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Chemical Physics Letters xxx (2008) xxx–xxx
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Contents lists available at ScienceDirect
Chemical Physics Letters
journal homepage: www.elsevier.com/locate/cplett
Intracellular quantification by surface enhanced Raman spectroscopy
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Ali Shamsaie, Jordan Heim, Ahmet Ali Yanik, Joseph Irudayaraj *
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Bindley Biosciences Center, Discovery Park, Purdue University, 225 S, University Street, 215 ABE Building, West Lafayette, IN 47907, United States
a r t i c l e
i n f o
a b s t r a c t
Article history:
Received 3 March 2008
In final form 21 June 2008
Available online xxxx
Metallic nanoparticles in a cellular environment have a tendency to aggregate which poses a major obstacle in extending in vivo surface enhanced Raman spectroscopy (SERS) applications beyond the qualification and into quantification domain. We introduce and demonstrate a novel SERS technique that will
enable precise quantification of exogenous chemicals in living human cells. Effective quantification of
the local concentrations of a dinitrophenol derivative (DAMP) based on a normalizing technique is shown
by utilizing gold nanoparticle entrapment in the lysosomal compartments in human cells. We believe
that the quantification technique developed here is general and can be extended to different environments utilizing different types of nanoparticles beyond the intracellular scheme proposed.
Ó 2008 Published by Elsevier B.V.
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Local optical fields of metallic nanoparticles can give rise to enhanced Raman signals that contain highly localized information
about the surrounding environment. Using this principle, it is possible to deploy these nanosensors inside living cells and collect
informative signals from the intracellular microcosm within seconds. Since the original work by Chourpa et al. [1], there have been
few studies on the intracellular applications of SERS spectroscopy.
In these studies, detection of both native cellular constituents and
exogenous molecules has been reported [2–5]. Major obstacles
prevent the extension of such applications beyond the qualification
and into the quantification domain. Conventionally, SERS signals
are highly irreproducible and do not render themselves to acceptable quantification of target intracellular constituents. In addition,
delivering SERS inducers (for example gold/silver nanostructures)
to points of interest inside cells is another major challenge. For
effective enhancement effects, gold nanoparticles used for SERS
studies must be larger than 20 nm in size [6,7]. When such particles are used for in vivo studies after passive uptake by the cells
gets caught in the endosomal/lysosomal pathway and is inevitably
trapped in these compartments [8]. Kneipp et al. in one of their latest works have rightly considered the lysosmal context of nanoparticle aggregates when interpreting the SERS bands [9]. The only
example of a semi quantitative intracellular application of SERS
was presented by Talley et al. where functionalized silver nanoparticles were employed as sensors to provide a rough estimate of
intracellular pH [10].
For the first time we introduce an experimental set-up that
takes advantage of the co-occurrence of gold nanoparticles and
an exogenous chemical (i.e., DAMP) inside the lysosomal compartment of a living human cell for quantifying the local concentration
of this chemical in living human cells. The focus of our report is not
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* Corresponding author. Fax: +1 765 496 1115.
E-mail address: [email protected] (J. Irudayaraj).
on SERS quantification, rather to suggest a simple normalization
strategy to quantifying a known analyte intracellularly. In order
to perform a consistent calibration, we have also developed a novel
normalization technique that uses the phonon/vibron/plasmon
modes inherent to the nanostructure as an internal standard, thus
eliminating the need for a second referencing analyte. For quantification studies we chose a nonfluorescent weak basic amine, a
derivative of dinitrophenol (DAMP1) because it has been shown
that this chemical selectively accumulates in the cellular compartments with low internal pH [11]. Conventionally DAMP is used as a
LysoTrackerTM probe to investigate the biosynthesis and pathogenesis of lysosomes through secondary fluorescent antibody imaging
techniques [12]. Fig. 1A depicts the structure of DAMP.
MCF10 epithelial cells were grown to confluence and incubated
overnight with 50 nm gold nanoparticles (at a concentration of
about 4.5 1010 particles/ml). Then the cell monolayer was
washed and incubated for 30 min with 30 lM DAMP solution
(Molecular Probes, Carsland, CA) at 37 °C. The washing step ensured that no ectopic binding (i.e., outside the cell) could have happened between gold nanoparticles and DAMP molecules. Cell
monolayers were washed with phosphate buffer saline (PBS, 7.4
pH, from Sigma Aldrich Inc., St. Louis, MO) before Raman measurements. SERS spectra were collected using a confocal Raman microscope (Senterra from Bruker Optics, Inc., Billerica, MA) fitted with a
60 water immersion objective and a 633 nm laser source, whose
excitation source was close to the surface plasmon resonance of
aggregates or aggregate intermediates. The system is fitted with
a pair of interference edge filters that pass longer wavelength
and reflect the laser line. For the 633 nm laser the cutoff is
<80 cm1. It should be noted that single particle resonance can
be observed at 530 nm, as particles aggregate the resonance
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DAMP: N-(3-((2,4-dinitrophenyl)amino)propyl-N-(3-aminopropyl)methylamine,
dihydrochloride.
