Quantitative ratiometric discrimination between
noncancerous and cancerous prostate cells
based on neuropilin-1 overexpression
Alessia Pallaoro, Gary B. Braun, and Martin Moskovits1
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510
Edited by* Richard P. Van Duyne, Northwestern University, Evanston, IL, and approved August 15, 2011 (received for review June 13, 2011)
A multiplexed, ratiometric method is described that can confi-
dently distinguish between cancerous and noncancerous epithelial
prostate cells in vitro. The technique is based on bright surface-
enhanced resonance Raman scattering (SERRS) biotags (SBTs)
infused with unique Raman reporter molecules, and carrying cell-
specific peptides. Two sets of SBTs were used. One targets the
neuropilin-1 (NRP-1) receptors of cancer cells through the RPARPAR
peptide. The other functions as a positive control (PC) and binds to
both noncancerous and cancer cells through the HIV-derived TAT
peptide. Point-by-point 2D Raman maps of the spatial distribution
of the two tags were constructed with subcellular resolution from
cells simultaneously incubated with the two sets of SBTs. Aver-
aging the SERRS signal over a given cell yielded an NRP/PC ratio
from which a robust quantitative measure of the overexpression
of the NRP-1 by the cancer cell line was extracted. The use of a
local, on-cell reference produces quantitative, statistically robust
measures of overexpression independent of such sources of uncer-
tainty as variations in the location of the focal plane, the local cell
concentration, and turbidity.
surface-enhanced Raman spectroscopy biomarker ∣ cancer cell
identification ∣ multiplexing ∣ silver nanoparticles
is a central goal in cancer research. Accordingly, seeking im-
provements in sensitivity and accuracy of detection and quanti-
fication through, for example, the development of cell-specific
diagnostic tools and biomarkers is an active enterprise. Although
it is known that such cells are present in body fluids, concentrat-
ing them to levels appropriate for confident identification and
quantification is still a serious challenge, despite significant ad-
vances that have been made by, for example, using microfluidics
(5). Once collected, the cells must be identified as either cancer-
ous or noncancerous within tolerable confidence limits, which
may be improved by, for example, using several nonredundant
biomarkers. Numerous promising identification methods have
been reported using antibodies. In this article, we report a tech-
nique based on surface-enhanced Raman spectroscopy (SERS)
and biotags that produce bright surface-enhanced resonance
Raman scattering (SERRS) signals, comparable to or exceeding
the intensities of fluorescent tags. The SERRS biotags (SBTs)
used in this study exploit peptides as recognition moieties. We
show that using SBTs ratiometrically can provide highly statisti-
cally significant, quantitative measures of neuropilin-1 (NRP-1)
overexpression insensitive to normal causes of uncertainty in
optical measurements such as variations in focal plane, cell con-
centration, and turbidity.
Normal Raman signals from cells and their infrared (FTIR)
absorbance patterns have been previously used to differentiate
between cancer and normal cells (6–9). SERS has been used as
an alternative immunohistochemistry (IHC) tool (10, 11) for the
detection of biomarkers in biological fluids or in vivo (12), and for
cancer detection from blood (13). However, Raman and FTIR
arly and rapid identification of malignant cells [ideally free-
flowing in biological fluids such as urine (1) or blood (2–4)]
signals measured directly from cells are typically much weaker
than those that are measurable with bright labels. Moreover, sam-
ple identification is often based on slight differences between
highly overlapping spectra. By contrast, numerous nonoverlap-
ping SERS and SBTs can be routinely synthesized (14) and simul-
taneously excited with a single, very low intensity laser source,
making the determination of the relative contribution of the in-
dividual SBTs to the overall spectrum tractable. SERS analysis is
also, in general, less time consuming than IHC protocols, because
cell labeling is carried out in a single step, without pre- or post-
treatments like washes, fixation, and permeabilization.
The central feature of SERS is its facile multiplexing capability
by using premade, encapsulated nanoparticle clusters that are
then infused with one of several highly Raman-active reporter
molecules. The SERS spectrum of an individual SBT acts as a
unique barcode that is easily differentiable in a composite SERS
spectrum originating from many tags. Several synthetic chal-
lenges have been overcome in the course of this study so that
their synthesis is now prescriptive. The SERS intensities achieved
are comparable to fluorescence (15). We have previously shown
that forerunners of the SBTs reported here are capable of direct
binding to, and uptake by, live cells (16).
