Chiral biosensing using terahertz twisted chiral metamaterial

: Subwavelength chiral metamaterials with tunable geometries and compositions are essential to advance the development of chiral biochemical samples detection. Here, we report a spatial symmetry breaking chiral terahertz (THz) metamaterial structure with stacked layers of L-shape arranged gold disks as the periodic unit cell. The chiroptical response can be adjusted on-demand by manipulating the number of stacking layers and the twisted angle of the periodic unit between adjacent array layers. We reveal that the chiroptical response originates from the optical resonances of the gold disks and the adjacent gold disks array layers via experiments and numerical simulation analysis. Furthermore, we find that this chiral metamaterial can realize label-free detection of proline in biological samples and label-free enantio-discrimination of chiral molecules. The change of the analyte concentration can also regulate the transmission circular dichroism (TCD) intensity of the chiral metamaterials. Our results not only provide new ideas into the design of functional chiral metamaterials, but also bring new strategies to develop chiroptical biosensing devices. results show that the chiroptical activity can be tuned by adjusting the number of L-shape-arranged gold disk array layers and the twisted angle between the units of adjacent layers. In addition, we demonstrated the chirality sensing capability of the twisted chiral metamaterials using two enantiomers of proline as the biological sample analytes. This new four-layer chiral metamaterial sensor can distinguish D- and L-proline with label-free. The change of the proline concentration can also regulate the TCD intensity of the chiral metamaterials. These new THz chiral metamaterials and sensor devices pave the way to applications in chiral sensing and label-free enantioselective biosensing in the THz regime.


Introduction
Chiral objects are common in our daily life. Many biological substances such as sugars, peptides, and amino acids are chiral and play important roles in medicine, biology, and chemistry [1][2][3][4][5][6][7]. The research of the chiral materials can be traced back to the 1890s, Bose experimentally demonstrated the optical properties of three-dimensional (3D) chiral materials for the first time [8]. Recently, THz chiral metamaterials with intriguing chiroptical properties have attracted wide attention in many emerging applications, such as chiral sensing and enantio-discrimination detection [9][10][11][12][13][14][15]. Same as chiral objects, chiral metamaterials appear in the form of mirror-symmetric enantiomers A and B, which cannot coincide with each other by simple rotation, scaling, or translation [16,17]. Structural chirality within metamaterials results in anisotropic interaction with right-handed circularly polarized (RCP) and left-handed circularly polarized (LCP) electromagnetic waves [18][19][20]. The chiroptical response of chiral metamaterials usually depends on their composition and the arrangement of their periodic arrays [21][22][23][24]. In recent years, a large number of chiral metamaterials have been demonstrated to manipulate transmission circular dichroism (TCD) response [25][26][27][28][29][30][31][32][33][34]. As an example, the TCD can be adjusted by changing the thickness of the azimuthally graded-depth of the plasmonic metamaterials [10], or by changing the thickness of the interval film of the double-layer chiral metamaterial [35]. One of the most important characteristics of chiral metamaterial is that its asymmetry can be artificially changed, which could effectively enhance the TCD responses [36][37][38]. Conventional passive control methods of chiral metamaterials usually rely on changing the size of the structural unit or the thickness of the metamaterials, and a large number of chiral metamaterials have been reported to manipulation polarization conversion and optical activity by using conversion passive control methods [39][40][41][42][43][44][45].
However, the works related to the THz chiral biosensing of the chiral metamaterials have rarely been reported yet.