0009-2614/$ - see front matter Ó 2008 Published by Elsevier B.V.
doi:10.1016/j.cplett.2008.06.064
Please cite this article in press as: A. Shamsaie et al., Chem. Phys. Lett. (2008), doi:10.1016/j.cplett.2008.06.064
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837 cm-1
Intensity
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sphere has been studied and classified according to their symmetry
groups (l,m) using spherical harmonic functions Y lm by Lamb [16].
More recently, using symmetry arguments, Dulvar has shown that
only the symmetric l = 0 and quadrupolar l = 2 spheroidal modes
are Raman-active [17]. The radial wave vector of the surface modes
are roughly at kn = pn/L where n = 0, 1, 2,... in an increasing order for
a fixed angular shape (l,m). Accordingly, peaks are expected at
shift ¼ ðSl V p =2DcÞ n where Sl is a constant of the order of unity
depending on the symmetry of the mode and Vp is the longitudinal/transverse sound velocity. Low-frequency shifts observed in
our measurements are extremely large for any resonant vibration
modes (fundamental frequencies) of nanoparticles with/without
analytes.
For excitations close to the surface plasmon resonances
(kexct = 633 nm), the electronic cloud is strongly polarized and
may interact strongly with the optical-phonon vibrations leading
to a clear low-frequency Raman peak. This peak is not well investigated in the literature; in fact in a majority of the SERS studies in
biology the spectra below 400 cm1 is often truncated/ignored.
Alternatively, we suggest that one can utilize this band to obtain
a quantitative measure of the amount of the metallic nanoparticles
or particle aggregates which gives rise to SERS. This could pave the
pathway to formulating a simple protocol for intracellular quantification using SERS which to this date has been compounded by a
lack of control of the formation of consistent nanoparticle aggregates. This is a major concern since the measured Raman signal
is spatially averaged including hot/cold areas with dense/few gold
particles. Therefore it is desirable to adjust the detected Raman signals relative to the metallic nanoparticle aggregates involved in the
measurement. In this article, we have used the vibron/plasmon
couplings to normalize the SERS spectra to prove that the precision
of the calibration and predication steps could be improved. Both
band intensity and band integrals were tried for normalization
but the best result was achieved with the band intensities. In Fig.
2C, it is shown that for a fixed concentration of DAMP the normalized SERS signal at the characteristic band (837 cm1) is independent of the location where measurement are taken.
The behavior of the plasmon–vibron modes with respect to gold
nanoparticle concentration was also investigated. Experiments on
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shifts to higher wavelengths which is desirable not only for resonance coupling to maximize enhancement but also to minimize
the effect of fluorescence [13]. Laser power was set to 10 mW
and the laser spot size was estimated to be close to 2 lm and spectral resolution was 3–5 m1. Collection times were set to 10 s in
our experiments and the OPUS software was used to chop the spectra to reveal Raman bands in the 400 cm1 to 1800 cm1 range and
baseline correction was done by the rubber band method using a
‘rubberband’ like string icon stretched between the endpoints of
the spectrum to provide the spectrum minima. Spectra were collected both in a random fashion and also specifically from the dark
spots believed to be gold nanoparticle aggregates. TEM images
confirm the entrapment of gold nanoparticles in the endosmal/
lysosomal compartments (Fig. 1B) and Fig. 1C shows a complete
spectrum of DAMP with the key peaks that can be used as signature for intracellular studies. The characteristic band at 837 cm1
assigned to the ring breathing vibration modes of this molecule
was used for quantification in this study.
Fig. 2A presents the intracellular SERS spectra of DAMP at varying concentrations (8 mM and 130 mM) of this chemical consistent
with the in vitro raman fingerprint for this chemical (Fig. 1C). One
important feature consistently observed in our measurements (Fig.
2A) is the Raman shift at lower frequencies (100–250 cm1), extremely low to be attributable to any molecular vibrational modes.
