Method and device for analyzing chiral biomolecule mixture

Abstract

The invention provides a method and a device for analyzing a chiral biomolecule mixture, and relates to terahertz spectrum analys.The method comprises the steps that midpoints of two identical long metal bars are perpendicularly connected, four ends of each long metal bar are perpendicularly connected with four identical short metal bars respectively, and narrow-band unit cells are formed; the lengths of the long metal rods and the short metal rods are changed in an equal-step-length mode, so that a plurality of narrow-band unit cells with gradually-changed resonant frequencies are obtained; the narrow-band unit cells with different resonant frequencies are arranged according to the size to form the gradient metasurface with broadband reflection spectrum resonance; the method comprises the following steps: grinding and mashing a chiral biomolecule mixture to be detected, mixing the mixture with deionized water, coating one surface, with unit cells, of the gradient metasurface, and drying to obtain the gradient metasurface loaded with a sample to be detected; a reflection CD spectrum of the gradient metasurface carrying a sample to be measured is measured by using a normal incidence terahertz reflection time domain polarization spectrum technology, and chiral fingerprint intrinsic information in a multi-component system can be quantitatively analyzed with high precision according to the reflection CD spectrum.

Description

Technical Field

[0001] This invention relates to the field of terahertz spectroscopy analysis technology, and more specifically, to a method and apparatus for analyzing mixtures of chiral biomolecules. Background Technology

[0002] Chirality is a ubiquitous phenomenon in nature, referring to the asymmetric characteristic of an object that cannot be superimposed on its mirror image through translation and rotation. Almost all amino acids exhibit opposite chirality (L-enantiomers and D-enantiomers), and these chiral isomers exhibit different toxicological and pharmacological properties, playing important roles in the food and biomedical industries. Chiral spectroscopic analysis of various enantiomers in biomolecules has yielded groundbreaking techniques based on magnetic / electrical mechanisms, such as vibrational circular dichroism (VCD), electronic CD (ECD), photoelectron CD (PECD) techniques, and second harmonic optical rotation dispersion (SHG-ORD) measurements. Among these, VCD and ECD spectroscopy are commonly used methods for analyzing the different responses of enantiomers under left-handed circularly polarized (LCP) and right-handed circularly polarized (RCP) incident light.

[0003] Terahertz (THz) waves (0.1–10 THz), located between the microwave and infrared bands, can be used to detect the vibrational and rotational eigenmodes of biomolecules. In existing studies, the sensitivity of biomolecule intrinsic chiral phonon CD detected by conventional terahertz CD spectroscopy is low, only a few degrees. To improve sensitivity, plasmonic chiral metasurfaces with strong near-field optical chirality have been introduced. However, since the total CD signal detected is a mixture of the biomolecule's intrinsic CD and unwanted metasurface background noise CD, it is difficult to directly identify the type and chirality of biomolecules using chiral metasurfaces, requiring additional data processing to separate and detect the intrinsic chiral spectral features of biomolecules. On the other hand, existing studies have identified biomolecule components by detecting the resonance frequencies of the absorption, transmission, or reflection spectra of linearly polarized (LP) incident THz achiral metasurfaces. However, these LP spectra cannot resolve the intrinsic chiral information of the analytes. Furthermore, most LP spectroscopy studies are based on narrowband resonances, which limits the ability to distinguish the intrinsic information of different enantiomers in biological mixtures. Biomolecular chirality, classified into L-type and D-type enantiomers with opposite chirality, plays a crucial role in biomedicine and the food industry due to its close correlation with drug toxicity and pharmacological activity. In developing this invention, the applicant discovered that the detection and analysis of biomolecular chirality is inevitably limited by the circular dichroism (CD) spectra of traditional ultraviolet (UV), visible, and infrared (VIR) light. In the UV band, naturally occurring chiral biomolecules typically exhibit a weak intrinsic electronic CD response (approximately tens of millidegrees), and chiral molecules are prone to photolysis under UV irradiation. In the VIR band, while enhancing the induced molecular vibrational CD using nanostructure resonances can avoid photolysis under long-term UV radiation, these enhanced VIR CD responses cannot directly reflect the intrinsic chirality information of biomolecules because biomolecules lack corresponding intrinsic chiral (quasi-)particles in the VIR band. The ability to sensitively and directly detect intrinsic information such as the type and chirality of molecules without damaging biomolecules, and to directly and quantitatively analyze the intrinsic information of chiral fingerprints in multi-component systems, has always been a technological bottleneck. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art, and discloses a method and apparatus for analyzing chiral biomolecule mixtures, which can quantitatively analyze the intrinsic information of chiral fingerprints in multi-component systems with high precision.

