Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters

Article abstract of DOI:10.1039/c0cc05265e

SERS nanotags have been prepared to accomplish the multiplex detection of cancer cells. Herein we evaluated the adequacy of lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA) to function as multiplex partners with a triphenylmethine Raman reporter (B2LA) under a single excitation wavelength. SERS experiments enabled the multiplex recognition of two different cancer cells with antibody-conjugated nanotags that were derivatized with optimized cyanine and triphenylmethine reporters.

Full text of DOI:10.1039/c0cc05265e

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COMMUNICATION
Multiplex cancer cell detection by SERS nanotags with cyanine and
triphenylmethine Raman reporterswz
Kaustabh Kumar Maiti,a Animesh Samanta,b Marc Vendrell,a Kiat-Seng Soh,a
Malini Olivoacd and Young-Tae Chang*ab
Received 30th November 2010, Accepted 15th January 2011
DOI: 10.1039/c0cc05265e
SERS nanotags have been prepared to accomplish the multiplex
detection of cancer cells. Herein we evaluated the adequacy of
lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA)
to function as multiplex partners with a triphenylmethine Raman
reporter (B2LA) under a single excitation wavelength. SERS
experiments enabled the multiplex recognition of two different
cancer cells with antibody-conjugated nanotags that were deri-
vatized with optimized cyanine and triphenylmethine reporters.
were capable to detect both cell lines using a single excitation
wavelength.
Our group recently reported the excellent properties of
triphenylmethines as Raman signature molecules,7 and
demonstrated the improved stability of their SERS signal
upon chemisorption on AuNPs using a lipoic acid (LA) linker.
A triphenylmethine derivative (B2LA) was identified as an
outstanding reporter in terms of both SERS signal intensity
and long-term stability.8 In order to study the multiplexing
capabilities of B2LA SERS nanotags, we evaluated their
compatibility with different Raman-active chemical structures,
such as cyanine molecules. Cy3 and Cy5 are well-known
cyanine compounds with absorption properties in the red/
far-red region of the visible range,9–12 and we envisioned that
they may be sensitive Raman reporters under the 633 nm laser
that is required for the excitation of B2LA SERS nanotags. To
adapt Cy3 and Cy5 structures for chemisorption, we designed
the synthesis of lipoic acid-containing cyanine derivatives
(Cy3LA and Cy5LA, respectively) so that they could be
attached on AuNPs using thiol-based chemistry (Scheme 1).
The chemisorption of Raman reporters is a critical step to
ensure the stability and reproducibility of SERS nanotags over
In recent years surface enhanced Raman spectroscopy (SERS)
has captured much attention due to the ability to provide
molecular information with high sensitivity using the sharp
and distinguishable spectral features of gold or silver nano-
particles (AuNPs or AgNPs).1–4 SERS nanotags have several
advantages, mainly high spectral specificity, improved contrast
and multiplexing capabilities.5,6 The multiplexing potential of
SERS nanotags relies on the narrow bandwidths of the vibra-
tional Raman spectra of the reporter molecules, and allows the
simultaneous recognition of closely related targets. The con-
current detection of defined multiple targets can facilitate the
development of accurate diagnostic probes, yet the identification
of multiplex reporter pairs that are compatible under the same
experimental conditions is difficult. Herein we prepared multi-
plex SERS nanotags after optimizing a new pair of Raman
reporters based on triphenylmethine and cyanine compounds.
After conjugation to antibodies (e.g. anti-EGFR and anti-
HER2) that recognize different types of cancer cells (OSCC
and SKBR-3, respectively), these multiplex SERS nanotags
a Singapore Bioimaging Consortium, Agency for Science, Technology
and Research (A*STAR), 11 Biopolis Way, 138667 Singapore
b Department of Chemistry & MedChem Program of Life Sciences
Institute, National University of Singapore, 117543 Singapore.