Importantly for biomedical applications, SERS employs tissue-
penetrating lasers in the red to near-infrared range resulting
in low autofluorescence; the high signal enhancement and sharp
peaks make it possible to distinguish SERS and especially
SERRS in biological specimens at low laser powers (16). Finally,
several versions of the biotags can be incubated simultaneously
to probe a variety of cell-specific markers taking advantage of
the multiplexing capability of SERS.
In this paper, we describe a SERRS-based approach to discri-
minate between prostate cancer cells (PPC-1) and noncancerous
prostate epithelial cells (RWPE-1) in vitro using cell-specific pep-
tides. Multiplexed SBTs consisting of polymer-encapsulated silver
particles each labeled with a unique Raman reporter molecule
(14) display either a receptor-specific peptide, or a general cell-
penetrating peptide (CPP). The SBTs are incubated with cells,
SERS mapped, and the signal from the various SBTs deconvo-
luted to identify the cells while in suspension, simulating their
capture from blood. The central idea being that tags displaying
the CPP act as a cell-resident calibrant against which the cancer-
indicating tags can be ratioed, thereby greatly reducing the uncer-
tainty in the quantitative measure of the cancer-indicative marker
that can result from, for example, variability in focal plane,
changes in turbidity, cell concentration, or abbreviated measure-
ment time. The use of a cell-resident reference would be parti-
Author contributions: A.P., G.B.B., and M.M. designed research; A.P. and G.B.B. performed
research; A.P. and G.B.B. analyzed data; and M.M. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1109490108PNAS ∣ October 4, 2011 ∣ vol. 108 ∣ no. 40 ∣ 16559–16564
cularly useful when a cancerous cell is identified on the basis of
the degree of overexpression of a biomarker that may also be
present (but to a lesser extent) on the noncancerous cell. The
ratiometric approach, in essence, provides a quantitative local
measure of the extent of expression of biomarkers associated with
the cancerous modifications of a specific cell, essentially provid-
ing a basis for quantifying overexpression.
Results and Discussion
Two types of SBTs functionalized with cell-targeting peptides
were synthesized. The first carries Cys-TAT (sequence CGRKK-
RRQRRR,cys added simply for conjugation purposes),a peptide
derived from the HIV-1 TAT protein and known CPP (17, 18).
TAT was used as a general targeting peptide because it binds
rapidly and uniformly on the surface of cells (Fig. S1), and also
enters many types through endocytosis (19). The second SBTuses
the peptide Cys-RPARPAR, which specifically binds to a pocket
in the biomarker protein NRP-1, (20) a receptor expressed on the
outer plasma membrane of certain types of cancer cells, which is
involved in angiogenesis, a hallmark of cancer growth, and tumor
cell migration (21). The binding ability of the RPARPAR peptide
to the NRP-1 receptor is given by the amino acid sequence that
follows the so-called C-end rule (CendR): a C-terminal arginine
(or lysine) in a general sequence of the type R/KXXR/K (where
X indicates a generic amino acid) is fundamental for the binding
activity of the peptide (20).
Cys-TAT is small and highly cationic. The exact mechanism
that allows this peptide to enter cells is still uncertain; however,
it is generally agreed that it relies heavily on the initial attachment
to the negatively charged moieties on most membranes. When
TAT is used to transport nanoparticles in cells, the main uptake
mechanism has been shown to be lipid-raft macropinocytosis
(22). TATalso has a cryptic CendR R/KXXR/K motif and could
compete with binding of RPARPAR (20) particles. This, however,
was not observed, likely because of the low concentrations of
SBTs. Our group has previously achieved rapid cellular uptake
of hollow gold nanoshells through the self-assembly of a TAT–
lipid layer around them (23). For the present application, TAT
is bound directly to a protein on the surface of the SBT.
