Surface-Enhanced Raman Spectroscopy-Based ... - ACS Publications

Chapter 13

Surface-Enhanced Raman Spectroscopy-Based Optical Labels Deliver Chemical Information from Live Cells 1,2

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Downloaded by UNIV OF ARIZONA on August 6, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0963.ch013

Janina Kneipp , Harald Kneipp , and Katrin Kneipp

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Wellman Center for Photomedicine, Harvard Medical School, Boston, M A 02114 Federal Institute for Materials Research and Testing, FG I.3, D-12489 Berlin, Germany Harvard-MIT Division of Health Sciences and Technology, Cambridge, M A 02139

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We demonstrate the application of surface-enhanced Raman scattering (SERS) in robust and sensitive optical probes for measurements in living cells. The hybrid probes consist of gold or silver nanoaggregates with an attached reporter species, e.g. a dye. They are detected based on the SERS signature of the reporter molecule. This approach results in several advantages compared to other optical labels, such as improved contrast, high spectral specificity, multiplex capabilities, and photostability. SERS probes do not only highlight targeted cellular or other biological structures through the specific reporter spectrum, SERS in the local optical fields of the gold or silver nanostructures also provides sensitive and spatially localized molecular structural information on the cellular environment.

© 2007 American Chemical Society

In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction Since more than a decade ago, it has repeatedly been proven that the fingerprint-like information in Raman microspectra can add to our understanding of the biochemical background of various regular, induced or pathological changes in eukaryotic cells(7 - 3). This is a basic research goal and a major prerequisite for progress in the areas of molecular medicine and nanobiotechnology. However, Raman investigations of living cells are not complication-free i f damage to the cells is to be avoided, because the applicable maximum intensity of the excitation laser is limited, but probe volumes are small and analyte concentrations low. In addition to the new challenges for Raman probing in live cells, current cell biology research generates a strong need for better optical labels(4). Although state-of-the-art fluorescence labels using dyes and quantum dots provide high sensitivity, the information they can deliver on chemical composition or molecular structure is very limited(5, 6). Therefore, improving optical labels regarding sensitivity, specificity, molecular structural information content, and spatial localization is another demanding subject in current biophotonics research. Here, we will show that both issues can be addressed by applying surface enhanced Raman scattering (SERS) in the local optical fields of noble metal nanoparticles. (for a more recent overview on SERS see refs (7 - 12)). Nanostructures from gold or silver lead to significant improvements of the Raman signals from the molecules in their nanometer-scaled environment. This suggests optical labels based on SERS signals of reporter molecules attached to gold and silver nanoparticles instead of using fluorescence tags (13 - 15). Moreover, aside from providing the specific SERS spectrum of the reporter dye, gold nanoparticles also enhance the Raman signatures of their environment and in this way enable sensitive chemical probing inside biological structures, such as inside living cells (16-19). In the following we investigate silver and gold nanoclusters and dyes commonly used in biological studies for their potential use in SERS hybrid probes. From two biocompatible components, the dye indocyanine green (ICG), complexed with serum albumin protein, and gold nanoparticles, we constructed a SERS probe and introduced it into cultured cells.

Experimental Silver and gold nanoparticles were produced in aqueous solutions by chemical preparation as described in ref (20). This preparation process results in isolated metal nanoparticles and small clusters comprised from 3-10


