Optical detection method for amyloid configuration based on infrared band multi-frequency resonance

Abstract

The application discloses an amyloid configuration optical detection method based on infrared waveband multi-frequency resonance, which comprises the following steps: obtaining an optical super-structure surface unit; adding an amyloid solution to be detected; irradiating incident light to realize electromagnetic field excitation; detecting the refractive index and layer thickness of a protein formation layer in a groove of a coupling complementary opening resonance ring working in a near-infrared waveband to obtain one-dimensional structure information of the amyloid formation layer to be detected; detecting the component molecule configuration in the groove of the coupling complementary opening resonance ring working in a mid-infrared waveband by using a characteristic absorption peak of a molecule to obtain structure information of the amyloid molecule to be detected; and determining the configuration analysis result of the amyloid to be detected and the relationship between the protein formation layer and the protein component molecule configuration by using the two aspects of information. The application realizes the measurement based on the medium refractive index and the measurement based on the molecular absorption spectrum on the same sensing surface, can realize the protein configuration analysis and organically connects the layer structure and the molecule configuration.

Description

Technical Field

[0001] This invention belongs to the field of optical detection, specifically relating to an optical detection method for amyloid protein conformation based on infrared multi-frequency resonance. Background Technology

[0002] Protein misfolding can lead to protein conformation disorders. Amyloid protein misfolding is closely associated with several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. The functional impairment caused by amyloid protein misfolding is irreversible, resulting in limited treatment effectiveness. Early detection and intervention are commonly used in clinical treatment. Measuring and analyzing amyloid protein conformation has become an effective auxiliary method for clinical diagnosis.

[0003] Existing methods for measuring and analyzing amyloid protein conformation include traditional methods and label-free optical sensing methods. Traditional methods include X-ray crystallography, nuclear magnetic resonance (NMR), and neutron reflectance spectroscopy. Their drawbacks are: high cost due to the use of large-scale equipment, long measurement time, and the inability to obtain static and steady-state structural information, making them unsuitable for monitoring dynamic changes in protein structure.

[0004] Label-free optical sensing methods are divided into label-free optical detection methods based on refractive index measurement and spectroscopic detection methods. Label-free optical detection methods utilize near-infrared light waves for detection. Because the volume of the light wave mode is close to that of protein molecules, the interaction between light and matter is strong, resulting in high sensitivity and signal-to-noise ratio. However, this method only provides one-dimensional structural information of the protein-forming layer. Based on the mechanism that "the protein-forming layer is uniquely determined by the protein molecule's conformation," information about the protein molecule's conformation is inferred from the structural information of the protein layer. Label-free optical detection methods specifically include the following two commonly used methods: 1) SPR (Surface Plasmon Resonance) technology, based on single-polarized light measurement, the output signal reflects the mass change of the analyte on the metal surface, but it provides limited information on the biomolecular structure and is usually used to study the dynamic characteristics of protein molecules interacting with other molecules. Furthermore, due to the ohmic loss of the metal, the resonant spectrum broadens, and the detection limit is 1 pg / mm². 22) DPI (Dual Polarization Interferometry) technology: This technology is based on Young's interference phenomenon of light, obtaining one-dimensional structural information of the protein molecular forming layer through dual-mode interference fringes. However, DPI-based equipment is relatively large and requires professional operation; moreover, the receiving distance between the imager and the spatial output light may cause blurring of the interference fringes, affecting detection sensitivity. Spectroscopic detection methods obtain the conformational information of protein molecules based on the characteristic peak spectra of functional groups. Commonly used methods include the following two: 1) FTIR (Fourier-transform Infrared Spectroscopy): This technology requires professional operation and standard library import; it is not suitable for testing aqueous samples in fluids because of its strong absorption and numerous overlaps at high frequencies. Therefore, it is not suitable for detecting liquid samples. 2) SERS (Surface Enhancement of Raman Scattering): The sensing surface of the metal particles in this technology is easily contaminated or oxidized, resulting in generally poor stability. Simultaneously, the metal particles may interact with other components of the sample, reducing the signal-to-noise ratio and specificity. Furthermore, the light field is limited to a few nanometers on the surface of the metal particles, making it suitable only for molecules with high vibrational frequencies.

