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Aug 5, 2011 - Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of tar...
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Immuno-Surface-Enhanced Coherent Anti-Stokes Raman Scattering Microscopy: Immunohistochemistry with Target-Specific Metallic Nanoprobes and Nonlinear Raman Microscopy Sebastian Schl€ucker,*,† Mohammad Salehi,† Gero Bergner,‡ Max Sch€utz,† Philipp Str€obel,§ Alexander Marx,§ Iver Petersen,^ Benjamin Dietzek,‡ and J€urgen Popp‡ †

Department of Physics, University of Osnabr€uck, 49069 Osnabr€uck, Germany Institute for Photonic Technology (IPHT), 07745 Jena, Germany § Institute of Pathology, University Hospital Mannheim, 68167 Mannheim, Germany ^ Institute of Pathology, University of Jena, 07743 Jena, Germany ‡

bS Supporting Information ABSTRACT: Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of target proteins in tissue specimens by conventional IHC is achieved with dye- or enzyme-labeled antibodies in combination with light microscopy. In this contribution, we demonstrate the proof-ofprinciple for IHC based on surface-enhanced coherent Raman scattering for contrast generation. Specifically, antibody-labeled metallic nanoshells in conjunction with surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy are employed for the selective, sensitive, and rapid localization of the basal cell protein p63 in normal prostate tissue. Negative control experiments were performed in order to confirm the selective binding of the target-specific metal nanoprobes and to disentangle the role of plasmonic (metal) and molecular (Raman reporter) resonances in this plasmon-assisted four-wave mixing technique.

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arious external labeling strategies for optical detection in bioanalytical chemistry and the life sciences are available, including molecular fluorophores and quantum dots for spectral multiplexing.13 Metal nanoparticles are also very attractive contrast agents for biological and medical applications; they have tunable size- and shape-dependent optical properties, combined with high photostability and chemical inertness in the case of noble metals.4,5 Contrast mechanisms for metal nanoparticles include light absorption as well as elastic and inelastic scattering. In particular, surface-enhanced Raman scattering (SERS), a plasmon-assisted vibrational spectroscopic technique based on inelastic light scattering,69 is nowadays increasingly employed for the selective detection of target molecules. Applications cover, for instance, the selective detection of DNA and proteins in SERSbased assays1014 and the selective localization of target proteins in tissue specimens by SERS microscopy1518 as well as targeted in vivo SERS.19 In addition to SERS, also, nonlinear spontaneous Raman techniques were demonstrated, in particular surface-enhanced hyper-Raman scattering (SEHRS).2022 Applications of coherent Raman techniques for the localization of target-specific metal nanoprobes in biological specimens, however, have not been reported so far. Coherent anti-Stokes Raman scattering (CARS) is a four-wave mixing process in which three incident photons generate a fourth photon at the anti-Stokes frequency ωAS= 2ω0  ωS via the r 2011 American Chemical Society

nonlinear third-order susceptibility χ(3) of the material. In contrast to spontaneous Raman scattering, vibrations with ωRaman are coherently driven under Raman-resonant conditions ωRaman = ω0  ωS, i.e., the difference frequency between pump (ω0) and Stokes (ωS) laser fields resonantly excites the corresponding nuclear motions.23,24 Raman-resonant and non-Raman-resonant (3) contributions to χ(3) are noted as χ(3) R and χNR, respectively. SECARS is surface-enhanced CARS; at least one or even all wavelengths (ω0, ωS, ωAS) involved in the CARS process are in electronic resonance with the plasmon band of the nanostructure. Both solid and colloidal nanostructures such as silver films and colloidal particles, respectively, were employed for early experimental demonstrations of SECARS obtained from benzene and other aromatic compounds.25,26 More recently, also molecules of biological interest were monitored by SECARS. For instance, the ultrasensitive SECARS detection of the nucleotides dAMP and dGMP was achieved with aggregated silver nanoparticles, picosecond laser excitation in a collinear geometry, and polarization control for background suppression.27 In contrast to label-free monitoring of analytes, the selective detection of target molecules relies on a ligand with specificity Received: May 20, 2011 Accepted: August 5, 2011 Published: August 05, 2011 7081