E-mail: chmcyt@nus.edu.sg; Fax: 65 6478 9957; Tel: 65 6478 8761
c Division of Medical Sciences, National Cancer Centre, 169610
Singapore
d School of Physics, National University of Ireland Galway, Ireland
w This article is part of a ChemComm web-based themed issue on
Surface Enhanced Raman Spectroscopy.
z Electronic supplementary information (ESI) available: Synthetic
procedures and characterization (NMR, MS); procedures for: nanotag
labeling, antibody conjugation, cell culture, Raman spectra determi-
nation and mapping experiments; TEM images and evaluation of the
SERS stability of prepared nanotags; additional data for SERS
measurements in cells. See DOI: 10.1039/c0cc05265e
Scheme 1 Synthesis of lipoic acid cyanine derivatives. Reagents and
conditions: (a) 1-iodopropane, CH3CN, 80 1C, 15 h; (b) 3-bromopropyl-
amine hydrobromide, 120 1C, 10 h; (c) di-tert-butyl dicarbonate,
DIEA, CHCl3, reflux, 4 h; (d) AcOH, acetic anhydride, pyridine,
110 1C; (e) TFA–DCM (1 : 9), r.t., 16 h; (f) lipoic acid activated ester
resin, CH2Cl2, r.t., 16 h.
c
3514 Chem. Commun., 2011, 47, 3514–3516
This journal is The Royal Society of Chemistry 2011

time.8,13 Starting from 2,3,3-trimethylindoline, we prepared
the intermediates 1 and 2 using reported procedures.14–16
Afterwards, 1 was condensed to the commercially available
bis-phenylimines 4 and 5 in acidic conditions, followed by the
subsequent addition of 3, the Boc-protected derivative of 2. As
a result, the Boc-derivatives of Cy3 and Cy5 (6 and 7,
respectively) were obtained with overall yields slightly over
50%. Deprotection of 6 and 7 with an optimized TFA-DCM
(1 : 9) solution afforded the corresponding free amine com-
pounds, which were treated with a lipoic acid-containing
activated ester resin17 to obtain Cy3LA and Cy5LA in good
purities and yields (see ESIz for characterization data).
anti-EGFR and Cy3LA anti-HER2 nanotags. EGFR is over-
expressed in diverse cancer cells (e.g. OSCC), and HER2 is a
well-known breast cancer marker with a high expression in
SKBR-3 cells.18 The antibody conjugation was verified by the
appearance of protein absorption peaks at 280 nm, and the size
of the fully functionalized nanotags was determined by trans-
mission electron microscopy (TEM) (Fig. S2, ESIz). Further-
more, we confirmed that the SERS signal intensities of both
nanotags were stable for several days (Fig. S3, ESIz).
In order to examine the multiplex differential recognition
of B2LA anti-EGFR and Cy3LA anti-HER2 nanotags in
cells, we incubated an equal amount of both nanotags in
OSCC cells (EGFR-positive and HER2-negative) and
SKBR-3 cells (HER2-positive and EGFR-negative). After
washings with PBS, the SERS measurement in OSCC cells
fully resembled the SERS spectra of B2LA (Fig. 2a) whereas
After chemisorbing B2LA, Cy3LA and Cy5LA on AuNPs,
we analyzed their SERS spectra under the 633 nm laser and
evaluated their multiplexing compatibility. Notably, a number
of peaks could be used to uniquely identify the three different
nanotags: 1617, 1374, 1366, 918 and 440 cmꢀ1 for B2LA-
AuNPs, 1589, 1383 and 613 cmꢀ1 for Cy3LA-AuNPs and
1596, 1501, 1404 and 1353 cmꢀ1 for Cy5LA-AuNPs (Fig. 1).
Whereas the discrimination between Cy3LA and Cy5LA-
derivatized nanotags may require further optimization, it
was apparent that the combination of triphenylmethine and
cyanine Raman reporters could be used as a basis for the
construction of multiplex SERS nanotags, and we explored
their application for the detection of related cancer cells.
