Letter pubs.acs.org/NanoLett
Detection of Nano-Oxidation Sites on the Surface of Hemoglobin Crystals Using Tip-Enhanced Raman Scattering Bayden R. Wood,*,† Mehdi Asghari-Khiavi,†,‡ Elena Bailo,§ Don McNaughton,† and Volker Deckert⊥,∥ †
Centre for Biospectroscopy, School of Chemistry, Monash University, 3800, Victoria, Australia Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § ISAS − Institute for Analytical Sciences, Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund, Germany ∥ Institut für Physikalische Chemie, Helmholtzweg 4, Friedrich-Schiller-Universität Jena, 07743 Jena, Germany ⊥ Institute of Photonic Technology IPHT, Albert-Einstein-Strasse 9, 07745 Jena, Germany ‡
S Supporting Information *
ABSTRACT: Hemoglobin nanocrystals were analyzed with tip-enhanced Raman scattering (TERS), surface-enhanced resonance Raman scattering (SERRS) and conventional resonance Raman scattering (RRS) using 532 nm excitation. The extremely high spatial resolution of TERS enables selective enhancement of heme, protein, and amino acid bands from the crystal surface not observed in the SERRS or RRS spectra. Two bands appearing at 1378 and 1355 cm−1 assigned to the ferric and ferrous oxidation state marker bands, respectively, were observed in both TERS and SERRS spectra but not in the RRS spectrum of the bulk sample. The results indicate that nanoscale oxidation changes are occurring at the hemoglobin crystal surface. These changes could be explained by oxygen exchange at the crystal surface and demonstrate the potential of the TERS technique to obtain structural information not possible with conventional Raman microscopy. KEYWORDS: Tip-enhanced Raman scattering, hemoglobin, surface oxidation 16, and another pair of β-like chains (β, ε, γ, and δ) encoded by a cluster of genes on the terminal portion of the short arm of chromosome 11.18 The conformational changes to the globin moieties induced by ligand binding are the basis of the cooperativity that results in the sigmoidal shape of the oxygen binding curve.18 When fully oxygenated, two of the heme groups in the Hb tetramer move approximately 100 pm toward one another, while the other two separate by about 700 pm.19 This movement was explained by Perutz20 who suggested that the binding of oxygen to the sixth coordination position on the Fe atom in the high spin ferrous state triggers a biochemical cascade that characterizes the T (tense-deoxygenated) to R (relaxed-oxygenated) state transition. In the T state, the sixth coordination sites of all four Fe atoms are unligated and the proximal histidine is constrained by intersubunit interactions thus resisting movement into the porphyrin plane and diminishing the O2 binding constant.18 When two dioxygen molecules bind to the Hb molecule, the quaternary structure “relaxes” to the R state, facilitating O2 binding to the two remaining subunits. The high and low-spin trigger mechanism was first suggested by Hoard21 and demonstrates how a simple chemical change can initiate a complex biochemical cascade.
T
ip-enhanced Raman scattering (TERS) exploits the nearfield enhancement generated by laser excitation of surface plasmons resulting from a strong electromagnetic field generated at the laser-irradiated tip apex of a metal or metallized scanning probe microscopy (SPM)-tip.1−6 The configuration simultaneously produces highly resolved topographic information and enhanced Raman scattering. Recently the technique has found application as a probe of biological molecules including RNA,7 DNA,8 collagen,9 and larger biological systems including membranes,10 cell walls,11 viruses12 mitochondria,13 bacteria,14 and hemozoin in malaria infected red blood cells.15 While the technique has been applied to some heme-based moieties, including cytochrome c16 and hemozoin,15 to the best of our knowledge there are no TERS studies on hemoglobin, which is surprising given the extent of resonance Raman studies on this important biomolecule. Recently, Kennedy et al.17 applied full IR absorption imaging at subdiffraction resolution of hemoglobin aggregates by coupling IR optics with an atomic force microscope. However, the spectral range was limited to the CH stretching region (3100−2800 cm−1). Hemoglobin (Hb) is involved in the most fundamental of biological processes, catalyzing a myriad of biochemical respiration reactions and playing an integral part in energy transfer processes. Hb is a tetramer consisting of four heme groups bound to one pair of α-like globin chains (α and ξ), encoded by a cluster of genes on the short arm of chromosome © 2012 American Chemical Society
Received: December 14, 2011 Revised: January 31, 2012 Published: February 10, 2012 1555
dx.doi.org/10.1021/nl2044106 | Nano Lett. 2012, 12, 1555−1560
Nano Letters
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Figure 1. (A) The typical optical response image of a silver-coated noncontact AFM tip used to analyze the hemoglobin crystal deposits. (B) AFM image of hemoglobin crystal deposits on mica. (C) x−z profile generated from the crystals marked by the red line in panel B.
ment is found, the tip is fixed at this position. The closed loop system of the AFM preserves the position with a precision of