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Analytical Chemistry toward the target. A prominent example is the specific interaction between an antibody and its corresponding antigen. The selectivity of the antibodyantigen recognition is the basis for immunohistochemistry (IHC), immunofluorescence, immunoRaman/SERS, and other immuno-based staining techniques. Central advantages of immuno-SECARS microscopy, the novel nonlinear vibrational methodology presented in this contribution, comprise (i) the higher spatial resolution of SECARS/CARS microscopy compared with standard light microscopy used in conventional IHC; (ii) the higher multiplexing capacity compared to conventional IHC, which is due to the small line width of vibrational Raman bands and the possibility to simultaneously detect several SERS particles; (iii) higher speed compared with conventional immuno-SERS microscopy since CARS microscopy can operate at μs integration times per pixel; (iv) higher robustness against tissue autofluorescence compared with conventional immunofluorescence since the generated SECARS signal is blue-shifted with respect to the pump laser wavelength, i.e., one-photon-excited fluorescence is simply not detected and does therefore not degrade image contrast. Here, we apply immuno-SECARS microscopy for localizing the basal cell marker p63 in prostate tissue sections by SERS-labeled p63 antibodies in conjunction with coherent Raman microscopy for read-out. Earlier work on the detection of p63 levels in histological screens includes enzyme-labeled p63 antibodies in conjunction with image-processing tools for quantification28,29 or conventional immuno-SERS microscopy using functionalized gold nanostars30 and gold/silver nanoshells.31

Figure 1. (Left) Extinction spectra of bare Au/Ag nanoshells (black), after incubation with a SAM (blue), and finally after conjugation to the antibody (red). (Right) TEM image for Au/Ag nanoshells (scale bar: 200 nm).

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’ RESULTS AND DISCUSSION SERS nanoprobes or nanotags comprise a metal colloid with Raman reporter molecules adsorbed onto its surface.18,32,33 In this study, gold/silver nanoshells optimized for red laser excitation are used as plasmonic substrates in order to minimize autofluorescence from the tissue specimen, which would degrade image contrast in immuno-SERS microscopy. The preparation of Au/Ag nanoshells follows procedures described by Xia and co-workers.34 Silver salts are reduced in a polyol process to yield silver nanospheres. HAuCl4 is added and reduced in a template-based replacement reaction, in which AgCl precipitates. The optical properties of gold/silver nanoshells can be calculated with modified Mie theory.35 For red (632.8 nm) laser excitation, the optimum plasmon resonance for maximum SERS signals is calculated to occur around 650 nm due to maximum enhancement of both the incident field at ω0 and the outgoing field at ωS = ω0  ωRaman. Figure 1 shows a TEM image of ∼6070 nm Au/Ag nanoshells (right) and the corresponding extinction spectrum (left). Figure 2 schematically shows the design and synthesis of the stabilized SERS labels (3) employed in this study.33 The gold/ silver nanoshells (1) are incubated with Raman reporter molecules containing hydrophilic spacer groups (2a, 2b), yielding a densely packed self-assembled monolayer (SAM) on the particle surface (3). The presence of a complete SAM has several advantages: the dense and uniform orientation of Raman reporter molecules leads to reproducible SERS signatures, while the complete surface coverage provides maximum signals. At the same time, coadsorption of other molecules from the environment, leading to unwanted spectral interferences, is avoided or at least minimized. The two SAM components 2a and 2b in Figure 2 are amides, obtained by forming the acyl chloride of 5,50 -dithiobis(2-nitrobenzoic acid) and coupling it to the corresponding amines containing either (2b) a short hydrophilic monoethylene glycol spacer with a nonreactive hydroxy terminus (H2NMEGOH) or (2a) a longer hydrophilic triethylene glycol spacer with a terminal carboxy group (H2NTEGCOOH).33 This design of SERS labels has several advantages.33 First, the hydrophilic spacers stabilize and render the entire SERS particle (3) water-soluble, independent of the terminal charge of a particular Raman reporter. Other approaches for stabilizing SERS particles include, for instance, encapsulation by a glass3640 or polymer shell.19,41,42 Second, the longer TEG spacer moieties ensure sterical accessibility of the COOH termini required for bioconjugation. Third, the stoichiometric ratio of Raman reporters

