uncorrected proof - Purdue University
Transkript
uncorrected proof - Purdue University
CPLETT 26216 No. of Pages 5, Model 5G ARTICLE IN PRESS 1 July 2008 Disk Used Chemical Physics Letters xxx (2008) xxx–xxx 1 Contents lists available at ScienceDirect Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett Intracellular quantification by surface enhanced Raman spectroscopy 3 Ali Shamsaie, Jordan Heim, Ahmet Ali Yanik, Joseph Irudayaraj * 4 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. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 EC 29 RR 28 CO 27 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 UN 26 TE 24 25 PR OO 8 1 6 4 9 10 11 12 13 D 5 F 2 * Corresponding author. Fax: +1 765 496 1115. E-mail address: josephi@purdue.edu (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 1 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 15 16 17 18 19 20 21 22 23 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 CPLETT 26216 1 July 2008 Disk Used 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 F 95 PR OO 94 TE 93 A B C 837 cm-1 Intensity 92 EC 91 RR 90 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 CO 89 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 UN 88 A. Shamsaie et al. / Chemical Physics Letters xxx (2008) xxx–xxx D 2 87 No. of Pages 5, Model 5G ARTICLE IN PRESS 400 600 800 1000 1200 1400 1600 1800 2000 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.2008.06.064 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 CPLETT 26216 No. of Pages 5, Model 5G ARTICLE IN PRESS 1 July 2008 Disk Used 3 EC TE D PR OO F A. Shamsaie et al. / Chemical Physics Letters xxx (2008) xxx–xxx UN CO RR 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 CPLETT 26216 1 July 2008 Disk Used 173 174 175 176 177 178 179 180 181 F 172 D 171 TE 170 EC 169 RR 168 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 CO 167 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- UN 166 A. Shamsaie et al. / Chemical Physics Letters xxx (2008) xxx–xxx PR OO 4 165 No. of Pages 5, Model 5G ARTICLE IN PRESS 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. Please cite this article in press as: A. Shamsaie et al., Chem. Phys. Lett. (2008), doi:10.1016/j.cplett.2008.06.064 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 CPLETT 26216 No. of Pages 5, Model 5G ARTICLE IN PRESS 1 July 2008 Disk Used 5 A. Shamsaie et al. / Chemical Physics Letters xxx (2008) xxx–xxx 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 Acknowledgment 240 This research was conducted at the Physiological Sensing facility at Purdue’s Discovery Park. 241 References 243 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] F 206 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. PR OO 205 D 204 235 [14] TE 203 EC 202 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] RR 201 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. CO 200 UN 199 Please cite this article in press as: A. Shamsaie et al., Chem. Phys. Lett. (2008), doi:10.1016/j.cplett.2008.06.064 236 237 238 239 242 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274