Mammalian cell surface imaging with nitrile-functionalized nanoprobes: Biophysical characterization of aggregation and polarization anisotropy in SERS imaging

Article abstract of DOI:10.1021/ja0670005

Functionalized silver nanoparticles (NPs) were synthesized with a cyano probe and utilized to label the surface of HeLa cells expressing a recombinant transmembrane domain of the platelet-derived growth factor receptor. Optical imaging, high-resolution Ra

Full text of DOI:10.1021/ja0670005

Published on Web 12/15/2006
Mammalian Cell Surface Imaging with Nitrile-Functionalized Nanoprobes:
Biophysical Characterization of Aggregation and Polarization Anisotropy in
SERS Imaging
Qingyan Hu,† Li-Lin Tay,*,‡ Matthew Noestheden,† and John Paul Pezacki*,†
Steacie Institute for Molecular Sciences and Institute for Microstructural Sciences, National Research Council
Canada, Ottawa, Ontario, Canada K1A 0R6
Received September 29, 2006; E-mail: lilin.tay@nrc-cnrc.gc.ca; john.pezacki@nrc-cnrc.gc.ca
Recently, Raman and surface enhanced Raman scattering (SERS)
spectroscopies have been applied to biosensing, as they offer high
information content about the analyte.1 Nanoparticle-based SERS
techniques have proven to be excellent methods for sensing DNA
hybridization2 and protein binding events.3 However, Raman and
SERS microscopies have seen limited applications in biological
imaging. Although high-resolution Raman microscopy yields high
information content cellular images, this technique is plagued by
poor scattering cross sections, which results in lengthy acquisition
times and high excitation power. The SERS effect, which is due
primarily to the enhancement of the electromagnetic field associated
with strong/localized surface plasmon resonances near the surface
of nanostructure assemblies,4 allows for greater sensitivity. Colloidal
gold or silver nanoparticles (NPs) are popular SERS active platforms
in most biological studies.3,5
An important frontier in molecular imaging is the utilization of
the enormous sensitivity of SERS to achieve specific detection of
cellular proteins in vivo.6 Very often proteins give rise to spectrally
complex finger print regions. This, coupled with the spectral
fluctuations inherent to SERS, makes the identification of specific
protein species extremely challenging. Therefore, we have designed
a biomolecular Raman label with a Raman signature that is unique
compared with those of cellular materials. Furthermore, our Raman
label possesses orthogonal functionality and is chemically inert
under live cell imaging conditions. We report here silver NPs (Ag-
NPs) with a ligand containing a cyano group as a Raman reporter
1 (Figure 1a). The syntheses of 1 and Ag-NPs coated with 1 (Ag-
Figure 1. (a) The chemical structure of Raman reporter 1; (b) Raman
NP-1) are in the Supporting Information. The unique vibrational
mode of the CtN stretch was used for vibrational contrast in
cellular imaging experiments. The probe molecule also has a
terminal hydrazide to achieve specific labeling of ketones conju-
gated to cell surface proteins. We successfully labeled HeLa cells
expressing the transmembrane domain of the platelet-derived growth
factor receptor (TM) with Ag-NP-1 using the method established
by Ting and co-workers (see Supporting Information).7
Figure 1c shows an optical image of a HeLa cell transfected
with TM containing an acceptor peptide (AP) tag that was
subsequently labeled with a ketone analogue of biotin7 and Ag-
NP-1. Its corresponding Raman intensity map from the CN vibration
band is depicted in Figure 1d. In the Raman map, three intense
spots (I, II, and III) are identified, and their corresponding Raman
spectra are shown in Figure 1b. Both spectra I and II exhibit obvious
CtN bands at 2230 cm-1. A much weaker CtN stretch was
observed at spot III. Intensity contrasts between the three SERS
hot-sites and regions without Ag-NPs are ∼3 orders of magnitude.
Inset of Figure 1b is a typical cellular Raman spectrum obtained
from spot IV of the same cell but in a region without Ag-NP-1
spectra of the CN vibration mode extracted from positions I, II, and III of
the cell shown in the optical image (c). Inset of (b) is a cellular Raman
spectrum taken from spot IV of the same cell. (d) Raman intensity map of
the CtN band of the same cell, and (e) the corresponding SEM image.
Inset in (e) showed the NPs in the lower right circle. (f) The group of NPs
as shown in the large oval of (e).
(Figure 1d). This cell spectrum consists of the following bands:
1660 (amide I), 1447 (CH2, CH3), 1285 and 1350 (amide III, NH,
CH), and 2990 (CH) cm-1 8
.
Membrane proteins often cluster onto microdomains called rafts,
which may help facilitate aggregation of NPs upon labeling.
Scanning electron microscopy (SEM) was performed on the same
cell to correlate the observed SERS hot-sites with the presence of
anchored NPs (Figure 1e). The SEM image revealed several NPs.
The inset of Figure 1e shows a pair of NPs located at the bottom
of the cell that consist of a single NP and a dimerized NP composed
of a nanorod and a spherical NP. Figure 1f shows the magnification