Figure 2. Synthesis of SERS-labeled antibody 4 starting from gold/silver nanoshells 1. The Raman reporter molecules 2a and 2b (1:100) are stabilized by hydrophilic spacer moieties.33 (Bottom right) SERS spectrum of 4. 7082

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Figure 3. White light (left) and SECARS (right) images of prostate biopsies incubated with SERS-labeled p63-antibodies. p63 is only abundant in the p63-(+) basal epithelium (bE, arrows) but not in the p63-() secretory epithelium (sE) and lumen (L). Figure 5. (Left) White light microscope image of prostate tissue. (Right) Conventional CARS microscopic image of the same tissue region. This experiment serves as a negative control for immuno-SECARS microscopy, which showed specific staining of the basal epithelium (cf. Figure 3 right). In contrast, no specific contrast is observed in conventional CARS microscopy (i.e., without the use of SERS-labeled antibodies).

Figure 4. (Left) Raw data from immuno-SECARS microscopic experiments (cf. Figure 2). (Middle) Negative control experiments with no time overlap between pump and Stokes pulses. (Right) Difference image.

containing short and longer spacer units (MEGOH/TEG COOH, here 100:1) allows one to control the extent of bioconjugation, including the maximum number of biomolecules which can be coupled to the SERS particle. For bioconjugation to antibodies directed against p63, the terminal carboxyl groups of the longer hydrophilic spacer moieties of SAM component 2a on the surface of the SERS particle (3) were activated with conventional EDC/ sulfo-NHS chemistry to the corresponding NHS ester. The reaction with primary amines such as lysine residues from the antibody yields the SERS-labeled antibody (4). The Raman spectrum of 4 in Figure 2 (bottom right) was obtained with the 632.8 nm line from a HeNe laser. The intense Raman peak at ∼1340 cm1 is assigned to the symmetric stretching vibration of the nitro moiety from the Raman reporter (see 2a and 2b); the other two peak bands arise from phenyl ring modes. Due to the SERS distance dependence, only modes from the Raman reporter but not the antibody are enhanced. The 1340 cm1 Raman peak was used as a marker for subsequent SECARS imaging experiments, i.e., the wavenumber difference between pump and Stokes laser was tuned to this Raman resonance. Both pump and Stokes laser wavelengths fulfill the plasmon resonance condition since the employed Au/Ag nanoshells with a diameter of ca. 60 nm exhibit a broad extinction band centered at λmax ∼ 645 nm (Figure 1). For immunohistochemistry with target-specific nanoprobes (4 in Figure 2) and coherent Raman microscopy,43 formalin-fixed and paraffin-embedded 5 μm thick prostate tissue sections from patients undergoing prostatectomy were employed. The white light image of the prostate biopsy in Figure 3 (left) exhibits the p63-(+) basal epithelium (bE, arrows) as well as the p63-() secretory epithelium (sE) and lumen. The SECARS image in Figure 3 (right) displays the selective abundance of p63 in the basal epithelium (bE): the red dots in Figure 3 (right) result from

