong>Chemistry</strong> & the ABCRF,<br />
University College Cork, Cork, Ireland and Biotechnology Research Institute, National Research Council Canada,<br />
Montreal, Quebec, Canada H4P 2R2<br />
Field-amplified sample stacking using a fused silica<br />
capillary coated with gold nanoparticles (AuNPs) embedded<br />
in poly(diallyl dimethylammonium) chloride (PDDA)<br />
has been investigated for the electrophoretic separation<br />
of indoxyl sulfate, homovanillic acid (HVA), and vanillylmandelic<br />
acid (VMA). AuNPs (27 nm) exhibit ionic and<br />
hydrophobic interactions, as well as hydrogen bonding<br />
with the PDDA network to form a stable layer on the<br />
internal wall of the capillary. This approach reverses<br />
electro-osmotic flow allowing for fast migration of the<br />
analytes while retarding other endogenous compounds<br />
including ascorbic acid, uric acid, catecholamines, and<br />
indoleamines. Notably, the two closely related biomarkers<br />
of clinical significance, HVA and VMA, displayed differential<br />
interaction with PDDA-AuNPs which enabled the separation<br />
of this pair. The detection limit of the three analytes obtained<br />
by using a boron doped diamond electrode was ∼75 nM,<br />
which was significantly below their normal physiological<br />
levels in biological fluids. This combined separation and<br />
detection scheme was applied to the direct analysis of these<br />
analytes and other interfering chemicals including uric and<br />
ascorbic acids in urine samples without off-line sample<br />
treatment or preconcentration.<br />
Indoxyl sulfate (IXS), a metabolite of tryptophan (TRP) and a<br />
dietary protein, is derived from intestinal metabolism and liver<br />
conjugation 1 and excreted in urine in high concentration. It is also<br />
an endogenous compound in mammals, in mouse plasma and<br />
brain samples, as detected by liquid chromatography/tandem<br />
mass spectrometry. 2 This circulating protein-bound uremic toxin<br />
stimulates glomerular sclerosis, interstitial fibrosis, and the<br />
progression rate of renal failure. IXS induces endothelial dysfunction<br />
by inhibiting endothelial proliferation and migration in vitro<br />
* To whom correspondence should be addressed.<br />
† Innovative Chromatography Group, Irish Separation Science Cluster (ISSC),<br />
Department of <strong>Chemistry</strong> & the ABCRF, University College Cork.<br />
‡ Biotechnology Research Institute, National Research Council Canada.<br />
(1) Dealler, S. F.; Hawkey, P. M.; Millar, M. R. J. Clin. Microbiol. 1988, 26<br />
(10), 2152–2156.<br />
(2) Wang, G.-F.; Korfmacher, W. A. Rapid Commun. Mass Spectrom. 2008,<br />
23 (13), 2061–2069.<br />
and its role in oxidative stress is implicated. 3 Homovanillic acid<br />
(HVA) is a major catecholamine metabolite, associated with the<br />
brain dopamine level. As a biomarker of metabolic stress of<br />
2-deoxy-D-glucose, the HVA level in the brain and the cerebrospinal<br />
fluid is indicative of pheochromocytoma and neuoroblastoma. 4<br />
Metanephrine, one of the hormones produced by the adrenal<br />
glands, breaks down to normetanephrine and vanillylmandelic acid<br />
(VMA) via the intermediate 4-hydroxy-3-methoxy-phenylglycol.<br />
Thus, VMA is always detected in the urine together with HVA<br />
and other catecholamine metabolites from pheochromocytoma or<br />
catecholamine-secreting chromaffin tumor cells. 5,6 Catecholamine<br />
levels can be found in blood samples; however, the urine test<br />
reflects the production rate of the catecholamines over the<br />
collection period. Even at abnormal levels, the elevated quantities<br />
of these catecholamines are still very low, i.e., highly selective<br />
and sensitive analytical methods are needed for such important<br />
biomarkers. The urinary VMA and HVA values are most useful<br />
and can be correlated with the stage of disease, management, any<br />
maturation of tumor, and prognosis from children with neuroblastoma.<br />
4 The urine HVA/VMA ratio could be a screening tool<br />
to support earlier detection of Menkes disease, a disorder that<br />
affects copper levels in the body, leading to copper deficiency. 7a<br />
The mechanism that removes HVA from the brain is still poorly<br />
understood, however, the efflux transport of HVA from the brain<br />
plays an important role in controlling the HVA level in the brain.<br />
This HVA efflux transport system is inhibited by several organic<br />
anions including IXS, and metabolites of monoamine neurotransmitters<br />
but not neurotransmitters per se. 7b Thus, it is of clinical<br />
importance to develop a rapid and sensitive method for simultaneous<br />
analysis of HVA, VMA, and IXS in urine and other biological<br />
samples.<br />
(3) Tumur, Z.; Niwa, T. Am. J. Nephrol. 2009, 29, 551–557.<br />
(4) Liebner, E. J.; Rosenthal, I. M. Cancer 2006, 32 (3), 623–633.<br />
(5) Eisenhofer, G.; Lenders, J. W. M.; Linehan, W. M.; Walther, M. M.;<br />
Goldstein, D. S.; Keiser, H. R. New Engl. J. Med. 1999, 340, 1872–1879.<br />
(6) Lenders, J. W. M.; Keiser, H. R.; Goldstein, D. S.; Willemsen, J. J.; Friberg,<br />
P.; Jacobs, M.-C.; Kloppenborg, P. W. C.; Thien, T.; Eisenhofer, G. Ann.<br />
Intern. Med. 1995, 123, 101–109.<br />
(7) (a) Menkes, J. H.; Alter, M.; Steigleder, G. K.; Weakley, D. R.; Sung, J. H.<br />
Pediatrics 1962, 29, 764–779. (b) Mori, S.; Takanaga, H.; Ohtsuki, S.;<br />
Deguchi, T.; Kang, Y.-S.; Hosoya, K.-I.; Terasaki, T. J. Cereb. Blood Flow<br />
Metab. 2003, 23, 432–440.<br />
(8) (a) Issaq, H. J.; Delviks, K.; Janini, G. M.; Muschik, G. M. J. Liquid<br />
Chromatogr. Relat. Technol. 1992, 15/18, 3193–3201.<br />
10.1021/ac101105q © 2010 American <strong>Chemical</strong> Society 6895<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
Published on Web 07/16/2010