[0005] Terminology Explanation:

[0006] CD (Circular Dichroism) represents the dichroism of circularly polarized light, that is, the differential absorption of left-handed and right-handed polarized light.

[0007] THz-TDS (THz time-domain spectroscopy) is a terahertz spectroscopy analysis method based on ultrafast laser technology. By measuring the time-domain electric field waveform of a terahertz pulse and performing Fourier transform, it obtains the frequency-domain optical parameters of the material (such as absorption coefficient, refractive index, dielectric constant, etc.), enabling qualitative and quantitative analysis of the physicochemical properties of the material.

[0008] THz-RTDPS (THz reflective time-domain polarization spectroscopy) is an advanced technique developed based on traditional terahertz time-domain spectroscopy (THz-TDS). By combining reflective detection and polarization resolution capabilities, it enables high-precision analysis of material chiral characteristics, anisotropy, and surface / interface properties.

[0009] AGMs (achiral gradient metasurfaces) are gradient supercells (metasurfaces) proposed in this invention, which are composed of narrow-band unit cell plasma metasurfaces with gradually changing structural parameters (gradually changing resonant frequencies). These supercells have broadband reflectance spectra, and the spectral range covers a variety of intrinsic fingerprint modes of chiral molecules.

[0010] TEFCS (THz eigenmode-fingerprint chiroptical spectroscopy) refers to the spectroscopic analysis method proposed in this invention. It utilizes the broadband resonance of AGM to simultaneously enhance the intrinsic mode fingerprint (CD) signals of different biomolecules / biomolecule mixtures. Based on the resonance frequency, sign, and amplitude of the CD signal of the molecular-AGM system, the intrinsic mode fingerprint characteristics (components, chirality, enantiomeric ratio) of biomolecules / biomolecule mixtures can be accurately and directly resolved. Because there is no interference from metasurface background noise (CD), the reflectance CD spectrum of the gradient metasurface carrying the analyte, measured by normal incidence terahertz reflection time-domain polarization spectroscopy (THz-RTDPS), only reflects the intrinsic fingerprint CD signal of the analyte itself. The resonant frequency of this CD spectrum corresponds to the intrinsic resonant frequency of the analyte. The components of the analyte biomolecular mixture can be directly analyzed based on the resonant frequency position of the reflectance CD spectrum. For example, if the measured resonant positions of the reflectance CD spectrum are around 0.88 THz and 1.2 THz, then the analyte contains histidine (intrinsic fingerprint resonant frequency of 0.88 THz) and glutamate (intrinsic fingerprint resonant frequency of 1.2 THz). The chirality of the analyte biomolecular mixture can be directly analyzed based on the line shape of the resonant CD spectrum and the sign of the CD value. For example, the measured line shape of the reflectance CD spectrum around 0.88 THz shows an initial resonant peak (positive peak value) followed by a resonant trough (negative trough value), and around 1.2 THz... If the THz region shows a resonant valley (negative valley value) followed by a resonant peak (positive peak value), then the chirality of histidine and glutamic acid in the analyte is levorotatory. By mixing the left- and right-handed enantiomers of the same amino acid in gradually varying ratios (4:0, 3:1, 2:2, 1:3, 0:4), the intensity of the measured CD spectrum will continuously and gradually change according to the ratio of the left- and right-handed enantiomer mixture. Based on the continuously and gradually changing reflectance CD spectrum, the ratio of left- and right-handed enantiomers in the analyte mixture can be determined. This confirms the potential of AGMs in quantitatively determining the enantiomer ratio in multi-component mixtures.