Thetwo SBTsets each carry a Ramanreporter: methylene blue
(MB) (Fig. 1A) on the FAM-Cys-TAT positive control set (called
PC-SBT) and thionin (Fig. 1B) on the FAM-Cys-RPARPAR
carrying particles (therefore called NRP-SBT). The absorption
maxima of the tags are approximately 670 nm for MB, and ap-
proximately 600 nm for thionin. Although the SERS enhance-
ment arises primarily on account of plasmonic excitation in the
silver nanoparticles, resonance Raman enhancement due to the
laser wavelength’s (633 nm) resonance with the two reporter
molecules likely contributes to their high overall SERRS cross-
section. The structure of the Raman reporter molecules and the
SERRS spectra of the SBTs are shown in Fig. 1. The number of
SBTs that gives rise to each of the spectra in Fig. 1C was estimated
to be approximately 4, assuming approximately 1 nM colloid con-
centration and a focal volume of 7 μm3for the 10× objective.
Signals arising from individual SBTs can be detected in deposited
samples mapped using the 100× objective (Fig. S2). We note in
passing that our purification step does leave some particles with
Ag monomer cores rather than Ag dimers. These do not produce
strong signals (14).
When a mixture of SBTs reside together in the laser’s focal
volume a composite spectrum results, which can be deconvoluted
ofeachcontributing speciestobedetermined (14, 24,25)provided
that reference spectra of the pure components are known. Spectra
were deconvoluted by assuming the composite spectrum to be a
linear combination of the individual components—a reasonable
assumption for stable, noninteracting particles. Concentrations
were derived from the individual spectroscopic contribution using
a calibration plot, constructed by deconvoluting SERS spectra
of solutions containing known concentrations of the two SBTs
(PC-SBT and NRP-SBT) (Fig. 2A). After subtracting the back-
ground, the spectra were normalized to the common band at
1;620 cm−1, assigned to the ring νðC-CÞ vibration mode of both
thionin and MB (26, 27). The data were fit (Fig. S3) using a
weighted least squares method (28). Fig. 2B shows the resulting
The cell lines chosen are RWPE-1 [prostate epithelium cells,
non tumorigenic, from normal tissue, immortalized by HPV
(29, 30)] and PPC-1 (epithelial, originated from a bone metastasis
of a prostate cancer patient, expressing the biomarker NRP-1).
After incubating the PC-SBTand NRP-SBT simultaneously with
each cell line suspended in DMEM supplemented with 10% FBS
at room temperature for 60 min, cells were placed on a micro-
scope slide and SERRS-mapped without fixing, by scanning the
focused laser beam (100× objective) in steps of 1.5 μm and
recording a full SERRS spectrum at each point (Fig. 3).
(270–1;700 cm−1) spectrum] were analyzed using Mathematica.
point comprised ofa full
polymer shell and functionalized with a modified BSA carrying several copies of a cell-specific or a universal peptide, then the SBTs are infused with a Raman
reporter (either thionin or MB). (D) Corresponding SERRS spectra obtained from an ensemble solution of SBTs (approximately 4 per focal volume). The spectra
have been shifted along the ordinate axis for clarity, and the dotted lines identify the bands of thionin that are nonoverlapping with bands in the MB spectrum.
The star indicates a common band. Laser 633 nm, 10× objective, power at sample approximately 0.11 mW, hole 400 μm, slit 400 μm, and exposure time 1 s × 3
Structural description of SBTsystem and spectra. (A) Thionin. (B) MB. (C) Schematic of a typical silver SBT: Two Ag monomers are encapsulated in a thin
www.pnas.org/cgi/doi/10.1073/pnas.1109490108Pallaoro et al.
The spectra were fit to a linear combination of the two tags’
individual reference spectra, and included sloping baseline cor-
rections: SðxÞ ¼ a?AðxÞ þ b?BðxÞ þ c þ d?x, where a, b, c, and d
are nonnegative adjustable fitting parameters; AðxÞ and BðxÞ
are the reference spectra as a function of the wavenumber value
x. The a and b coefficients together with the calibration plot
(Fig. 2B) were used to construct the composition maps.
Percent composition maps are depicted in Fig. 4 A and D and
colorized on the basis of the ratio of thionin (cancer marker NRP-
1) to MB (control). A high value of the NRP/PC ratio is colored
red; a low value of the ratio is green; intermediate values are red-
green color mixtures as shown in the bar at the right of Fig. 4.