of the larger group of NPs encircled in Figure 1e (large oval). Again,
the NPs identified in Figure 1f are made of small NP aggregates
(tetramer and trimer) and a few monomers. Comparing the SEM
images to the Raman intensity plot facilitated the assignment of
the NP aggregates responsible for all the observed intense SERS
† Steacie Institute for Molecular Sciences.
‡ Institute for Microstructural Sciences.
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C O M M U N I C A T I O N S
hot-spots in Figure 1d. Strong SERS signals observed at spots I,
II, and III were generated by tetramer, trimer, and dimer NPs,
respectively. Although the SEM image (Figure 1e) also revealed
another Ag aggregate located at the lower left of the cell, careful
examination of this cluster with SEM showed that the Ag cluster
is actually embedded inside the cell. Raman spectra taken from
this region do show enhancement, although much weaker in
intensity (∼50-fold) compared with those aggregates located on
the cell surface. Moreover, repeated cell surface labeling with Ag-
NP-1 indicated that only nanoparticle aggregates (dimers or greater)
showed measurable SERS signals. Generally, the SERS enhance-
ment factor is about 1010 (see Supporting Information).
Theoretical calculations have shown that SERS enhancement
factors from isolated NPs are orders of magnitude smaller than those
of aggregated NPs.9 The SERS enhancement factor of a dimerized
NP increases dramatically when the molecule of interest is situated
in the interstitial gap of the dimer NPs,9,10 implying that enhance-
ment depends strongly on the interstitial gap between the NPs. With
an interstitial gap of ∼5 nm, the SERS enhancement factor can
exceed 6 orders of magnitude and 10 orders of magnitude with a
1 nm interstitial gap.11 Trimers and other smaller NP aggregates
can be treated as ensembles of coupled dimer systems and therefore
can produce extremely intense SERS hot-sites provided sufficiently
small interstitial gaps are present. Although single NPs are capable
of field enhancement, their SERS intensity is simply too weak to
be observed in our imaging experiments.
We also observed that aggregated NPs exhibit strong polarization
anisotropy. Particles identified in the SEM images labeled as spots
I, II, and III (Figure 1e) all have their interparticle axes oriented
largely parallel to the incident electric field. This relationship is
necessary to achieve a capacitive field enhancement and hence
generate intense SERS hot-sites.4,10 If the incident light polarization
is perpendicular to the interparticle axes of the aggregated NPs,
the exclusion of the field between the NPs results in no enhancement
(see Supporting Information).
As the Raman images were obtained by rastering cells under a
tightly focused incident beam, light-induced damage is a legitimate
concern. To investigate this problem, we observed SEM images of
several cells before and after Raman imaging (Figure 2a). The
Raman and SEM image of a representative cell showed excellent
correlation between the locations of SERS and aggregated Ag-NP-
1. The same cell was examined under SEM after Raman imaging.
We observed localized rupturing of the cell membrane under several
aggregated Ag-NP-1 (Figure 2c,d) and a NP monomer (Figure 2b).
These results indicated that damage at NP aggregates was the most
extensive. Supporting this was the finding that, when we reduced
the imaging power density by 1 order of magnitude, no detectable
membrane damage was observed. Similar NP-induced photothermal
effects to cells have been reported12 and often result in irreparable
damage to cells.
In summary, we have demonstrated imaging of membrane
proteins on cells using a cyano-labeled SERS probe. Observed
SERS hot-sites correlate well with small aggregated NPs. Further-
more, the strongest SERS signals originate from aggregates oriented
in the appropriate direction with respect to the incident laser
polarization. These observations are critical for the design of future
molecular imaging agents for SERS imaging of cells and tissues.
Fortuitously, many cell surface receptors function as clusters which
may help to facilitate NP aggregation and orientation in cellular
Figure 2. (a) SEM image of a cell. Upper right inset: magnification of a
group of aggregated NPs. The scale bar is 200 nm. Lower left inset: the
corresponding Raman intensity image of the same cell obtained with a power
density of 105 W/cm2. Laser-induced damage to the cell is shown in (b)
the monomer (blue circle in a), (c) the aggregates, and (d) a pair of dimers.
and tissue imaging experiments.13 We are currently pursuing such
imaging studies.
Acknowledgment. We thank Prof. Alice Ting at MIT for
providing biotin ketone, as well as the AP-CFP-TM and BirA
plasmids. We gratefully acknowledge the help of Mr. Jeff Fraser
for SEM imaging, and Dr. Jean Lapointe for fabricating the
patterned Si substrate.
Supporting Information Available: Experimental details and
information on polarization anisotropy. This material is available free
of charge via the Internet at http://pubs.acs.org.
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