[0011] The first aspect of this invention discloses a method for analyzing a mixture of chiral biomolecules, comprising: designing a unit cell: two identical long metal rods are perpendicularly connected at their midpoints, and four identical short metal rods are perpendicularly connected to the four ends of the long metal rods respectively, all the metal rods being located on the same plane and integrated onto the surface of a quartz substrate to form a narrow-band unit cell; designing a gradient metasurface: keeping the shape of the narrow-band unit cell unchanged, the lengths of the long and short metal rods are changed in equal increments to obtain multiple narrow-band unit cells with gradually changing resonant frequencies; arranging multiple narrow-band unit cells with different resonant frequencies to form a gradient metasurface with broadband reflectance spectrum resonance; preparing a sample: grinding and crushing the chiral biomolecule mixture to be tested and mixing it with deionized water to coat the side of the gradient metasurface with unit cells, and then drying it to obtain a gradient metasurface carrying the sample to be tested; measuring the reflectance CD spectrum: measuring the reflectance CD spectrum of the gradient metasurface carrying the sample to be tested using normal incident terahertz reflection time-domain polarization spectroscopy (THz-RTDPS).

[0012] The method for analyzing chiral biomolecular mixtures disclosed in this invention preferably further includes: analyzing fingerprint features: determining the intrinsic modal fingerprint features of the chiral biomolecular mixture based on the resonance frequency, sign, and amplitude of the reflectance CD spectrum, wherein the intrinsic modal fingerprint features include: components, chirality, and enantiomer ratio.

[0013] In this technical solution, since there is no interference from metasurface background noise CD, the reflection CD spectrum of the gradient metasurface carrying the test sample, measured by normal incident terahertz reflection time-domain polarization spectroscopy (THz-RTDPS), only reflects the intrinsic fingerprint CD signal of the test sample itself. The resonant frequency of this CD spectrum corresponds to the intrinsic resonant frequency of the test analyte. The composition of the biomolecular mixture can be directly analyzed based on the resonant frequency position of the reflection CD spectrum. The chirality of the biomolecular mixture can be directly analyzed based on the line shape of the resonant CD spectrum and the sign of the CD value. When left- and right-handed enantiomers of the same type of amino acid are mixed in a gradually changing ratio, the intensity of the measured CD spectrum will continuously and gradually change according to the ratio of the left- and right-handed enantiomer mixture. The ratio of left- and right-handed enantiomers in the test mixture can be analyzed based on the continuously and gradually changing reflection CD spectrum.

[0014] According to the method for analyzing chiral biomolecule mixtures disclosed in this invention, preferably, the width of both the long and short metal rods is 6µm, the length of the short metal rod is 28µm shorter than the length of the long metal rod, and the length of the long metal rod is 40µm to 70µm.

[0015] According to the method for analyzing chiral biomolecule mixtures disclosed in this invention, preferably, the period of the narrow-band unit cell in the y-axis direction is 78µm; the period of the narrow-band unit cell in the x-axis direction is 48µm~78µm.

[0016] In the method for analyzing chiral biomolecule mixtures disclosed in this invention, preferably, the long and short metal rods are made of gold.

[0017] According to the method for resolving chiral biomolecular mixtures disclosed in this invention, preferably, the gradient metasurface comprises 11 narrow-band unit cells of different sizes.

[0018] According to the method for analyzing chiral biomolecule mixtures disclosed in this invention, preferably, the chiral biomolecule mixture to be tested is a mixture of multiple amino acids, including L-histidine, D-histidine, L-tyrosine, D-tyrosine, L-glutamic acid, D-glutamic acid, L-glutamine, and D-glutamine.