the nanoshells targeted against p63, a homologue of p53. This protein is abundant in the basal epithelium of the normal prostate, while it is absent in the neoplastic glands. An enlarged view of Figure 3 (right) is shown in the Supporting Information (Figure S1). The images presented in Figure 3 clearly demonstrate the capability of target-specific probes in conjunction with coherent Raman microscopy for the selective, sensitive, and rapid localization of tumor-relevant antigens in tissue specimens. Control experiments were performed in order to carefully check the validity of the results and their interpretation. The first negative control experiment (Figure 4), realized by introducing a time mismatch between pump and Stokes picosecond laser pulses, yielded no contrast from tissue specimens incubated with p63-SERS nanoshells. This negative result confirms that a nonlinear process, which requires the simultaneous presence of two laser pulses with different wavelengths, is responsible for signal generation. The second negative control experiment was regular CARS microscopy on blank tissue, i.e., without the addition of p63specific SERS-labeled antibodies. As expected, the intrinsic contrast for tissue was observed in this case (Figure 5), i.e., there is no specific contrast for p63 since no antibodies were employed. This negative result, therefore, confirms that the selective staining of the basal epithelium in Figure 3 (SECARS image, right) is due to the specificity of the p63 antibody, which is conjugated to the SERS particles. These two control experiments indicate that the contrast in Figure 3 arises from target-specific metal nanoprobes, which selectively recognize p63 in the basal epithelium of the normal prostate via the p63 antibody. In addition to these negative control experiments, it is further necessary to clearly demonstrate the contribution of molecular resonances to the experimentally observed SECARS signals since other contributions may be as well responsible for contrast generation. In conventional CARS, for instance, a non-Raman-resonant background contributes to the overall CARS signal. This background is due to purely electronic contributions (material parameter: nonlinear susceptibility χ(3) NR) and leads to complex dispersive CARS line shapes via the interference of contributions 24 In fourarising from χ(3)NR and its resonant counterpart χ(3) R . wave mixing experiments on gold nanostructures without molecules adsorbed onto their surface, a similar phenomenon is observed. In 7083

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Analytical Chemistry this case, the purely electronic contribution arises from the metal. The generation of coherent anti-Stokes scattering (CAS; no Raman process involved) has been observed from coupled pairs of gold nanospheres in near-field44 and from gold nanowires in far-field45 four-wave mixing experiments. One aspect of this study was, therefore, to unambiguously demonstrate the contribution of molecular resonances to the experimentally observed SECARS signals, i.e., to highlight the role of vibrational contrast from the Raman reporter molecules in addition to purely plasmonic contributions from the Au/Ag nanoshells. We, therefore, performed a series of control experiments in a two-channel microfluidic chip specifically designed for quantitative CARS experiments (Figures S2S4, Supporting Information)46 with the aim to directly compare two samples, each in a separate channel, under identical experimental conditions in the same microscopic field of view. Briefly, these control experiments on SERS particles, bare colloids, and Raman reporters in the two-channel chip (supporting text and Figures S2S4, Supporting Information) demonstrate that both plasmonic and molecular resonances contribute to the experimentally observed contrast. It is this vibrational contribution to the contrast which distinguishes SECARS microscopy as a plasmon-assisted nonlinear Raman imaging technique from other, purely plasmonic approaches.

’ CONCLUSION Combining the fast image acquisition rate of CARS microscopy with the selectivity and signal enhancement due to target-specific plasmonic nanoprobes allows the rapid detection of target molecules in complex biological specimens. Multicolor SECARS experiments can be envisioned, in which antibodies or other target-specific ligands are conjugated to spectrally distinct SERS labels and detected, for example, either by spectrally resolved detection schemes or through a rapid sequential tuning of pump/ Stokes laser wavelengths, e.g., by a spatial light modulator.47 This proof-of-principle study on immunohistochemistry with surface-enhanced coherent Raman microscopy and targeted nanoprobes may serve as a starting point for further investigations using this combined plasmonic/nonlinear vibrational imaging technique. ’ EXPERIMENTAL SECTION Chemicals. NaCl, Na2HPO4, KCl, KH2PO4, AgNO3, HAuCl4 hydrate, Tween 20, ethanol, anhydrous ethylene glycol, polyvinyl-pyrrolidone (PVP), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), N-(3-dimethyl-aminopropyl)-N0 -ethyl-carbodiimide (EDC), and N-hydroxy-sulfosuccinimide sodium salt (s-NHS) were purchased from Sigma/Aldrich/Fluka. Bovine serum albumin (BSA), Hepes, and Tris were purchased from Carl Roth, Germany. Phosphate-buffered saline (PBS) with pH 7.4 was prepared from the corresponding sodium and potassium salts. Tris-tween (TT) buffer was prepared using 50 mM Tris buffer, 0.15 M NaCl, and 0.05 vol % Tween 20 and adjusted with 0.1 N HCl to pH 6.8. Mouse antihuman p63 clone (Y4A3) and Hrprabbit antimouse IgG were purchased from antibodies online. Azide was removed by a desalting column (NAP5) from Thermofisher; BSA was removed by Melon Gel IgG Spin purification kit from Thermofisher. Target retrieval solution was obtained from Dako. In all reaction steps, ultrapure water was used. Synthesis of SAM-Based SERS Labels and Their Conjugation to Antibodies. Synthesis of Au/Ag Nanoshells. The preparation of

