SUPPLEMENTARY INFORMATION doi: 1.138/nnano.21.29 Gammadion fabrication The gammadions were designed using CAD software with a line width of 8 nm and a periodicity of 8 nm, figure S1. They were arrayed to cover a total area of 5x5 mm 2. The PCMs were fabricated on borosilicate glass slides 25x25x1 mm 3. The slides were cleaned for 5 min in acetone and 5 min in isopropanol both under ultrasonic agitation before being blown dry in a stream of nitrogen. A bilayer of PMMA was spun to a thickness of about 2 nm and baked at 18 C for 1 hour. A 1 nm NiCr layer was evaporated as a charge conduction layer during the electron beam lithography. The pattern was exposed in a Vistec VB6 UHR EWF lithography tool. After exposing the samples, the NiCr layer was removed in a chromium etch, rinsed in water and dried in a stream of nitrogen before development in IPA:MIBK (2.5:1). Prior to metal deposition, the samples were exposed for 1 minute at 6W to an oxygen plasma. 5 nm of titanium was used as an adhesion layer and followed by 1 nm of gold. The final patterns were achieved in a lift-off process by leaving the samples in acetone for about 3 hours. 4nm 8 nm 8 nm 4nm Figure S1. A schematic diagram of the gammadion structure of a RH-PCM is shown in the upper panel, in the lower panel are electron micrographs of: achiral crosses (A); right handed gammadion (B); and left handed gammadion (C) nature nanotechnology www.nature.com/naturenanotechnology 1
supplementary information doi: 1.138/nnano.21.29 Spectroscopic characterisation of PCMs In figure S2 are CD spectra collected, in water, from LH-PCMs that have Au thicknesses of 3, 6 and 1 nm. The absolute level of CD increases with the thickness of the gammadions, increasing by approximately an order of magnitude with every increase in thickness. In figure S3a are spectra collected from LH and RH-PCM that were immersed in water (refractive index 1.33333) and ethanol (refractive index 1.36242). In Figure S3b, the wavelength of the III mode of a 6 nm thick LH-PCM has a function of the refractive index of the surrounding liquid is plotted. As expected from equation 1 in the text, the wavelength of the III is linearly dependent upon the refractive index of the surrounding dielectric environment. 2-2 1 nm x.18 3 nm 6 nm x.11 4 5 6 7 8 9 Wavelength [nm] Figure S2. CD spectra collected in water from LH-PCM with thicknesses of 3, 6 and 1 nm of Au. The spectra have been scaled to aid comparison 2 nature nanotechnology www.nature.com/naturenanotechnology
doi: 1.138/nnano.21.29 supplementary information 6 a) b) 84-6 -12 12 6 LH-PCM RH-PCM LM wavelength [nm] 82 8 78 76-6 4 5 6 7 8 9 Wavelength [nm] 1.65 1.7 1.75 Refractive Index Figure S3 a) CD spectra collected from LH and RH-PCM in the presence of water (magenta) and ethanol (black). b) A plot of the wavelength of the III( LH-PCM (6 nm thick)) versus the refractive index of the surrounding liquid. The red line is the best linear fit nature nanotechnology www.nature.com/naturenanotechnology 3
supplementary information doi: 1.138/nnano.21.29 Table of protein data Pprotein Mr + Fold Quaternary Structure Dimensions 1 PDB code β-lactoglobulin 18,4 8-stranded dimer 7Å x 4Å x 35Å 1BEB (36,8) beta barrel Concanavalin A 26, 12-stranded tetramer * 8Å x 5Å x 3Å 1VLN (14,) beta sandwich OmpA 35,2 8-stranded monomer 45Å x 25Å x 25Å 1QJP beta barrel Haemoglobin ~17, 6 α-helices Tetramer 6Å x 5Å x 3Å 1J3Z (68,) Globin fold (2α,2β subunits) Myoglobin 16,7 6 α-helices monomer 4Å x 4Å x 2Å 2V1K Globin fold BSA 66,2 21 α-helices Orthogonal bundle monomer 9Å x 8Å x 6Å 1AO6 # + The relative molecular mass (Mr) of the protein is quoted for the peptide, together with any co-factors or modifications. The value in parentheses is the Mr of quaternary structure of the protein found in solution. *Concanavlin A can form dimers at acid ph ~ph 5.5 but tetramers at ph 7.5 and above so the form bound to the metal surface is not known unambiguously. # Ccoordinates are from the human serum albumin protein rather that the bovine form that has not been crystallised. 1 The dimensions of the proteins are obtained manually from the crystal structures and present a rough indication of the size of the quaternary structure of the protein in solution.. 4 nature nanotechnology www.nature.com/naturenanotechnology
