Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Anal. Chem. 2010, 82, 7035–7043
Quantitation, Visualization, and Monitoring of
Conformational Transitions of Human Serum
Albumin by a Tetraphenylethene Derivative with
Aggregation-Induced Emission Characteristics
Yuning Hong, † Chao Feng, ‡ Yong Yu, †,‡ Jianzhao Liu, † Jacky Wing Yip Lam, † Kathy Qian Luo, ‡,§
and Ben Zhong Tang* ,†,#
Nano Science and Technology Program, Department of Chemistry, Institute of Molecular Functional Materials,
Bioengineering Program, and Department of Chemical and Biomolecular Engineering, The Hong Kong University of
Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China, School of Chemical and
Biomedical Engineering, Nanyang Technological University, Singapore 637457, and Department of Polymer Science
and Engineering, Institute of Biomedical Macromolecules, Key Laboratory of Macromolecular Synthesis and
Functionalization of the Ministry of Education, Zhejiang University, Hangzhou 310027, China
Human serum albumin (HSA) is a major protein component
of blood plasma, and its assay is of obvious value to
biological research. We, herein, present a readily accessible
fluorescent bioprobe for HSA detection and quantitation.
A nonemissive tetraphenylethene derivative named
sodium 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene
(BSPOTPE) is induced to emit by HSA, showing
a novel phenomenon of aggregation-induced emission
(AIE). The AIE bioprobe enjoys a broad working range
(0-100 nM), a low detection limit (down to 1 nM), and a
superior selectivity to albumins. The fluorescent bioassay
is unperturbed by the miscellaneous bioelectrolytes in the
artificial urine. The AIE luminogen can also be used as a
rapid and sensitive protein stain in gel electrophoresis for
HSA visualization. Utilizing the AIE feature of BSPOTPE
and the Förster resonance energy transfer from HSA to
BSPOTPE, the unfolding process of HSA induced by
guanidine hydrochloride is monitored, which reveals a
multistep transition with the involvement of molten globule
intermediates. Computational modeling suggests that
the AIE luminogens dock in the hydrophobic cleft between
subdomains IIA and IIIA of HSA with the aid of hydrophobic
effect, charge neutralization, and hydrogen bonding
interactions, offering mechanistic insight into the
microenvironment inside the hydrophobic cavity.
Human serum albumin (HSA) is the most abundant protein
in the circulatory system and plays multiple biological functions
in the human body. 1 For example, it regulates water balance
between blood and tissues and serves as a physiological carrier
for various endogenous and exogenous substances that are
* To whom correspondence should be addressed. E-mail: tangbenz@ust.hk.
† Nano Science and Technology Program, Department of Chemistry, Institute
of Molecular Functional Materials, and Bioengineering Program, HKUST.
‡ Department of Chemical and Biomolecular Engineering, HKUST.
§ Nanyang Technological University.
# Zhejiang University.
(1) Peters, T., Jr. Adv. Protein Chem. 1985, 37, 161.
partially soluble in the bloodstream, such as bilirubin, fatty acids,
and drugs. 2-5 The albumin is synthesized in the liver. A low level
of albumin in the blood serum, known as hypoproteinemia, may
indicate liver failure, cirrhosis, and chronic hepatitis. 6 When blood
passes through healthy kidneys, the kidneys filter out waste
products but keep the substances the body needs, such as albumin
and other proteins. Appearance of an excess amount of proteins
in urine is a sign of chronic kidney disease, which may result in
diabetes, high blood pressure, and problems associated with
inflammation in the kidneys. 7 Many studies have been done on
diabetic nephrosis, with microalbuminuria being identified as an
early sign. 8 Microalbuminuria is commonly diagnosed by elevated
protein concentration (30-300 mg/L) in the urine on at least two
occasions. A higher albumin value is regarded as albuminuria.
The urine-testing dipsticks impregnated with pH sensitive dyes
are normally used for daily health monitoring. 9 The level of
albumin caused by microalbuminuria, however, cannot be accurately
assayed by the dipsticks due to their low sensitivity. 10 It
is, thus, of clinical value to develop effective methods for urinary
protein detection and quantitation.
Colorimetric methods, such as Brandford and Lowry assays,
have traditionally been used for protein quantitation in solutions. 11,12
These methods, however, generally lack sensitivity and accuracy,
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Lancet 1996, 347, 1299.
(7) Hoogenberg, K.; Sluiter, W. J.; Dullaart, R. P. F. Acta Endrocrinol. 1993,
129, 151.
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H. H.; Passa, P.; Steffes, M. W.; Striker, G. E.; Viberti, G. C. Lancet 1995,
346, 1080. (b) Nomata, S.; Haneda, K.; Moriya, T.; Katayama, S.; Iwamoto,
Y.; Sakai, H.; Tomino, Y.; Matsuo, S.; Asano, Y.; Makino, H. Jpn. J. Nephrol.
2005, 47, 768.
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(10) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem.,
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10.1021/ac1018028 © 2010 American Chemical Society 7035
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Published on Web 07/20/2010