In this work, we studied a new type of chiral metamaterials with broken symmetry that could enhance TCD by using the synergy between twisted morphology and local plasmonic response. It consists of several layers of chiral periodic arrays, where each one has unit cells made of four gold disks with different radii arranged in an L-shape and is rotated by a twisted angle with respect to the next layer in the stack. In the THz regime, the geometry-dependent chiral optical activity of the unit cell was systematically investigated by sequentially changing the twist angle of helical stacks. The experimental results show that by stacking up four layers of chiral periodic arrays and superimposing the L-shaped units with a twisted angle of 22.5°, the TCD intensity of the proposed chiral metamaterials can reach a higher level. By using this new four-layer chiral metamaterial a sensing platform, we carried out quantitative concentration measurements of proline solution and label-free enantio-discrimination between its D-and L-enantiomers. We found that the TCD intensity of chiral metamaterials increases with the concentration of proline. This chiral metamaterial and associate study can support the design of THz polarization sensor that has great potential for quantitative detection and chiral discrimination of biological materials. Figure 1 shows the proposed twisted and chiral metamaterial. Each layer of periodic unit is made up of four gold disks with different radii arranged in an L-shape. The size of the entire metamaterial array is 6.6 mm × 7.2 mm. The layered metamaterial structures are fabricated via photolithography on a 500 µm thick SiO 2 . The disks are made of 40 nm-thick gold on a 5 nm chromium adhesion layer. Gold is used due to its large numbers of free electrons, which is essential for plasmon generation. The metamaterials are built in two enantiomer forms (A and B), as shown in Fig. 1. They are mirror images of each other relative to the y-z plane. This stacked form, going from one-layer to a four-layer can be obtained straightforwardly by the aforementioned photolithography technique. An 11 µm polyimide (PI) is spin-coated between each layer as a dielectric spacer as shown in Figs. S1 and S2. The preparation processes are described in detail in the supplementary. An optical microscope image is shown in Fig. 2(a).

Structure and design
COMSOL Multiphysics simulations show the electric field and surface current distributions of the chiral metamaterials. In the simulation, the permittivity of gold is set in the Drude model with a plasma frequency ω p = 1.37 × 10 16 rad/s and collision frequency ω c = 4.07 × 10 13 rad/s. The refractive index of SiO 2 is set to 2.2 [46,47]. The polyimide (PI) with 11 µm thickness was described by a dielectric constant of 2.93 with loss tangent delta of 0.044 [48]. The circularly polarized incident wave is perpendicular to the metamaterial surface.
The fabricated twisted chiral metamaterials are characterized by a transmission-type THz time-domain spectroscopy system (THz-TDS) as shown in Fig. S3. The femtosecond laser oscillator used for the broadband THz beam has a repetition rate of 80 MHz and is purchased from Coherent. The spectral bandwidth is set to 70 nm. The pulsed laser beam with 800 nm center wavelength is focused onto a photoconductive switch. The transmission coefficients for RCP and LCP waves and the TCD are converted from linear polarization with four wire-grid polarizers. This system has a bandwidth of 0.1 THz to 2.0 THz by Fourier transform. To avoid the effects of moisture in air, the entire system is purged with dry nitrogen during measurements.

Results and discussion
TCD is a parameter describing the difference between transmission coefficients of RCP and LCP waves. It is defined as (1) where T RCP and T LCP are the total transmittances of the metamaterials under RCP and LCP waves, T ij is the i-polarized transmittances under j-polarized incidence with {i, j} ∈ {+, −} and + representing RCP while -representing LCP. t ij is the corresponding transmission coefficients. The data process is described in the supplementary The TCD responses of the chiral metamaterials were first characterized using simulations. Figure 2(b) shows the simulated maximum intensity of TCD (|TCD max | in the interested frequency rge of 0.5 to 0.6 THz) of the four-layer enantiomer A for varying twisted angles γ from 0 to 56.25°with a step of 11.25°, where γ is the twisted angle of the periodic unit between adjacent consecutive array layers. It is seen that the strongest TCD occurs at γ = 22.5°. Figures 2(d)-2(f) illustrate three TCD spectra of enantiomer A and enantiomer B with different γ. Due to the symmetry inversion, the RCP and LCP responses of enantiomer A will be equal to the LCP and RCP responses of enantiomer B. For a monolayer L-shape arranged gold disks array, the TCD is very weak, as shown in Fig. 2(c). ranging from 1 to 5 layers, as the layer number increases, the maximum intensity of TCD appears when the layer number equals 4.