Points corresponding to intensities below 100 counts per second
were assumed not to contain SBTs and set to black. This thresh-
old was based on the consideration of noise and the observation
that individual SBTs deposited out of a dilute solution onto a
glass slide produced well-resolved signals above 100 counts per
second (Fig. S2). Bright field images of the same cell groups
are shown in Fig. 4 B and E. The SERRS and bright field images
are overlaid in Fig. 4 C and F to show that signals arise from the
cells and not from the glass substrate. It is gratifying to note that
the PPC-1 cells, which are expected to bind both peptides, are
indeed stained as a mosaic with a mean ratio of approximately
1.0. By contrast, the SERRS images of the noncancerous prostate
cell RWPE-1 show very little red, indicating very low NRP/PC
ratios with the few red points situated primarily outside the cells
(mean of approximately 0.3 including outer rim), likely indicating
nonspecifically bound SBTs. The maps and their histograms
(Figs. S4 and S5) agree well with the histograms of average-cell
signals discussed below (Fig. 5A).
To discriminate confidently between these different types of
cells, we analyzed the average spectrum arising at both the entire
cell level and in maps with subcellular resolution (Figs. S6 and
S7), extracting ratios of NRP/PC. Sixteen PPC-1 cells and 24 con-
trol RWPE-1 cells were analyzed, and the resulting histograms of
the NRP/PC ratio are shown in Fig. 5A. Ninety-six percent of the
RWPE-1 cells were found to have NRP/PC ratios below 0.6,
whereas 100% of the PPC-1 were found to have ratios above this
value. Interestingly, the distribution for the PPC-1 cells is skewed
toward high ratio values, likely reflecting the heterogeneity of cell
expression of NRP-1 and particle affinity, binding/uptake rates,
or receptor recycling during prolonged incubation. We found that
changing the concentration of the SBT mixture did not signifi-
cantly affect the ratios.
The nonnormal distribution depicted by the histogram (Fig. 5A)
was converted into a normal distribution through a logarithmic
transformation. The statistical analyses are summarized in
Fig. 5B. Student’s t test was also performed on transformed data.
spectra of mixtures of the two SBTs: green trace, 100% PC-SBT; red trace,
100% NRP-SBT. (B) Calibration curve derived from the relative least-squares
coefficients for the two components corrected for the known concentrations
of MB or thionin SBT present in the solution. This calibration curve was used
to calculate the relative quantity of each tag resident on a given cell.
Calibration plots for the mixed-SBT deconvolution series. (A) SERRS
NRP- and PC-SBTs are synthesized, combined, and added
to either noncancerous or cancer cells suspended in DMEM
supplemented with 10% FBS. These are incubated for
60 min at room temperature. Cells are placed on a micro-
scope slide, and their SERRS spectrum is acquired using mi-
croRaman. The sample is mapped along the x and y axes in
1.5-μm steps, 5-μW power at sample, 1-s exposure time. The
SERRS spectrum averaged over the whole cell area is decon-
voluted into the individual components.
Schematic of the SERRS cell mapping experiment.
Pallaoro et al. PNAS
October 4, 2011
In Fig. 5B, the boxes define the interquartile range (IQR) (i.e. the
range thatcontainsthemiddle50% ofthe data inthedistribution),
the black square dots are the means of the distributions, and the
whiskers determine the upper and lower inner fences outside
which suspected outliers (extreme values) lie. These limits are
located, respectively, at Q1 ðlower quartileÞ − 1.5 × IQR and
Q3 ðupper quartileÞ þ 1.5 × IQR. No extreme values were ob-
served for the PPC-1 group, whereas the RWPE-1 group has two.
OneisintheupperfenceatlnðNRP∕PCÞ ¼ −0.15 andtheotherin
the lower fence at lnðNRP∕PCÞ ¼ −2.01; however, neither were
rejected as outliers. The higher extreme value observed was likely
due either to nonspecifically bound SBTs (Fig. S8) or glass-bound
SBTs located coincidentally close to a cell. The lower extreme is
likely caused by a few nonspecifically bound tags. This is clearly
illustrated in Fig. S8, which shows that for a noncancerous cell
the body is uniformly green (PC-dominated) with the few red
points (indicating NRP contribution) at the periphery. The loca-
tion of the outliers at the cells’ periphery suggests that the high
degree of confidence in distinguishing between cancerous and
noncancerous cells based on statistics can be further augmented
through microscopic observation of the cells. RWPE-1 cells show
weak but nonzero RPARPAR binding (resulting in a mean ob-
served NRP/PC ratio of approximately 0.3, somewhat greater
than zero—the expected value if all interactions were specific).