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gold/silver nanoshells with an extinction maximum at 645 nm is based on the template-engaged replacement reaction between silver nanoparticles and an aqueous HAuCl4 solution. Silver nanospheres were obtained in a polyol process at 160 C employing PVP and AgNO3 dissolved in ethylene glycol as described elsewhere.34 Synthesis of Hydrophilically Stabilized SERS Labels with a Complete SAM. The centrifugate of 2 mL of a colloidal solution containing gold/silver nanoshells was dissolved in 1 mL of a 1 mM solution containing the Raman-active reporter unit attached to the short spacer unit (DTNBMEG) and 10 μL of a 1 mM solution containing the Raman-active reporter unit attached to the longer spacer unit (DTNBTEGCOOH). The mixture was stirred for 12 h. The hydrophilically stabilized Raman-active reporter molecules are dissolved in a solution of PVP in ethanol (1 g of PVP solved in 40 mL of ethanol). After formation of a complete SAM, monitored by the maximum SERS signal for the Raman reporter molecule, the colloid was washed twice with PVP in ethanol. Biofunctionalization of Hydrophilically Stabilized SERS Labels. Five hundred μL of the SERS labels dispersed in 50 mM Hepes buffer (OD = 2.5) were activated with 50 μL of a 0.2% s-NHS solution and 50 μL of a 0.3% EDC solution. After incubation for 20 min, the nanoparticles were centrifuged and suspended in 300 μL of Hepes buffer. One μg anti p63 (IgG) was added to the activated SERS labels for an incubation time of 60 min at room temperature. The colloid was washed two times with a mixture of PBS, 0.05% Tween 20, and 0.2% BSA. Finally, the SERS-labeled antibodies were dispersed in TT buffer with 0.2% BSA. The average number of antibodies on each SERS nanoparticle was determined using an enzyme-based assay using a conjugate of Hrp-rabbit antimouse IgG and SERS nanoparticles. The enzyme activity was measured in 96 well plates. For quantification, a calibration curve obtained from a Hrp-rabbit antimouse IgG dilution series (no SERS particles) was established. The number of colloidal particles was estimated using the LambertBeer law together with the corresponding extinction coefficients.38 On the basis of the enzyme assay, we estimate that the number of antibodies is ∼234 ( 23 (N = 4) for solid 40 nm Au spheres and ∼762 ( 76 (N = 4) for hollow 70 nm Au/ Ag spheres. Immunohistochemistry. Prostate tissue biopsies were obtained from healthy donors. Formalin-fixed and paraffin-embedded samples were cut into 5 μm thick slices with a microtome. Paraffin removal and rehydration were performed by subsequent washing with xylene, ethanol, ethanol/water mixtures, and finally distilled water. Antigen retrieval was performed using a commercially available target retrieval solution. The tissue was blocked with 2% BSA in TT buffer for 20 min before incubation with the SERS-labeled antibodies (250 μL, OD 0.2) for 20 min. Unbound SERS particles were removed by rinsing several times with TT buffer. Raman Microspectroscopy and CARS Microscopy. SERS experiments on colloids in a quartz cuvette (d = 2 mm) were performed with a WiTec alpha 300 R microspectrometer. The 632.8 nm line from a HeNe laser was used for excitation of Raman scattering. A grating monochromator (f = 30 cm, 600 lines/mm) equipped with a Peltier-cooled CCD camera was employed for recording the SERS spectra. The experimental setup for CARS microscopy used in this study comprises a frequency-doubled Nd:Vanadate laser, a Ti:Sa oscillator, two nonlinear optical parametric oscillators, and a laser scanning microscope.43 The SECARS wavelengths were 647 nm (pump laser) and 708 nm (Stokes laser), respectively. 7084