Experiments have also shown that this band exists even in the absence of the analyte and in the background spectra collected from
bare gold nanoparticles (Fig. 2B). In Fig. 2B, SERS signals are compared in the absence (shown as ‘background’ spectrum) of gold and
presence bare gold nanoparticles. Our comparison confirms that
the secondary peak observed is clearly due to the presence of gold
nanoparticles. It is also shown that the location of the secondary
peak depends on the excitation signal wavelength (633–785–
830 nm) and the Raman shift increases with decreasing wavelength of the excitation frequency as reported in literature [14,15].
It is widely accepted that these low-frequency shifts are due to
the vibrational (acoustic phonons) mode couplings to the electromagnetic signal through the dipoles created by the modulation of
the surface polarization charges (surface plasmon–polaritons). The
spherical and torsional (vibration) modes of a homogeneous elastic
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Raman Shift (cm-1)
Fig. 1. (A) Chemical structure of DAMP, (B) TEM image (magnification: 27 500) showing the entrapment of gold nanoparticles in the membrane bound lysosomal/
endosomal compartments, and (C) Raman spectrum of DAMP showing the ring breathing vibration mode peaks at 837 cm1.
Please cite this article in press as: A. Shamsaie et al., Chem. Phys. Lett. (2008), doi:10.1016/j.cplett.2008.06.064
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Fig. 2. (A) Samples of the intracellular SERS signals of DAMP collected in vivo. (B) In vitro background spectrum of nanoparticle aggregates without DAMP denoting the
presence of the first/secondary peak at low wave-numbers due to the phononic–plasmonic couplings. (C) The normalization was done by dividing the 837 cm1 peak
intensity with the signal intensity for the first and the second peak for a fixed amount of DAMP concentration.
Fig. 3. Three in vitro SERS spectra of DAMP at 130 mM concentration incubated with nanoparticles of three different concentrations (a, b, and c) spotted on glass sides of
approximately 5 lm2 in cross-sectional area.
Please cite this article in press as: A. Shamsaie et al., Chem. Phys. Lett. (2008), doi:10.1016/j.cplett.2008.06.064
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ibration experimental model was designed. To simulate the intracellular condition the following procedure was followed: a 20 ll
droplet of 50 nm gold nanoparticle colloidal solution was allowed
to dry on a glass slide to which different concentrations of DAMP
solutions (prepared in water or buffer) were applied. A glass coverslip was placed on the top and SERS signals were collected with the
same settings mentioned above. SERS spectra of DAMP obtained
from this platform contained a high degree of irreproducibility
(RSD% of band intensity >20) as expected and used for
quantification.
The inconsistent nature of nanoparticle aggregation in cells is
one of the main cited reasons behind the lack of reproducibility
of spectral signals rendering quantification impossible. Loren et
al. [18,19] used a second analyte (a self assembled monolayer consisting of a thiol derivative of Dabcyl tethered to gold colloids) as
an internal standard to normalize the SERS signals to reduce the
varying enhancements originating from the chaotic assemblies of
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raman spectra from different surfaces, for example plain glass
slide, gold slide as well as glass and gold slides incubated with gold
nanoparticle clearly show that the presence of the plasmon–vibron
peaks below 400 cm1 is due to the plasmon modes of the spherical particles.
The variation of the signal intensity with respect to gold nanoparticle densities collected from 5 lm2 spots deposited on a glass
slide is depicted in Fig. 3. The signal intensities of the two normalizing peaks (100 cm1 and 230 cm1) varying in proportion to
the gold particle concentration (4.5 102, 4.5 105, and
4.5 1010 particles/ml respectively denoted by ‘a’, ‘b’, and ‘c’) is
demonstrated.
The efficiency of this normalization approach can be verified by
a decrease in the RSD% (percentage of relative standard deviation)
of the SERS signal intensities tested over a range of analyte concentration (Fig. 4). To quantify the local concentration of DAMP in the
vicinity of gold nanoparticle aggregates, an in vitro univariate cal-
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Fig. 4. (A) In vitro calibration based on the normalized intensities of 837 cm1 band of DAMP SERS spectra. Error bars represent two standard deviations. (B) Decrease in the %
RSD values of SERS intensities (n = 10) after normalization. The hashed pair on right is related to the intracellular data.
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Acknowledgment
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This research was conducted at the Physiological Sensing facility at Purdue’s Discovery Park.
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References
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[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
F
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I. Chourpa, H. Morjani, J.F. Riou, M. Manfait, FEBS Lett. 397 (1) (1996) 61.
K. Kneipp et al., Appl. Spectrosc. 56 (2) (2002) 150.
J. Kneipp, H. Kneipp, W.L. Rice, K. Kneipp, Anal. Chem. 77 (8) (2005) 2381.
C. Eliasson, A. Loren, J. Engelbrektsson, M. Josefson, J. Abrahamsson, K.
Abrahamsson, Spectrochim. Acta, Part A – Mol. Biomol. Spectrosc. 61 (4)
(2005) 755.