doi: 1.138/nnano.21.29 supplementary information α-helical proteins β-sheet proteins Amino acid 12 8 Myoglobin 12 8 Omp A 12 Tryptophan 4-4 4-4 8-8 -8 4-12 12 8 BSA -12 12 8 Concanavlin A CD[mdeg] 4-4 4-4 -4-8 -8-8 -12 5 6 7 8 9 Wavelength [nm] -12 5 6 7 8 9 Wavelength [nm] -12 5 6 7 8 9 Wavelength [nm] Figure S4. For α-helical (myoglobin, BSA), β-sheet (concanavlin A and Omp A) and tryptophan. The red spectra were collected in Tris buffer prior to protein adsorption (solid line LH-PCM, dashed line RH-PCM) and the black were collected in protein / amino acid solutions. Spectra collected from achiral crosses in solutions of the chiral materials are shown in blue. 2 nature nanotechnology www.nature.com/naturenanotechnology 5
supplementary information doi: 1.138/nnano.21.29 Quantifying the amount of protein adsorption: SPR data Tryptophan Outer membrane protein A Concanavalin A β-lactoglobulin BSA Myoglobin Haemoglobin 1 2 3 4 λ AV [nm] 1 2 3 4 SPR response Figure S5 (left panel) λ AV values obtained from the TM. (right panel) SPR response induced by solutions of identical concentration to that used to produce data in left panel. 21 6 nature nanotechnology www.nature.com/naturenanotechnology
doi: 1.138/nnano.21.29 supplementary information To illustrate the relationship between the observed λ AV and the amount of material adsorbed we have performed surface plasmon resonance (SPR) measurements on solutions of the tryptophan and the six proteins (SPR measurements could not be performed on the β-lactoglobulin solution at elevated temperatures), at a concentration of 1 mg / ml on a bare Au SPR chip. In fig S5 the SPR data are compared with the corresponding λ AV values. There is good qualitative agreement between the relative size of SPR response and the λ av shift data, apart from the case of BSA where the λ AV shift is larger than would be expected based on the SPR response. Given that a single SPR response unit is 1 pgmm -2 we can calculate estimates of coverage, in terms of monolayers, of proteins and tryptophan (see table below). The SPR and λ AV shifts data may be expected to differ slightly, since the latter arises not only because of adsorption onto the gold surfaces of the gammadions but can also have small contributions from chiral material that is adsorbed on to the glass substrate and is within the vicinity of the gammadions and the electric field they generate. Consequently, since the chiral materials may have different adsorption behaviour on glass and gold, the effective coverage detected by the LSPRs of the gammadions may differ slightly from the coverage of the chiral materials determined by SPR, and account for disparities between the two data sets. Table of coverages estimated from SPR data Measured Coverage / Calculated Coverage / picogmm -2 monolayers Haemoglobin 3329.5 Myoglobin 1247.39 BSA 364.9.15 β-lactoglobulin 19.6.2 Concanavlin A 1281 1.66 OMP A 1936.26 Tryptophan 186.39 nature nanotechnology www.nature.com/naturenanotechnology 7
supplementary information doi: 1.138/nnano.21.29 Determination of estimates for the disymmetry factor g. We have calculated disymmetry factors using the λ shifts for the II LSPR. To calculate the g factors we have used the coverages determined (ppgmm -2 ) from SPR and calculated an average area occupied by the proteins and the tryptophan. We have also assumed a l d value of 1 nm. Given that we are estimating key parameters, there is a level of uncertainty in the calculated g values, however the values are only intended has a guide to the order of magnitude of the disymmetries. Table of g factors g factor Haemoglobin.7±.6 Myoglobin.8±.6 BSA.5±.6 β-lactoglobulin.677±.6 Concanavlin A.28±.6 OMP A.46±.6 Tryptophan -.236±.6 8 nature nanotechnology www.nature.com/naturenanotechnology