188 nanoparticles. Gold colloidal solution containing 60 nm gold spheres was also purchased from Polysciences Inc. (Eppelheim, Germany/ Warrington, PA). Both kinds of gold nanoparticles gave the same SERS spectra and provide almost the same enhancement factors. Methylene Blue, Hoechst33342, and indocyanine green (ICG) (all purchased from Sigma) were prepared in 10" - ΙΟ" M stock solutions in water or, for applications in cells, in 5 mg/ml aqueous solution of human serum albumin (HSA). These stock solutions were added to the aqueous solution of silver and gold nanoclusters for final concentrations between 10" and 10- M. For application in cells, a SERS nanoprobe consisting of 1 0 ' M indocyanine green (ICG) complexed with human serum albumin (HSA) on 60 nm gold nanoparticles was delivered into cultured cells of a metastatic Dunning R3327 rat prostate carcinoma line ( M L L ) (donated by Dr. W. Heston, Memorial Sloan-Kettering Cancer Center, New York, N Y ) . After overnight incubation, nanoparticles that were endocytosed must be included in lysosomes. In accordance with this assumption we found gold accumulations in the range of 100-1000 nm in lysosomes by light and electron microscopy after incubation for 20 hours and longer. The presence of gold particles in the cells could also be verified by the appearance of SERS signals collected from the cells. Microscope inspection provided evidence that the cells were dividing after incubation with the ICG-SERS probes. While incubated with the nanosensors, the cells were visibly growing, and no evidence of cell death was found. A slightly lower density after 20 hours incubation with the diluted medium was observed when compared with control cells growing in undiluted medium, probably resulting from the dilution of nutrients. Controls in diluted medium with ICG/NaCl and NaCl-diluted culture medium showed growth rates similar to those incubated with the gold particles and with the ICG-SERS nanoprobes. For testing the SERS probes, SERS spectra were measured from aqueous solutions (25 μΐ droplets) of silver and gold nanoclusters loaded with different concentrations of the reporter dye. Excitation at different wavelengths (680 nm, 786 nm and 830 nm) was applied. (For detailed experimental parameters see Figure captions.) A 60x microscope water immersion objective was used for focusing the excitation laser to a probed volume of -50 fL. The same microscope objective was also used to collect the Raman scattered light. A single stage spectrograph with holographic edge or notch filters in front of the entrance slit arid a liquid nitrogen-cooled C C D detector were used for spectral dispersion and collection of the scattered light. Raman spectra were acquired from the living cells in the physiological environment (PBS buffer) using laser intensities of - 2 mW in accumulation times of 1 sec and less. 5

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189 Raster scans over single living cells were carried out with a computercontrolled x,y-stage in 2 μιη steps at a laser spot size of ~ 4 x l 0 ' c m . A l l experiments on the cell culture were carried out at 830 nm excitation. 8

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Results and Discussion A key question in creating hybrid SERS probes for biological applications is a nanostructure that provides a high level of electromagnetic field enhancement. This is the prerequisite for an intense and stable spectroscopic signature of a reporter but also for an efficient and sensitive probing of the cellular environment. Moreover, the nanostructure should be biocompatible in order to enable probing of cells and other biological structures without influencing their physiological state or inducing any damage. So far, aggregates formed by silver or gold nanoparticles provide the highest enhancement level observed in SERS experiments, yielding enhancement factors up to 14 orders of magnitude (72, 27, 22). Figure 1 shows typical nanoclusters formed by silver and gold nanoparticles and their plasmon absorption. Interestingly, and very useful for biological applications, extremely high enhancement factors for aggregates can be obtained at near infrared excitation despite the fact that the plasmon resonance for isolated nanoparticles can be found around 400 nm for silver and 520 nm for gold. As reporter molecules to be attached to these nanoaggregates we chose dyes known and approved in biological and medical applications as fluorescence markers. However, instead of using the broad and relatively non-specific fluorescence signals of these dyes, here we rely on their specific (surface enhanced) Raman signature. Figure 2a displays SERS spectra measured from two biocompatible dyes, Methylene Blue and Hoechst33342, attached to silver nanoaggregates. Both dyes show very specific SERS spectra comprised from several narrow lines. The idea to create an optical label based on these SERS signals provides several advantages over other labels. The characteristic, fingerprint-like pattern of many narrow Raman bands of the label molecule rather than, e.g., one broad fluorescence band, provides a high specificity, which has been used in multiplexing experiments unprecedented so far by fluorescence studies (75). Moreover, the near infrared excitation intensity is not in resonance with the electronic transitions of the two dyes. This prevents photobleaching and results in a high label stability. SERS experiments performed on silver and gold nanoaggregates at N I R excitation show that nanoclusters of both metals exhibit comparably good SERS enhancement factors (Figure 2b). Figure 2b compares SERS spectra of the dye indocyanine green on silver and gold nanoaggregates. The favorable enhancing



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Figure 1. Extinction spectra and EM images of typical silver and gold nanoaggregates usedfor SERS studies in cells.