This is likely due to nonspecific adsorption of NRP-SBT on the
control cells’ membrane.
The statistics indicate that the ratiometric SERS multiplexing
approach we used does an excellent job in distinguishing cancer-
ous from noncancerous epithelial prostate cells. Referring to
Fig. 5B, the two cell populations form distinct clusters along
the ratiometric axis, well separated both visually and numerically.
Applying the t test to the two populations also shows them to be
highly distinct (p < 0.001), suggesting that with proper choice of
tags and peptides the ratio (or rather, its natural logarithm) easily
discriminates between noncancerous and cancerous cells.
The multiplexed, ratiometric method described was shown to
successfully distinguish between cancerous and noncancerous
epithelial prostate cells. The technique is based on deconvoluting
SERRS spectra arising from two sets of bright SBTs simulta-
neously bound to the cells. One of the two sets of SBTs was func-
tionalized to target the NRP-1 receptors of cancer cells through
the RPARPAR CendR peptide. The other functions as a positive
control and binds to both noncancerous and cancer cells through
the HIV-derived TAT peptide. Each was infused with a unique
Raman reporter molecule. Averaging the SERRS signal over a
given cell yielded an NRP/PC ratio that functioned as a statisti-
cally robust quantitative measure of the overexpression of the
NRP-1 by the cancer cell line. This indicative ratio is independent
of variations in the location of the focal plane, the local cell con-
centration, and turbidity. The technique also benefits from the
very low laser intensities (100 μW) needed for good signal to
noise and the use of a single wavelength to excite all SBTs.
Materials and Methods
Materials. All chemicals for the synthesis of the SBTs were purchased from
Sigma except where specified. Silver nitrate (AgNO3) 99.9999%; trisodium
citrate dihydrate (Fisher); hexamethylenediamine (HMD), 98%; polyvinylpyr-
rolidone (PVP), molecular weight 40,000; bovine serum albumin (BSA),
powder; potassium chloride (Mallinckrodt Baker); thionin acetate salt (dye
content approximately 90%); MB (dye content ≥82%); N-succinimidyl 3-(2-
pyridyldithio)propionate (SPDP), Thermo Fisher Pierce no. 21857; Fluores-
cein-C-X-GRPARPAR-OH (where X is an aminohexanoic linker) designated
FAM-Cys-RPARPAR was a gift from Erkki Ruoslahti’s group at Sanford–Burn-
ham Medical Research Institute, University of California, Santa Barbara, CA;
cates the NRP/PC ratio extracted by deconvolution of the point-by-point
SERRS spectra across the scanned area of a group of (A) PPC-1 cells and a
group of (D) RWPE-1 cells. Bright field image of (B) the same PPC-1 and
(E) RWPE-1 cells. (C and F) The corresponding image overlays. Color code in-
dicates the NRP/PC ratio ranging from 0.1 (green) to 10 (red). Scale bar, 5 μm.
Two-dimensional mappings for cancer and normal cells. Color indi-
populations. (A) The histogram represents the percentage of either RWPE-1
(blue) or PPC-1 (orange) cells that have the values of the NRP/PC ratio indi-
cated. (B) Box plots of the NRP/PC ratio for both PPC-1 and RWPE-1 popula-
tions after application of the logarithmic transformation to render both
distributions normal. The experimental data (orange dots for PPC-1 cells
and blue dots for RWPE-1 cells) are depicted on the left of each box as a visual
aid. The middle line of each box represents the median, the solid black square
is the mean, and the whiskers define the inner upper and lower fences be-
yond which possible outliers are found. The boxes do not overlap, indicating
statistically excellent differentiation between these two populations; t test
n1¼ 16 and n2¼ 24, p < 0.001.