[0019] A second aspect of the present invention discloses an apparatus for resolving mixtures of chiral biomolecules, comprising a gradient metasurface with broadband reflectance spectrum resonance as provided in any of the above-described technical solutions.

[0020] The apparatus for analyzing mixtures of chiral biomolecules disclosed in this invention preferably further includes: a terahertz reflection time-domain polarization spectroscopy detection system.

[0021] According to the apparatus for analyzing chiral biomolecular mixtures disclosed in this invention, preferably, the terahertz reflection time-domain polarization spectroscopy detection system specifically includes: a terahertz generator, which generates terahertz pulses by exciting a GaAs photoconductive antenna at 780 nm using a fiber femtosecond laser, wherein the spot size of the terahertz field completely covers the entire gradient metasurface; a beam splitter, disposed in the terahertz pulse optical path, for splitting the terahertz light into two beams, one beam directed towards the terahertz detector and the other towards the gradient metasurface; a first polarizing mirror, disposed in the terahertz pulse optical path between the terahertz generator and the beam splitter; a second polarizing mirror, disposed in the optical path between the beam splitter and the terahertz detector; and a terahertz detector, for receiving the terahertz pulses reflected back from the gradient metasurface.

[0022] The beneficial effects of this invention include at least the following: Utilizing the phonon-photon coupling mechanism between chiral molecules and metasurfaces in the terahertz band, a sensitive terahertz intrinsic fingerprint chiral spectroscopy (TEFCS) technique based on terahertz broadband achiral gradient metasurfaces (AGMs) is proposed to distinguish various biomolecules. Through this invention, by utilizing the phonon-photon coupling between biomolecules and metasurfaces, the composition, chirality, and enantiomer ratio of various chiral molecule / chiral biomolecule mixtures can be directly and accurately determined by the resonance frequency, sign, and amplitude of the CD spectrum. Compared with existing technologies, this invention improves detection sensitivity, enabling direct measurement of chiral phonon eigenin information in the THz band; the operating band of AGMs covers the entire molecular fingerprint spectrum, allowing direct resolution of chiral fingerprint eigenin information in multi-component systems, and has the potential to promote new advances in medical diagnostics, drug development, and the food industry. Attached Figure Description

[0023] Figure 1 A schematic diagram of a gradient metasurface structure and a narrow-band unit cell structure according to an embodiment of the present invention is shown.

[0024] Figure 2 A schematic diagram of a gradient metasurface structure according to an embodiment of the present invention is shown.

[0025] Figure 3 A schematic diagram of the optical path of a terahertz reflective time-domain polarization spectroscopy detection system according to an embodiment of the present invention is shown. Detailed Implementation

[0026] To better understand the above-described objects, features, and advantages of the present invention, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention; however, the invention may be practiced in other ways different from those described herein, and therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] One embodiment of the present invention discloses a spectroscopic method for resolving chiral biomolecular mixtures based on a terahertz broadband achiral gradient metasurface, comprising: designing a unit cell structure: two identical long metal rods are perpendicularly connected at their midpoints, and four identical short metal rods are perpendicularly connected to the four ends of the long metal rods respectively, all metal rods being located on the same plane and integrated onto the surface of a quartz substrate to form a narrowband unit cell; designing a gradient metasurface: keeping the shape of the narrowband unit cell unchanged, the lengths of the long and short metal rods are changed in equal increments to obtain multiple narrowband unit cells with gradually varying resonant frequencies; and combining multiple narrowband units with different resonant frequencies... Cellular arrangement forms a gradient metasurface (AGM) with broadband reflectance spectrum resonance; Sample preparation: The chiral biomolecule mixture to be tested is ground and crushed, mixed with deionized water and coated on the cell-containing side of the gradient metasurface, and then dried to obtain the gradient metasurface carrying the sample to be tested; Reflectance CD spectrum measurement: The reflection CD spectrum of the gradient metasurface carrying the sample to be tested is measured using normal incidence terahertz reflection time-domain polarization spectroscopy; Fingerprint characteristics analysis: The intrinsic mode fingerprint characteristics of the chiral biomolecule mixture are determined based on the resonance frequency, sign and amplitude of the reflection CD spectrum. The intrinsic mode fingerprint characteristics include: composition, chirality and enantiomeric ratio.