properties along with their biocompatibility suggest gold nanoaggregates as very promising SERS nanosensors for intracellular studies (72). Figure 2c illustrates the detection limits for the reporter dye I C G bound to H S A in gold colloidal solution. The dye is widely used in medical imaging and surgical applications based on its fluorescence signal. Usually in these applications, local I C G concentrations are in the micromolar (10" M) range. Spectra A to D in Figure 2c were collected from gold colloidal solutions containing I C G in 1 0 " M to 1 0 ' M concentration, respectively. Spectrum D demonstrates that the strong Raman line at 945 cm' is still detectable at an I C G concentration of 1 0 " M . This corresponds to ~3 dye molecules in the probed 50 fL volume. Surface enhanced Raman lines of I C G in such low concentration experiments can be understood in terms of very high SERS enhancement factors in the hot spots of the nanoaggregates (72, 22). Comparing the signal count rate per molecule for the strongest surface enhanced Raman lines of I C G in such low concentration experiments with the count rate of the fluorescence per "free" ICG molecule, the number is one order of magnitude higher for the SERS lines. 6

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Figure 2. a) SERS spectra of the biocompatible dyes Methylene Blue (A) and Hoechst 33342 (B) on silver nanoaggregates. The signals were collectedfrom ~ 5000 molecules in the probed volume, 3 mW 830 nm excitation, collection time 1 second.



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Figure 2. b) SERS spectra oflO~ M indocyanine green in silver (A) and gold (B) colloidal solutions obtained with 5 mW 786 nm excitation. SERS spectra are shown after fluorescence background correction. Reprinted with permission from (\%) Copyright 2005 ACS.

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Figure 2. c) SERS spectra of ICG bound to HSA in gold colloidal solution. Spectra A to D were collectedfrom gold colloidal solutions containing ICG in I0' M to Iff Mconcentration, respectively, at 15 mW, 830 nm excitation, 1 second collection time Reprinted with permission from (18) Copyright 2005 ACS

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194 As has long been practiced in various cell biology applications (23 - 25) gold nanoparticles can be delivered into live cells (see also Experimental section). Figure 3 illustrates the basic concept of intracellular SERS probes. Large nanoparticle aggregates can easily be visualized by standard bright field microscopy inside the cells, here, in the lysosomal structures of a fibroblast cell. Raman measurements with laser intensities of ~ 1 0 W cm' and accumulation times of 1 sec and less, and excitation with 830 nm (out of resonance) preclude from the observation of non-SERS spectra from the cells. However, at positions in the cells where gold nanoparticles are present, surfaceenhanced Raman spectra can be measured in the living cells in their physiological environment. Figure 4 shows the detection and imaging of a SERS label made from gold nanoparticles and ICG in a single live cell using the 945 cm" SERS signal (see also Figure 2). In addition to the SERS signals of the reporter ICG, at these positions, also the Raman signatures from the cellular components in the immediate surroundings of the gold nanostructures experience surface enhancement. This enables sensitive chemical probing of the particles' vicinity in very short times. Figure 5 illustrates this ultrasensitive molecular probing using SERS nanosensors made from gold nanoparticles and ICG. Spectrum A represents the spectrum of pure ICG in the physiological environment. Spectrum Β shows the SERS signature of the reporter ICG along with SERS lines that originate from the cellular surroundings of the gold nanoparticles. After subtracting the SERS signal from the reporter ICG (trace A ) , trace C displays the SERS spectrum of the cellular components. The Raman lines in spectrum C can be assigned to vibrations characteristic for protein and nucleotide molecular groups, such as C - H deformatioi^ending modes at 1450 cm" , C - N deformation at 1166 and 1229 cm" , possible contributions from Phe and Tyr -1207 cm" , as well as cytosine and adenosine ring vibrations and/or protein amide II contributions around 1540 cm' (tentative assignments after refs. (26 - 29). As described above, SERS takes place in the local optical fields of metal nanostructures and is therefore restricted to the immediate vicinity of the gold nanoparticles (see also Figure 3 for a schematic). Thereby, SERS probes enable the acquisition of Raman signals not only at high sensitivity but also from nanometer scaled volumes. This local confinement of the SERS effect has several advantages over regular Raman experiments: For SERS studies in cells, SERS nanoprobes can be positioned at discrete locations, e.g., in a specific cellular compartment, and the spectral information is obtained only from the nano-environment of these probes and hence that particular compartment. This is different from the spectral information collected in a "normal" Raman microspectroscopic experiment, where all positions in a whole cell are probed. This also means that the maximum lateral resolution in such a Raman experiment


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Figure 3. Basic concept of intracellular SERS probes: Gold nanoparticles are transferred into cells. Aggregates, which provide optimum SERS enhancement and are typically utilized in the live cell experiments are shown in the transmission electron micrograph and the schematic drawing. During excitation with laser light in the near-infrared (h vj, such gold nanoaggregates provide enhanced local optical fields in their nm-scaled vicinity, leading to surface-enhanced Stokes (h v$) andanti Stokes Raman signals (h v ) of the attached reporter molecules as well as of different cellular molecules (see also Figure 5).