Statistics of the SERRS ratios obtained from cancer and normal cell
www.pnas.org/cgi/doi/10.1073/pnas.1109490108 Pallaoro et al.
Cys-TAT (CGRKKRRQRRR-OH), Anaspec; Fluorescein-NHS (#46410, Pierce);
polyoxyethylene (20) sorbitan monolaurate solution 10% in H2O (Tween
20, T20); D-PBS (Gibco), Syringe filters Millex-GV, 0.22 μm, PVDF, 13 mm,
ethylene oxide sterilized.
Peptide Labeling. Cys-TAT was labeled with NHS-Fluorescein (FAM-NHS,
Pierce). FAM-NHS first was dissolved at 5 mg∕mL in DMSO and stored at
−20°C. Cys-TAT was dissolved in water (Hyclone) to a concentration of
1 mg∕mL and also stored at −20°C. The two were combined, 300 μL peptide
and 25 μL dye, and reacted for 3 h at room temperature and stored at −20°C.
BSA Modification. BSA was reacted with SPDP (Thermo Fisher Pierce #21857).
BSA was dissolved at 22 mg∕mL in water, and 50 μL 10× PBS pH 7.2 (Invitro-
gen) was added. SPDP was dissolved in DMSO (100 mg∕mL) and added to the
BSA, shaking for 3 h at room temperature. The protein was dialyzed over-
night using a Slide-a-Lyzer with a molecular-weight cutoff of 20 kDa, against
0.1× PBS pH 7.4, containing 0.02% NaN3with three changes of the buffer.
The volume increased during the dialysis because of the change in salt con-
ditions. The solution was recovered and passed through a 0.22-μm syringe
filter. Final concentration of the protein was determined by UV-visible spec-
troscopy to be 6.5 mg∕mL with a content of approximately 5 SPDP per BSA,
found through pyridyl reduction (extinction at 343 nm of 8;080 M−1cm−1).
BSA modified by the previous reaction of the sulfo-NHS group on SPDP
with lysines interacts with the silver surface of the colloid. The BSA binds
strongly through the SPDP molecule with the pyridine group appearing in
the SERS spectrum prior to Raman reporter being added. Each protein carries
several free hanging SPDP groups that can interact with the cys-peptides,
thus holding peptides on the outside of the SBT coating for interacting with
Ag SBTs Synthesis. The silver colloid was synthesized according to the Lee and
Meisel protocol (31): 500 mL of deionized water (DI, resistivity 18 MΩ) with
1 mM silver nitrate were brought to a boil. Then, 10 mL of 1% sodium citrate
was added. The mixture was kept at boiling temperature for about 90 min
until the color turned dark green/gray. Aliquots of the colloid were taken
and centrifuged at 0.8 × g to remove the smallest particles. The yellow super-
natant was discarded, and the pellet was resuspended in DI. The pellet was
diluted until the absorbance of the band at 406 nm was 0.3 at 0.1-mm path
length. The resulting colloid was called Ag03. NRP and PC-SBTwere then pre-
pared by adding to every 100 μL of Ag03 3.5 μL phosphate buffer (250 mM,
pH 7.5), 4 μL HMD (0.4 mg∕mL in DI, pH 4.0), waiting for 2 min, then adding
4 μL 1% PVP 40 kDa in DI and 100 μL DI, and finally waiting for 5 min before
proceeding with further functionalization. The particles resulting from this
controlled aggregation stage were called AgPHPand were then used to
prepare NRP and PC-SBTs: For PC-SBT, to every 200 μL AgPHPwe added
2 μL BSA-SPDP (6.5 mg∕mL in 0.1× PBS), waited 15 min, then added 4 μL
MB (600 μM in DI) and 2 μL KCl (500 μM) and incubated at room temperature
for 30 min; then, we added 2 μL FAM-Cys-TAT (500 μM in DI), incubated for
15 min, and backfilled with 1 μL BSA 5% (0.1× PBS). For the NRP-SBT, to every
200 μL of AgPHP, we added 2 μL BSA-SPDP (6.5 mg∕mL in 0.1× PBS), waited
15 min, added 4 μL of thionin (0.2 mg∕mL in DI), incubated for 30 min at
room temperature, added 2 μL BSA-SPDP (6.5 mg∕mL in 0.1× PBS), waited
for 15 min, then added 1 μL BSA 5% (in 0.1× PBS) and 5 μL FAM-C-X-GRPAR-
PAR-OH (200 μM in DI). After adding 0.005% final concentration of Tween-20
(T20), the SBTs were washed by centrifugation (10 min at 0.8 × g), the super-
natant was discarded (to remove most of the non-SERS bright single silver
nanoparticle biotags), and the pellet resuspended in 1/20th the initial volume
in 0.1× PBS/0.1 % BSA/0.005% T20.