Nanotags derivatized with B2LA and Cy3LA were encapsulated
with a mixture of thiol polyethyleneglycol (PEG-SH) and
carboxylic acid-containing PEG-SH that allowed the covalent
linkage to the free amine groups of antibodies.3
After encapsulation, we conjugated monoclonal antibodies
against two different epidermal growth factor receptors
(EGFR (Erb-B1) and HER2 (Erb-B2)) to render B2LA
Fig. 2 Multiplex SERS spectra upon incubation of both B2LA
anti-EGFR and Cy3LA anti-HER2 nanotags with: (a) OSCC cells,
(b) SKBR-3 cells, (c) co-cultured cells. Spectra were measured with
Fig. 1 Normalized SERS spectra of B2LA, Cy3LA and Cy5LA after
chemisorption on AuNPs. Spectra were measured in a Raman micro-
scope (633 nm laser excitation, 6.2 mW laser power, acquisition time:
10 s) and plotted as average intensities (n = 3). The most distinctive
peaks from every reporter are highlighted in yellow.
a
Raman microscope (633 nm excitation wavelength, 6.2 mW
laser power, acquisition time: 10 s) and plotted as average intensities
(n = 3).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 3514–3516 3515

triphenylmethine and cyanine Raman reporters. The SERS
compatibility under a single excitation wavelength between a
selected triphenylmethine and different lipoic acid-containing
cyanine reporters was examined. Nanotags derivatized with a
selected pair (B2LA and Cy3LA) were derivatized with anti-
EGFR and anti-HER2 antibodies, and proved to specifically
recognize the respective cancer cells (e.g. OSCC and SKBR-3)
with non-overlapping SERS peaks. After confirming the perfor-
mance of these nanotags in co-culture conditions and determin-
ing their localization by SERS mapping experiments, we
demonstrated that B2LA and Cy3LA are fully compatible
Raman reporters for the preparation of multiplex SERS nanotags
and can be used for the concurrent detection of related
cancer cells.
The authors gratefully acknowledge the A*STAR Cross
Council Office (CCO), Singapore (Grant CCOGA02_005_2008),
and the National University of Singapore (NUS) (Young
Investigator Award: R-143-000-353-101) for their financial
support.
Fig. 3 Bright field and SERS mapping images of: (a) B2LA anti-
EGFR nanotag-treated OSCC cells (1615 cmꢀ1), (b) Cy3LA anti-HER2
nanotag-treated SKBR-3 cells (1468 cmꢀ1). All mapping images (size:
30 ꢁ 30 mm2) were scanned at an interval of 2 mm (633 nm excitation
wavelength) and the intensities were normalized between the lowest (0)
and the highest color (1) values.
Notes and references
the SERS signal of SKBR-3 cells coincided with the spectra
of Cy3LA (Fig. 2b). Moreover, as negative controls, we did
not observe significant SERS signals in OSCC or SKBR-3
cells after incubation with antibody-free B2LA and Cy3LA-
nanotags (Fig. S4, ESIz). Altogether, these results clearly
indicated that: (1) B2LA anti-EGFR and Cy3LA anti-HER2
nanotags specifically recognized OSCC and SKBR-3 cells
with non-overlapping SERS peaks, (2) the SERS signals
derived from any possible non-specific binding of the two
nanotags were negligible. Finally, we demonstrated that
B2LA and Cy3LA-nanotags could be used as a multiplex
platform by recognizing both OSCC and SKBR-3 cells after
they were co-cultured in the same wells. Upon incubation
with an equal proportion of both B2LA and Cy3LA-nano-
tags, the SERS signals of the co-cultured cells showed clearly
separable peaks from the two reporter molecules: 1615, 1363,
917 and 437 cmꢀ1 for B2LA anti-EGFR, and 1585, 1465,
1380, 1268, 1118, 931, 612 and 556 cmꢀ1 for Cy3LA- anti-
HER2 (Fig. 2c). With this data, we attested that B2LA and
Cy3LA are fully compatible Raman reporters for the pre-
paration of multiplex SERS nanotags.
1 C. L. Zavaleta, B. R. Smith, I. Walton, W. Doering, G. Davis,
G. B. Shojaei, M. J. Natan and S. S. Gambhir, Proc. Natl. Acad.
Sci. U. S. A., 2009, 106, 13511–13516.
2 X. M. Qian, X. Zhou and S. Nie, J. Am. Chem. Soc., 2008, 130,
14934–14935.
3 X. Quin, X.-H. Peng, D. O. Ansari, Q. Yin-Geon, G. Z. Chen,
D. M. Shin, L. Yang, A. N. Young, M. D. Wang and S. Nie, Nat.
Biotechnol., 2007, 26, 83–90.