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Figure 4. Spectral map of ICG in a cell based on the 945 cm' SERS line of the molecule (see also Figure 2b). Intensities are shown in gray scale (highest value in white). A photomicrograph of the cell, indicating the mapped area, is shown for comparison. Scale bar: 28 microns.



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Figure 5. SERS spectra measured inside living cells incubated with ICG on colloidal gold: SERS spectrum of ICG (A), SERS spectrum of ICG and cell environment (B), difference spectrum C displaying SERS features of cell components. For the assignment of the bands see text.

is no longer limited by the excitation wavelength; instead, it is influenced by the metal nanostructure used to provide the enhancement.

Conclusions To summarize, we have demonstrated a new type of optical probe based on SERS that is stable, specific, and biocompatible, and can be used for applications in live cells. SERS nanosensors can be detected and imaged based on the unique spectroscopic signature of the SERS signal of a reporter molecule attached to the gold nanostructure. In addition to its own detection by the characteristic SERS spectrum of the reporter, the probe we are proposing delivers surface enhanced Raman signatures of the cell components in its vicinity. This provides the capability of ultrasensitive chemical characterization of nanometer scaled units in single live cells. Due to the large effective Raman scattering cross section, SERS probes fulfill the requirements of dynamic in vivo systems -the use of very low laser powers and very short data acquisition times.


198 Acknowledgement This work was supported in part by D O D grant # A F O S R FA9550-04-10079, N I H grant # PO1CA84203, and by the generous gift of Dr. and Mrs. J.S. Chen to the optical diagnostics program of the Massachusetts General Hospital Wellman Center for Photomedicine.


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199 17. Zeiri, L . ; Bronk, Β. V . ; Shabtai, Y . ; Czege, J.; Efrima, S. Colloids and Surfaces a-Physicochemical and Engineering Aspects 2002, 208, 357-362. 18. Kneipp, J.; Kneipp, H . ; Rice, W. L.; Kneipp, K . Analytical Chemistry 2005, 77, 2381-2385. 19. Premasiri, W. R.; Moir, D. T.; Klempner, M. S.; Krieger, N.; Jones, G.; Ziegler, L . D . Journal of Physical Chemistry Β 2005, 109, 312-320. 20. Lee, P. C.; Meisel, P. Journal of Physical Chemistry 1982, 86, 3391-3395. 21. Kneipp, K . Experimental Technique of Physics 1988, 36, 161. 22. Kneipp, K . ; Wang, Y.; Kneipp, H . ; Perelman, L . T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667. 23. Feldherr, C.; Kallenbach, E.; Schultz, N. J Cell Biol. 1984, 99, 2216-2222. 24. Tkachenko, A . G.; Xie, H . ; Liu, Y . L . ; Coleman, D.; Ryan, J.; Glomm, W. R.; Shipton, M. K . ; Franzen, S.; Feldheim, D. L . Bioconjugate Chemistry 2004, 15, 482-490. 25. Chithrani, B . D.; Ghazani, Α. Α.; Chan, W. C. W. Nano Letters 2006, 6, 662-668. 26. Thomas Jr., G.; Prescott, B.; Olins, D . Science 1977, 197, 385-388. 27. Parker, F. S. Applications of Infrared, Raman, and Resonance Raman Spectroscopy in Biochemistry, Plenum Press: New York and London, 1983; Vol. 28. Peticolas, W. L . ; Patapoff, T. W.; Thomas, G . Α.; Postlewait, J.; Powell, J. W. Journal of Raman Spectroscopy 1996, 27, 571-578. 29. Puppels, G. J.; De Mul, F.; Otto, C.; Greve, J.; RobertNicoud, M.; ArndtJovin, D. J.; Jovin, T. Nature 1990, 347, 301-303.


Surface-Enhanced Raman Spectroscopy-Based ... - ACS Publications

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