In the following discussion, we focus on the THz polarization response of twisted four-layer enantiomer A excited by linearly polarized (LP) THz waves and circularly polarized (CP) THz waves. When the incident waves are LP, the polarization state of the transmitted wave changes owing to its broken symmetry. As shown in Fig. 3(a), there is a resonance at 0.46 THz under y-LP incidence, and a resonance at 0.58 THz under x-LP incidence. Then, we use polarization rotation angle (PRA) ψ and polarization ellipsoid angle (PEA) ε to characterize the polarization state conversions. They can be derived as follows [35] tan2ψ = tan2βcos∆δ (2) where tanβ = T x /T y , ∆δ = δ xδ y . T x and T y are the transmission under two orthogonal LP incidence. δ x and δ y are the phases for two orthogonal LP signals. PRA represents the rotation angle of the polarization direction of the output wave relative to the incident wave. The positive value of PRA means that the rotation angle is clockwise direction, and the negative value means that the rotation angle is counterclockwise. PEA reflects the chirality ofhe output wave, that positive values mean right-handed rotation and the negative values mean left-handed rotation. As shown in Fig. 3(c), the peak values of PRA and PEA spectra are 44°and 15.6°at 0.51 THz, which means that the output wave exhibits right-handed rotation chirality with the rotation angle in the clockwise direction. When the incident waves are CP, we are more concerned about the chiroptical response. Figure 3(b) shows the polarization analysis of the transmission spectra in CP waves of the four-layers enantiomer A. Due to the asymmetry of the chiral structure unit cell, T ++ and T − are different. T ++ spectrum has a trough at 0.46 THz, and T − spectrum has a trough at 0.57 THz. Figure 3(d) shows the simulated and measured TCD spectra, the peaks of the TCD spectra are at 0.46 THz and 0.57 THz. Figure 3(e) shows experimental results of the evolution of the TCD along the increasing layer number of enantiomer A. It is found that the TCD gradually increases at 0.46 THz and 0.57 THz. The corresponding measured RCP and LCP transmissions can be seen in Fig. S4. Additional studies were performed to reveal the influence of the asymmetry of the L-shape units on the chiroptical responses. We compared the TCD efficiency of gold disks with the same radius (r = 30 µm) in the four layers to the configuration with different radii (r 1 = 25 µm, r 2 = 30 µm, r 3 = 35 µm, r 4 . = 40 µm), as shown in Fig. 3(f). The metamaterial with the same radii has weak spatial asymmetry, which produces weaker chiral responses at 0.57 THz. Therefore, the strong TCD response efficiency in L-shape chiral metamaterials also originates from the spatial asymmetry of the unit cell. We analyze the electric field and surface current density distributions of enantiomer A at 0.57 THz when illuminated with CP waves, as shown in Fig. 4. The electric dipole moments induced by the flowing currents are illustrated with color scales for the electric field intensity distributions, and the red arrows indicate the direction of oscillating currents. For LCP incidence, the electric field intensities on the first to fourth layer reveal that the local plasmonic resonance could lead to strong electric field enhancement in the gap of the adjacent gold disks, which results in a transmission dip. For RCP incidence, the electric field intensity is much smaller than that of the LCP wave due to the spin-dependent coupling effect. Therefore, the spin-selectivity can be identified by the distribution of the electric field intensity corresponding to the resonance frequency and the coupling frequency of each layer. This difference in the distribution of electric fields between RCP and LCP waves corresponds to the TCD response. Figures 4(e) and 4(j) show the surface current density distributions at the bottom periodic unit cell. The currents flow in a helical pattern and generate the local plasmonic modes.