Cell Culture. PPC-1 cells were a generous gift from Erkki Rouslahti’s group
(Sanford–Burnham Medical Research Institute, University of California, Santa
Barbara, CA). They were grown in DMEM/high glucose (HyClone) supplemen-
ted with 10% FBS. RWPE-1 cells (ATCC) were grown in Keratinocyte serum
free medium (Invitrogen) supplemented with bovine pituitary extract
(0.05 ng∕mL) and recombinant EGF (5 ng∕mL). Both were incubated at
37°C in a 5% CO2atmosphere. For SERRS mapping experiments, cells were
plated in multiwell plates and after either 24 or 48 h harvested using a none-
nzymatic cell dissociation buffer (Invitrogen) that does not disrupt the mem-
brane receptors. Cells were then washed by centrifugation for 2 min, and
the pellet was resuspended in the appropriate volume of DMEM þ 10%
FBS in order to obtain a concentration of 1.2–1.3 × 105cells∕100 μL. SBTs
were then added to cell suspension (with PC versus NRP ratio of 1∶2 or
1∶7 vol∕vol) and incubated for 60 min at room temperature; then, a 10-μL
aliquot was placed on a microscopy glass slide, covered with a coverslip,
sealed with nail polish, and mapped by SERS. For microscopy characteriza-
tion, cells were plated in multiwall plates and after either 24 or 48 h incu-
bated with SBT for 120 min at 37°C in a 5% CO2atmosphere, then imaged.
SERS Measurements. All SERS measurements (live cell mapping and composi-
tion calibration) were carried out using a Horiba Jobin–Yvon LabARAMIS
instrument equipped with a 633-nm laser, confocal microscope with 10×,
50×, and 100× objectives.Measurements for concentration calibration onSBT
ensembles were carried out using a 10× objective (100 μW power at sample),
633 laser line, hole 400 μm, slit 400 μm, exposure time 1 s, averaged three
Live cell mapping was performed in point scan mode, with hole 600 μm,
slit 300 μm, using a 100× objective and 633-nm laser (power at sample 5 μW);
exposure time 1 s, step size 1.5 μm.
Data Analysis. Initial data analysis (mapping, whole cell averaging of signal,
baseline correction, normalization) was performed with the help of the on-
board program LabSpec. The spectral deconvolution and fitting of single
spectra for the composition calibration was done using a weighted least-
squares algorithm in IgorPro (WaveMetrics Inc.), whereas the deconvolution
and NRP/PC ratio calculations for point-to-point maps were done using
Mathematica’s FindFit, a nonlinear least-squares-fitting algorithm, and the
SBT pure spectra, taken from Fig. 2, were processed by a customized note-
book. The statistics on the whole cell average data were performed using
OriginPro (Version 8.1). Student’s t test was performed, and p < 0.001 was
considered highly significant.
ACKNOWLEDGMENTS. We are grateful to Professor Erkki Ruoslahti for provid-
ing us with advice and samples of the FAM-C-X-GRPARPAR-OH peptide. We
thank Professor Norbert Reich at University of California, Santa Barbara, and
Dr. Tambet Teesalu in the Ruoslahti lab for many helpful discussions. This
work was supported by the Institute for Collaborative Biotechnologies
through Grant DAAD19-03-D-0004 from the US Army Research Office and
made extensive use of the Materials Research Laboratory Central Facilities
at University of California, Santa Barbara, supported by the National Science
Foundation under Awards DMR-0080034 and DMR-0216466 for the high-
resolution transmission electron microscopy/scanning transmission electron
microscopy. G.B.B. was also supported in part by a fellowship from the Santa
Barbara Cancer Center.
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