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’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Homepage: biophotonik. physik.uos.de.

’ ACKNOWLEDGMENT Financial support from the German Research Foundation (DFG; SFB 630, Teilprojekt C1), the Fonds der Chemischen Industrie, the Th€uringer Kultusministerium/EFRE (TKM, B578-06001), and the EU/Nieders€achsisches MWK (EFRE, W2-80111700) is acknowledged. ’ REFERENCES (1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (2) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (3) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (4) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025–1102. (5) Huang, X.; Neretina, S.; El-Sayed, M. A. Adv. Mater. 2009, 21, 4880–4910. (6) Aroca, R. Surface-Enhanced Vibrational Spectroscopy; Wiley: New York, 2006. (7) Kneipp, M.; Moskovits, H.; Kneipp, H. Surface-Enhanced Raman Scattering: Physics and Applications. In Topics in Applied Physics; Springer: Berlin, 2006; Vol. 103. (8) Le Ru, E. C.; Etchegoin, P. G. Principles of Surface-Enhanced Raman Spectroscopy; Elsevier: Amsterdam, 2009. (9) Stiles, P. L.; Dieringer, J. A.; Shah, N. C.; Van Duyne, R. P. Annu. Rev. Anal. Chem. 2008, 1, 601–626. (10) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536–1540. (11) Graham, D.; Mallinder, B. J.; Whitcombe, D.; Smith, W. E. ChemPhysChem 2001, 2, 746–748. (12) Grubisha, D. S.; Lipert, R. J.; Park, H.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936–5943. (13) Han, X. X.; Zhao, B.; Ozaki, Y. Anal. Bioanal. Chem. 2009, 394, 1719–1727. (14) Schl€ucker, S.; Kiefer, W. Selective Detection of Proteins and Nucleic Acids with Bio functionalized SERS Labels. In Frontiers of Molecular Spectroscopy; Laane, J., Ed.; Elsevier: Amsterdam, 2009. (15) Schl€ucker, S.; K€ustner, B.; Punge, A.; Bonfig, R.; Marx, A.; Str€obel, P. J. Raman Spectrosc. 2006, 37, 719–721. (16) Kim, J.; Kim, J.; Choi, H.; Lee, S.; Jun, B.; Yu, K.; Kuk, E.; Kim, Y.; Jeong, D. H.; Cho, M.; Lee, Y. Anal. Chem. 2006, 78, 6967–6973. (17) Sun, L.; Sung, K.; Dentinger, C.; Lutz, B.; Nguyen, L.; Zhang, J.; Qin, H.; Yamakawa, M.; Cao, M.; Lu, Y.; Chmura, A. J.; Zhu, J.; Su, X.; Berlin, A. A.; Chan, S.; Knudsen, B. Nano Lett. 2007, 7, 351–356. (18) Schl€ucker, S. ChemPhysChem 2009, 10, 1344–1354. (19) Qian, X.; Peng, X.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. Nat. Biotechnol. 2008, 26, 83–90. (20) Kneipp, H.; Kneipp, K. J. Raman Spectrosc. 2005, 36, 551–554. (21) Kneipp, J.; Kneipp, H.; Kneipp, K. Chem. Soc. Rev. 2008, 37, 1052–1060.

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