The above experimental results prove that by stacking up four layers of chiral periodic arrays and superimposing the L-shaped units with a twisted angle of 22.5°, the TCD intensity of the proposed chiral metamaterials can reach a higher level. Therefore, the chiral metamaterial sensors we will discuss next are all based on the chiral metamaterial with a twisted angle of 22.5°and four array layers. We demonstrated the chiral sensing capability of the four-layers twisted metamaterial using proline, which is an essential amino acid with a molecular weight of 115.13 g/mol. It has two enantiomers, D-proline and L-proline. These two prolines are purchased from Damasbeta Company with purity of 99%. In the experiment, deionized water is used to prepare the proline solution with different concentrations. The volume of the proline solution added on the surface of the chiral metamaterials is 20 µL. The proline solution crystallized at the temperature of 60°C to form a proline film with a thickness of about 0.38 mm. Images of the crystallized biological samples on different substrates can be found in Fig. S7. We characterize the polarization states by PRA and PEA parameters. Figure 5(a) shows the experimental PRA spectra of the D-proline with different solution concentrations. All the peaks of PRA spectra reach to a maximum of 44°f or each sample concentration, but the abrupt frequency corresponding to the peak values of PRA spectra red-shifted significantly with the increase of the D-proline concentration. Figure 5(b) shows the PEA spectra of the D-proline. The peak frequency moves to lower frequency with the increase of D-proline concentration. Figure 5(c) shows the PRA and PEA spectra of the D-and L-proline with the same solution concentrations, respectively, and the PRA or PEA spectra are almost the same around the abrupt frequency of 0.42 THz. Although the above polarization conversion methods can be used to quantitatively determine the concentration of proline, it is difficult to distinguish D-proline and L-proline chiral enantiomers. As a result, we begin to study the chiroptical response of the proline sample on the four-layer twisted enantiomer A. In this case, the information carried by the amplitude and phase of the transmitted CP wave is essential to obtain the TCD spectra of the proline on the enantiomer A. Figure 6(a) shows the TCD spectra of chiral metamaterial without proline, D-proline on SiO 2 substrate, and D-proline on chiral metamaterial, respectively. It can be seen that the chiral intensity of proline sample is very low in the absence of chiral metamaterial enhancement. The TCD spectra of D-proline with the chiral metamaterials are shown in Fig. 6(b), which indicates that the peak of the TCD spectra is at about 0.54 THz, and the peak redshifts while enhances as the D-proline concentration increases.   Fig. 6(a). From Fig. S8, it can be seen that the variation trend of the D-proline TCD intensity of the SiO 2 substrate is similar to that of the four-layer metamaterial in enantiomer A (blue line), indicating the similar chirality of the four-layer metamaterial enantiomer A and the D-proline sample. But their intensity is different. Therefore, the asymmetry of the metamaterial enantiomer A can be improved after being coated with D-proline [49,50]. The TCD spectra of D-and L-proline without metamaterial exhibit handedness-flipped chiroptical response [49]. The chirality of the four-layer metamaterial enantiomer A and the L-proline sample is opposite, thus the asymmetry of the metamaterial enantiomer A reduces after being coated with L-proline. We measured the peak of TCD spectra (|D-TCD max | and |L-TCD max | for D-proline and L-proline, respectively) on the four-layer metamaterial enantiomer A and calculated the dissymmetric factor ∆|TCD max |=|D-TCD max | -|L-TCD max | at different concentrations ( Fig. S9(a)). It can be seen the ∆|TCD max | are positive, which means that the |TCD max | of D-proline is always larger than the L-proline at different concentrations, then, it is easy to distinguish the chiral enantiomers between D-and L-proline intuitively from the values of dissymmetric factor ∆|TCD max |. As shown in Fig. 6(c) and S9(b), we also see that there is a°prominent difference in chirality between two peaks for D-and L-proline within a certain frequency band. Due to the increased ratio of proline in a fixed volume of the proline solution expanded the proline film thickness, the peak frequency of TCD spectra is red-shifted significantly with the increase of D-proline concentration, while the peak frequency of TCD spectra is blue-shifted significantly with the increase of L-proline concentration. In order to verify its universality of the responses of such a design to other chiral molecule, the numerical simulations have been conducted [51], which nearly perfect reflect the experimental observations, details are included in the supplemental materials.

Conclusion
In summary, a new type of chiral metamaterial has been designed by twisting the unit cell of adjacent layers along the vertical axis with a certain angle to break the symmetries. Our results show that the chiroptical activity can be tuned by adjusting the number of L-shapearranged gold disk array layers and the twisted angle between the units of adjacent layers. In addition, we demonstrated the chirality sensing capability of the twisted chiral metamaterials using two enantiomers of proline as the biological sample analytes. This new four-layer chiral metamaterial sensor can distinguish D-and L-proline with label-free. The change of the proline concentration can also regulate the TCD intensity of the chiral metamaterials. These new THz chiral metamaterials and sensor devices pave the way to applications in chiral sensing and label-free enantioselective biosensing in the THz regime.