Chiral Plasmonic Fields Probe Structural Order of Biointerfaces

The structural order of biopolymers, such as proteins, at interfaces defines the physical and chemical interactions of biological systems with their surroundings and is hence a critical parameter in a range of biological problems. Known spectroscopic methods for routine rapid monitoring of structural order in biolayers are generally only applied to model single-component systems that possess a spectral fingerprint which is highly sensitive to orientation. This spectroscopic behavior is not a generic property and may require the addition of a label. Importantly, such techniques cannot readily be applied to real multicomponent biolayers, have ill-defined or unknown compositions, and have complex spectroscopic signatures with many overlapping bands. Here, we demonstrate the sensitivity of plasmonic fields with enhanced chirality, a property referred to as superchirality, to global orientational order within both simple model and “real” complex protein layers. The sensitivity to structural order is derived from the capability of superchiral fields to detect the anisotropic nature of electric dipole–magnetic dipole response of the layer; this is validated by numerical simulations. As a model study, the evolution of orientational order with increasing surface density in layers of the antibody immunoglobulin G was monitored. As an exemplar of greater complexity, superchiral fields are demonstrated, without knowledge of exact composition, to be able to monitor how qualitative changes in composition alter the structural order of protein layers formed from blood serum, thereby establishing the efficacy of the phenomenon as a tool for studying complex biological interfaces.


EM Simulations
The electromagnetic simulations were performed using the COMSOL Multiphysics software (v4. 4) with the Wave Optics module. COMSOL uses the finite element method to solve Maxwell's equations over a specified geometry for linearly polarized input light, with the reflection being measured at a surface above the structure. Periodic boundary conditions at the vertical boundaries were used, creating an infinite array of structures to approximate the periodicity of the nanostructure array. We used values of Au permittivity from Johnson et al [1]. The values for refractive index of polycarbonate were taken from Sultanova et al [2].
= 1 for all the materials.
The chiral dielectric is realised using the constituent equations for a chiral dielectric medium [3]: Here, is the permittivity of free space, is the relative permittivity, 0 is the permeability of free space, is the relative permeability, is the complex electric field, is the complex magnetic flux density, is the magnetic field, is the electric displacement field and is a second rank tensor describing the chiral property of a molecular layer. , the chirality tensor, is only non-zero for a chiral For the isotropic chiral dielectric, the electric displacement D, magnetic field strength H and the timevarying derivative of the magnetic field / were changed to those given in equations S3-S5. Only the equations for the x components are shown. Similar changes have to be made for the y and z, using the corresponding and chirality tensor components. (S10) (S11) S5

Chirality Plots from Simulation
The spatial distribution of optical chirality of the LH and RH structures for the isotropic and anisotropic chiral layers is shown in figure S4. Qualitatively the isotropic and anisotropic cases seem very similar, but the difference can be discerned by calculating the optical chirality at the maximum points (seen as the darkest areas on the chirality maps) which are noted in the figure. Figure S4. Optical chirality maps for the matched and linear combinations at the left peak (~693nm), dip (~703nm) and right peak (~711nm).

Chirality Calculation of ξ
is a local parameter describing the chirality of a chiral dielectric, the sign of which defines the handedness of the chiral dielectric. [6] The value for molecular chiral dielectrics can be estimated using the general form given by: Derivation and detailed description is available in work by Govorov et al. [3] Here is an intrinsic coefficient that determines the magnitude of chiral properties. Also ħ0 (where ħ is the reduced Planck's constant, h/2π, and 0 is the absorption frequency) and  are the energy and intrinsic width of the S6 resonant chiral excitation of the dielectric. Consequently, the value of is wavelength dependent. This equation can also be written as: = ( 1 hc/λ + hc/λ 0 + Γ 12 + 1 hc/λ − hc/λ 0 + Γ 12 ) (S13) Using the following parameters: hc = 1.23984193 eV.μm λ0 = 0.28 μm.
= 0.4 eV βc= 4.5x10 -4 eV Proteins absorb in the UV region, so we use an estimate of λ0 = 280 nm (IgG abosption). Figure S5 shows the plot of for λ0 = 280 nm. As our resonance lies at ~700nm, we make the approximation that the value over the entire plasmonics resonance region is equal to the value at 700nm; we calculate that ~ 1 × 10 −4 . The magnitude is used as the imaginary part is negligible here. This value is approximate based on generic biomolecules absorbing in the UV region for an isotropic layer. The value used in the manuscript for an isotropic dielectric ( = 1.7 × 10 −4 ) has been identified as the optimum value to match the ΔΔS values of the experimental results. We expect these differences between the above model and a real system occur due to the thickness of a real layer that influences the effective value.

Templated Plasmonic Substrates: Fabrication and Properties
The TPSs are made using an injection moulding machine (ENGEL), following the technique explained by Gadegaard et al [4]. The master shim for this is made using e-beam lithography. To create the master, 100 nm of PMMA is spin coated onto a Si wafer and baked for an hour at 180 ˚C. The resist is patterned using a VB6 UHR EWF lithography tool (Vistec). The exposed resist is developed in IPA and Methyl Isobutyl Ketone, MIBK (3:1 ratio) for 60 secs. Ni is electroplated onto the surface and removed from the wafer to provide the Nickel shim that is then used as the master in a tool placed in the injection moulder. Polycarbonate pellets are thermally heated and pushed into the tool to create small plastic slides with the nanostructures indented on the surface. These slides are then coated with 100 nm of Au in an e-beam evaporator at a rate of ≈0.3 nm s -1 .
The nano patterns are indentations in the surface and have a depth of ~80 nm, are 500 nm in length from arm to arm, and have a pitch of 700 nm. When gold is evaporated onto the surface, it takes the shape of the indentation and forms a hybrid plasmonic structure constituting an inverse structure at the S8 top and a solid one at the bottomsee Figure 2 in the main text. For more information on the plasmonic behaviour of the TPS refer to Karimullah et al [5].