[0028] like Figure 1 and Figure 2As shown, according to the above embodiment, the long metal rod 3 and the short metal rod 4 are further made of gold and integrated onto the surface of the same quartz substrate 1. The width of both the long and short metal rods is 6µm, the length of the short metal rod is 28µm shorter than the length of the long metal rod, and the length of the long metal rod is 40µm~70µm. The period of the narrow-band unit cell in the y-axis direction is 78µm; the period of the narrow-band unit cell in the x-axis direction is 48µm~78µm. The long metal rod decreases from 70µm to 40µm in steps of 3µm, while the corresponding short metal rod decreases from 42µm to 12µm in steps of 3µm, forming 11 different narrow-band unit cells. This gradient metasurface is composed of these 11 narrow-band unit cells of different sizes.

[0029] According to another embodiment of the present invention, a practical application process of the gradient metasurface provided in the above embodiments is also disclosed:

[0030] Step 1: Design a narrow-band plasma-induced achiral anisotropic metasurface (structure shown in...) Figure 1 );

[0031] Step 2, reduce by equal step size Figure 1 The rod length l1 shown is used (correspondingly, the period of the unit cell in the x-direction also decreases accordingly). Eleven narrowband unit cells with gradually varying resonant frequencies are designed, and these eleven unit cell structures are used to form an achiral gradient metasurface (AGM) with broadband reflection spectrum resonance. The structure is shown in [reference needed]. Figure 2 This broad spectrum can cover a variety of intrinsic fingerprint patterns of biomolecules;

[0032] Step 3, prepare different amino acid samples:

[0033] (1) Weigh 0.3g of different amino acids respectively, crush them in a mortar with deionized water, and coat the samples onto AGMs and the substrate respectively;

[0034] (2) Mix any two of the eight amino acids (L-histidine, D-histidine, L-tyrosine, D-tyrosine, L-glutamic acid, D-glutamic acid, L-glutamine, and D-glutamine), with each amino acid weighing 0.15g and the total mass of the mixed amino acids being 0.3g. Crush the different amino acid mixtures separately in a mortar with deionized water, and then coat the mixtures onto AGMs and the substrate respectively;

[0035] (3) Mix L-histidine and D-histidine in ratios of 4:0, 3:1, 2:2, 1:3 and 0:4, and mix L-tyrosine and D-tyrosine in ratios of 4:0, 3:1, 2:2, 1:3 and 0:4, wherein the total mass of the mixed amino acids is 0.3g. Crush the different amino acid mixtures separately in a mortar with deionized water, and coat the mixtures onto AGMs respectively;

[0036] (4) Weigh 0.025g-0.3g of L-histidine and L-tyrosine respectively, crush them in a mortar with deionized water, and coat the samples onto AGMs respectively.

[0037] For all the above samples, in order to evaporate the moisture and fix the amino acids on the AGMs / substrate, the AGMs / substrate containing amino acids were dried in an oven at 50°C for 1 hour.

[0038] Step 4: The reflectance spectra of broadband AGMs and the reflectance CD spectra of all amino acid samples were measured using normal incident terahertz reflection time-domain polarization spectroscopy (THz-RTDPS). Comparison of the reflectance CD spectra of the molecule-AGM system and the molecule-substrate system revealed that the intrinsic chiral fingerprint CD signal of the molecule-AGM system can be significantly enhanced by up to 10-fold. Furthermore, the reflectance CD spectra detected by this method not only have high amplitude but also sufficient sensitivity, reaching 10-10. 24 degree / (mol / nm 3 It can directly analyze the intrinsic fingerprint characteristics (components, chirality, enantiomeric ratio) of biomolecules / biological mixtures.