A. Shamsaie, M. Jonczyk, J. Sturgis, J.P. Robinson, J. Irudayaraj, J. Biomed. Opt.
12 (2) (2007) 020502.
K. Kneipp, H. Kneipp, J. Kneipp, Acc. Chem. Res. 39 (7) (2006) 443.
A. Wei, B. Kim, B. Sadtler, S.L. Tripp, Chemphyschem 2 (12) (2001) 743.
B.D. Chithrani, A.A. Ghazani, W.C.W. Chan, Nano Lett. 6 (4) (2006) 662.
J. Kneipp, H. Kneipp, M. McLaughlin, D. Brown, K. Kneipp, Nano Lett. 6 (10)
(2006) 2225.
C.E. Talley, L. Jusinski, C.W. Hollars, S.M. Lane, T. Huser, Anal. Chem. 76 (23)
(2004) 7064.
R.G.W. Anderson, J.R. Falck, J.L. Goldstein, M.S. Brown, Proc. Natl. Acad. Sci.
USA – Biol. Sci. 81 (15) (1984) 4838.
R.P. Haugland, The Handbook, Invitrogen Corp., Carlsbad, 2005. p. 580.
K. Faulds, R. Littleford, D. Graham, G. Dent, E. Smith, Anal. Chem. 76 (2004)
592.
D.A. Weitz, T.J. Gramila, A.Z. Genack, J.I. Gersten, Phys. Rev. Lett. 45 (5) (1980)
355.
J.I. Gersten, D.A. Weitz, T.J. Gramila, A.Z. Genack, Phys. Rev. B 22 (10) (1980)
4562.
H. Lamb, Proc. London Math. Soc. 13 (1882) 187.
E. Duval, Phys. Rev. B 46 (9) (1992) 5795.
A. Loren, J. Engelbrektsson, C. Eliasson, M. Josefson, J. Abrahamsson, K.
Abrahamsson, Nano Lett. 4 (2) (2004) 309.
A. Loren, J. Engelbrektsson, C. Eliasson, M. Josefson, J. Abrahamsson, M.
Johansson, K. Abrahamsson, Anal. Chem. 76 (24) (2004) 7391.
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We have proposed and experimentally shown a quantification
method which can be extended to SERS-based measurements in
different environments with different nanoparticles. Lower detection limits might be achievable using chemicals with a high Raman
cross-section, e.g. using a SERRS tag.
[15]
[16]
[17]
[18]
[19]
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gold nanoparticles [18]. The use of a linker (or a second chemical)
fingerprint necessitates additional chemical steps and interferes
with the fingerprint of the target analyte. In addition approaches
that utilize a second analyte (or a chemical monolayer) as an internal standard are not conducive for intracellular quantification. We
propose utilizing the Raman-active band inherent to the nanostructure responsible for enhancement to serve as an intrinsic
internal standard for in vivo quantification of analytes in cells.
Experiments were conducted in the concentration range between 8 mM and 130 mM correlating the Raman intensity dependent concentration of DAMP in vitro to the intracellular
concentration of DAMP in the lysosome of MCF10 cells. Calibration
coefficient improved from 53% (not shown) to 92% after applying
our proposed normalization (Fig. 4A). Here, the normalization
was done by dividing the 837 cm1 band intensity/integral by
the excitation dependent plasmon (180–250 cm1) or the back
scattered light peak, observed around 100 cm1 band. Since the
plasmon and the back scattered peak are intrinsic to the enhancing
metal, these appear in the analyte spectra which lie in the
enhancement range of the metal. The direct proportionality of
these intrinsic peaks (first peak depicting the back scattering and
the second depicting the Plasmon) used as a normalizing standard
(Fig. 2C) for the same concentration (50 nM) of the analyte from
eight different measurements show that the normalization is consistent (Fig. 2B). The random SERS spectra of DAMP from these
eight different measurements were surprisingly close (±10%). The
same normalization protocol was also applied to the intracellular
SERS signals with similar improvements in the reproducibility
(%RSDs were 36% without normalization and 9.7% with normalization respectively, Fig. 4B). Based on the above approach, the local
concentration of DAMP surrounding the gold nanoparticle aggregates (right most pair in Fig. 4A) was estimated to be 44 mM.
In summary, in this study we have for the first time demonstrated a novel SERS strategy that enables nonfluorescent intracellular concentration determination in a quantitative manner using
the inherent plasmon peak of gold nanoparticles in the spectra.
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