doi: 1.138/nnano.21.29 supplementary information Numerical electromagnetic simulations. We performed numerical simulations of electromagnetic fields in the vicinity of the nanoparticles using a commercial finite-element package (Ansoft HFSS version 11.) with a mesh size of 4. nm. Permittivity values for gold, glass substrate and surrounding water were taken from E. D. Palik, Handbook of optical constants of solids (Academic Press, New York, 1985). We then calculated the CD spectrum by calculating the optical rotation of linearly polarized light according to [C.R.Cantor and P.R.Schimmel, Biophysical Chemistry, Vol.2, Chapter 8 (198)]. In fig. S6(a) we compare the CD spectra calculated for left handed and right handed gammadions. As is observed in experiment, the CD of LH particles is essentially the negative of the CD for RH particles. We also plot the CD spectra calculated for cross particels, which practically zero for all wavelengths. Achiral structures such as crosses do not exhibit any significant chiral optical effects. In figs. S6(b) and (c) we plot the local optical chirality, C, as defined in equation 3, normalized by the values magnitudes for circularly polarized plane waves. This is calculated at the substrate interface of the and for the chirality at its maximum value (i.e. for an excitation wavelength ~75 nm). We see enhanced optical chirality for all three particle shapes under excitation by LH and RH circularly polarized light. However, the enhancement is rather small for cross particles (lower panels), and for RHCP light with LH particles and LHCP light with RH particles. For these circumstances the optical chirality carries the same sign as the excitation light (i.e. positive for LHCP light and negative for RHCP light) and the weak enhancement arises from the enhanced electromagnetic fields in the vicinity of the particles. For the excitation of LH particles with LHCP light and the excitation of RH particles with RHCP light we see strong enhancement of the local optical chirality, amounting to 1-2 orders of magnitude over that for a circularly polarized plane wave. Interestingly, for these combinations the local optical chirality is opposite in sign to the excitation light. Clearly, the symmetry of the structure is critical i.e. the local optical chirality is dominated by the symmetry of the metallic structure rather than the light which excites it. This makes the enhancement in chiral phenomena described in this paper very different (in both magnitude and concept) from that reported by Baev et al [Opt. Express. 15, 573 27] and Lieberman et al. [Angewandte Chemie 12, 4933 28], who demonstrated that CD spectra from a biomacromolecule can be enhanced in the presence of an achiral plasmonic particle. Such a resonant enhancement, as reported nature nanotechnology www.nature.com/naturenanotechnology 9
supplementary information doi: 1.138/nnano.21.29 by Baev and Liebermann, arises when an electronic transition of the molecular system overlaps with the plasmonic resonance of the particle. The fundamental difference between the physics responsible for our observations is demonstrated by the fact that the optical chirality is determined by the symmetry of the particle, and that the chiral enhancements observed in our experiments are ~1 times greater than those reported by Baev/Lieberman. Figure S6 Finite element modelling of our PCMs. (a) Comparison of modelled CD spectra of different particle arrays. (b, c) Plots of the local optical chirality in the vicinity of our PCMs, as defined in equation 3, normalized by the values magnitudes for circularly polarized plane waves. The left hand plots are for excitation by left hand circularly polarized light while the right hand plots are for right hand circularly polarized light. 1 nature nanotechnology www.nature.com/naturenanotechnology