[0039] In this embodiment, the schematic diagram of the experimental optical path for normal incidence THz-RTDPS (THz reflective time-domain polarization spectroscopy) is as follows: Figure 3 As shown, a fiber femtosecond laser was used to excite a GaAs photoconductive antenna (PCA) at 780 nm to generate a terahertz pulse, with an excitation time of 80 fs. The terahertz field spot size was approximately 2 cm × 2 cm, which could completely cover the entire metasurface (1.5 cm × 1.5 cm). All experiments were conducted in a relatively dry room temperature environment (humidity maintained at 10%). When probing the metasurface reflection spectrum, only the reflection signal of the LP normally incident on the metasurface needed to be detected; therefore, both THz polarizers (first polarizer and second polarizer) were rotated to +90°. However, when probing the reflection CD spectrum of different biomolecule-AGM systems, a circularly polarized wave with normal incidence was required; therefore, the first polarizer was rotated to +90° to obtain an LPTHz incident wave that could be decomposed into LCP and RCP THz waves. After the LP THz wave was normally incident on the sample surface, it was reflected normally to the terahertz detector after passing through a beam splitter and the second polarizer rotated to ±45°. Among them, the two sets of reflected LP signals corresponding to the second polarizer being rotated to ±45° can be synthesized into reflected LCP and RCP light.

[0040] like Figure 1As shown, another embodiment of the present invention discloses an apparatus for analyzing mixtures of chiral biomolecules. This apparatus uses a gradient metasurface as disclosed in any of the above embodiments to carry the analyte for detection. The overall structure and unit cell structure of the gradient metasurface are as follows: the gradient metasurface is a plasma-induced achiral anisotropic metasurface; the substrate 1 is z-cut quartz; and chiral molecules 2 cover the structured side of the metasurface. The narrow-band unit cells have periods p1 and p2 along the y and x directions, respectively; the short gold rod (short metal rod 4) has a length of l2, the long gold rod (long metal rod 3) has a length of l1, and all rods have a width of w = 6µm, p1 = 78µm; the length of p2 is l1 + 8µm; and the length of l2 is l1 - 28µm.

[0041] like Figure 3 As shown, according to the above embodiment, the apparatus for resolving chiral biomolecule mixtures further includes: a terahertz generator, which generates terahertz pulses by exciting a GaAs photoconductive antenna at 780 nm using a fiber femtosecond laser, with an excitation time of 80 fs, and the size of the terahertz field spot completely covers the entire gradient metasurface; a beam splitter, disposed in the terahertz pulse optical path, for splitting the terahertz light into two beams, one directed to the terahertz detector and the other directed to the gradient metasurface; a first polarizing mirror, disposed in the terahertz pulse optical path between the terahertz generator and the beam splitter; a second polarizing mirror, disposed in the optical path between the beam splitter and the terahertz detector; and a terahertz detector, for receiving the terahertz pulses and the terahertz pulses reflected back from the gradient metasurface.

[0042] In summary, by utilizing the phonon-photon coupling mechanism between chiral molecules and metasurfaces in the terahertz band, this invention proposes a sensitive terahertz eigenmode fingerprint chiral spectroscopy (TEFCS) technique based on terahertz broadband achiral gradient metasurfaces (AGMs) to distinguish various biomolecules. Through this invention, by utilizing the phonon-photon coupling between biomolecules and metasurfaces, the composition, chirality, and enantiomer ratios of various chiral molecule / chiral biomolecule mixtures can be directly and accurately determined using the resonance frequencies, signs, and amplitudes of CD spectra.