4 S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens and
S. S. Gambhir, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
5844–5849.
5 J. Kneipp, H. Kneipp, B. Wittig and K. Kneipp, Nanomed.:
Nanotechnol., Biol. Med., 2010, 6, 214–226.
6 A. Matschulat, D. Drescher and J. Kneipp, ACS Nano, 2010, 4,
3259–3269.
7 S. J. Cho, Y. H. Ahn, K. K. Maiti, U. S. Dinish, C. Y. Fu,
P. Thoniyot, M. Olivo and Y. T. Chang, Chem. Commun., 2010,
46, 722–724.
8 K. K. Maiti, U. S. Dinish, C. Y. Fu, J. J. Lee, K. S. Soh,
S. W. Yun, R. Bhuvaneswari, M. Olivo and Y. T. Chang, Biosens.
Bioelectron., 2010, 26, 398–403.
9 K. Jia, Y. Wan, A. D. Xia, S. Y. Li, F. B. Gong and G. Q. Yang,
J. Phys. Chem. A, 2007, 111, 1593–1597.
10 A. Toutchkine, P. Nalbant and K. M. Hahn, Bioconjugate Chem.,
2002, 13, 387–391.
11 H. J. Gruber, G. Kada, B. Pragl, C. Riener, C. D. Hahn,
G. S. Harms, W. Ahrer, T. G. Dax, K. Hohenthanner and
H. G. Knaus, Bioconjugate Chem., 2000, 11, 161–166.
12 A. Iqbal, L. Wang, K. C. Thompson, D. M. J. Lilley and
D. G. Norman, Biochemistry, 2008, 47, 7857–7862.
13 M. Gellner, K. Kompe and S. Schlucker, Anal. Bioanal. Chem.,
2009, 394, 1839–1844.
14 B. Chipon, G. Clave, C. Bouteiller, M. Massonneau, P. Y. Renard
and A. Romieu, Tetrahedron Lett., 2006, 47, 8279–8284.
15 C. Bouteiller, G. Clave, A. Bernardin, B. Chipon, M. Massonneau,
P. Y. Renard and A. Romieu, Bioconjugate Chem., 2007, 18,
1303–1317.
16 A. Toutchkine, D. V. Nguyen and K. M. Hahn, Bioconjugate
Chem., 2007, 18, 1344–1348.
17 J. W. Lee, Y. Q. Louie, D. P. Walsh and Y. T. Chang, J. Comb.
Chem., 2003, 5, 330–335.
18 H. Zhang, A. Berezov, Q. Wang, G. Zhang, J. Drebin, R. Murali
and M. I. Greene, J. Clin. Invest., 2007, 117, 2051–2058.
19 P. Monaghan, M. G. Ormerod and M. J. O’Hare, Int. J. Cancer,
1990, 46, 935–943.
20 W. Ueda, S. Wang, N. Dumont, J. Y. Yi, Y. Koh and
C. L. Arteaga, J. Biol. Chem., 2004, 279, 24505–24512.
To confirm the recognition properties of B2LA anti-EGFR
and Cy3LA anti-HER2 nanotags and analyze their localization
in OSCC and SKBR-3 cells, we performed SERS mapping
experiments in both cell lines. As shown in Fig. 3, images of
nanotag-treated OSCC cells and SKBR-3 cells displayed intense
SERS signals at two distinguishable frequencies (e.g. 1615 and
1468 cmꢀ1 respectively) in the cell surface region. Non-treated
OSCC and SKBR-3 cells showed negligible SERS signals at both
frequencies (Fig. S5, ESIz). These mapping pictures confirmed
that the interaction between B2LA anti-EGFR and Cy3LA anti-
HER2 nanotags and their respective receptors was mainly
localized at the cell surface, which corresponds well with the
high expression of EGFR and HER2 at the plasma membrane
of cancer cells.19,20
In summary, we developed a novel multiplex SERS platform
for cancer cell detection based on the combination of
c
3516 Chem. Commun., 2011, 47, 3514–3516
This journal is The Royal Society of Chemistry 2011