[0043] The broadband resonance-enhanced TEFCS proposed in this invention benefits from superior spectral overlap across the entire molecular fingerprint spectrum and direct photon-phonon coupling between the terahertz broadband AGM and chiral biomolecules. Compared to existing biomolecular spectroscopy techniques, this method offers higher CD signals and sufficient sensitivity, and can accurately and directly resolve the composition and intrinsic fingerprint characteristics (components, chirality, enantiomeric ratio) of biomolecules / biomixtures based on the resonance frequency, sign, and amplitude of the CD signal. It has the potential to advance advancements in medical diagnostics, drug development, and the food industry.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for analyzing mixtures of chiral biomolecules, characterized in that, include: Design unit structure unit: The midpoints of two identical long metal rods are perpendicularly connected to each other, and the four ends of the long metal rods are perpendicularly connected to four identical short metal rods respectively. All metal rods are located on the same plane and integrated into the surface of the quartz substrate to form a narrow band unit cell. Designing a gradient metasurface: Keeping the shape of the narrowband unit cell unchanged, the lengths of the long and short metal rods are changed in equal increments to obtain multiple narrowband unit cells with gradually changing resonant frequencies; multiple narrowband unit cells with different resonant frequencies are arranged to form a gradient metasurface with broadband reflection spectrum resonance. Sample preparation: The mixture of chiral biomolecules to be tested is ground and crushed, mixed with deionized water and coated on the cell-containing side of the gradient metasurface. After drying, a gradient metasurface carrying the sample to be tested is obtained. Measurement of reflection CD spectrum: The reflection CD spectrum of a gradient metasurface carrying the sample to be tested is measured using normal incidence terahertz reflection time-domain polarization spectroscopy.

2. The method for analyzing mixtures of chiral biomolecules according to claim 1, characterized in that, Also includes: Fingerprint characteristics analysis: The intrinsic pattern fingerprint characteristics of the chiral biomolecule mixture are determined based on the resonance frequency, sign, and amplitude of the reflected CD spectrum. The intrinsic pattern fingerprint characteristics include: components, chirality, and enantiomer ratio.

3. The method for analyzing mixtures of chiral biomolecules according to claim 1, characterized in that, The width of both the long metal rod and the short metal rod is 6µm. The length of the short metal rod is 28µm shorter than that of the long metal rod, and the length of the long metal rod is 40µm to 70µm.

4. The method for analyzing mixtures of chiral biomolecules according to claim 1, characterized in that, The period of the narrowband unit cell in the y-axis direction is 78µm; the period of the narrowband unit cell in the x-axis direction is 48µm~78µm.

5. The method for analyzing mixtures of chiral biomolecules according to claim 1, characterized in that, The long metal rod and the short metal rod are made of gold.

6. The method for analyzing mixtures of chiral biomolecules according to claim 1, characterized in that, The gradient metasurface comprises 11 narrowband unit cells of different sizes.

7. The method for analyzing mixtures of chiral biomolecules according to any one of claims 1 to 6, characterized in that, The chiral biomolecule mixture to be tested is a mixture of multiple amino acids, including L-histidine, D-histidine, L-tyrosine, D-tyrosine, L-glutamic acid, D-glutamic acid, L-glutamine, and D-glutamine.

8. An apparatus for resolving mixtures of chiral biomolecules, characterized in that, Including a gradient metasurface with broadband reflection spectrum resonance as described in any one of claims 1 to 6.

9. The apparatus for analyzing mixtures of chiral biomolecules according to claim 8, characterized in that, Also includes: Terahertz reflective time-domain polarization spectroscopy detection system.

10. The apparatus for analyzing mixtures of chiral biomolecules according to claim 9, characterized in that, The terahertz reflection-type time-domain polarization spectroscopy detection system specifically includes: The terahertz generator uses a fiber femtosecond laser to excite a GaAs photoconductive antenna at 780nm to generate terahertz pulses. The excitation time is 80fs, and the size of the terahertz field spot completely covers the entire gradient metasurface. A beam splitter, located in the terahertz pulse optical path, is used to split the terahertz light into two beams, one of which is directed toward the terahertz detector and the other toward the gradient metasurface. A first polarizing mirror is disposed on the terahertz pulse optical path between the terahertz generator and the beam splitter; A second polarizing mirror is disposed in the optical path between the beam splitter and the terahertz detector; The terahertz detector is used to receive the terahertz pulses reflected back from the gradient metasurface.

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