[0005] Therefore, how to comprehensively, quickly, and easily measure and analyze amyloid protein conformation is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides an optical detection method for amyloid protein conformation based on multi-frequency resonance in the infrared band. The technical problem to be solved by this invention is achieved through the following technical solution:

[0007] An optical detection method for amyloid protein conformation based on infrared multi-frequency resonance includes:

[0008] Obtain a pre-constructed optical metasurface unit; wherein, the optical metasurface unit comprises, from top to bottom, a metal aluminum layer, an aluminum oxide layer, a metal gold layer and a silicon substrate layer; the surface of the metal aluminum layer is formed with multiple pairs of coupled complementary open resonant rings by etching grooves, and the multiple pairs of coupled complementary open resonant rings operate in the near-infrared band and the mid-infrared band respectively;

[0009] An amyloid protein solution to be tested is added to the optical metasurface unit, causing the amyloid protein solution to be tested to accumulate in the grooves corresponding to the multiple pairs of coupled complementary open resonant rings;

[0010] Electromagnetic field excitation is achieved by irradiating the optical metasurface unit with incident light.

[0011] For a coupled complementary open resonator operating in the near-infrared band, the refractive index and layer thickness of the protein-forming layer in the groove are detected, and the one-dimensional structural information of the amyloid protein-forming layer to be tested is obtained based on the obtained refractive index and layer thickness of the protein layer.

[0012] For coupled complementary open resonant rings operating in the mid-infrared band, the characteristic absorption peaks of molecules are used to identify the molecular configuration of the components in the groove, thereby obtaining the structural information of the amyloid protein molecule to be tested.

[0013] Using the one-dimensional structural information of the amyloid-forming layer and the structural information of the amyloid molecule, the conformational analysis results of the amyloid protein and the relationship between the conformation of the protein-forming layer and the protein component molecules are determined.

[0014] In one embodiment of the present invention, the plurality of pairs of coupled complementary open-ended resonant rings include a first subunit operating in the mid-infrared band and a plurality of second subunits operating in the near-infrared band; the operating resonant frequencies of the first subunit and the second subunits do not interfere with each other; each subunit includes a pair of coupled complementary open-ended resonant rings of unequal size, spaced apart and arranged side by side along the length direction on the surface of the aluminum layer, the opening direction of the two rings being on the side away from the center of the two rings, the two rings having their own inherent resonant frequencies and reaching the critical coupling condition; the length direction of the surface of the aluminum layer corresponds to the X direction, and the width direction corresponds to the Y direction;

[0015] The first subunit is located in the central region of the surface of the aluminum metal layer; the plurality of second subunits are divided into two groups, located on one side of the surface of the aluminum metal layer corresponding to the wide side, and the plurality of second subunits on the same side are arranged periodically along the Y direction; the plurality of second subunits have the same size, and the size of each second subunit is smaller than the size of the first subunit.

[0016] In one embodiment of the present invention, the thicknesses of the aluminum layer, the aluminum oxide layer, the gold layer and the silicon substrate are 0.1 μm, 0.25 μm, 0.6 μm and 725 μm, respectively.

[0017] In one embodiment of the present invention, for the optical metasurface unit, the length L of the aluminum layer surface is... MIR It is 14μm in diameter and has a width of W. MIR It is 5μm;

[0018] In the first subunit, the radius R of the large ring MIR1 The radius R of the small ring is 1.49 μm. MIR2The diameter of the first subunit is 1.34 μm, the width W1 of both rings is 0.37 μm, the opening width A1 of both rings is 0.24 μm, the coupling spacing G1 between the two rings is 0.4 μm, and the period length L of the first subunit is 1.34 μm. NIR The length L corresponding to the surface of the aluminum layer MIR The periodic width of the first subunit corresponds to the width W of the surface of the aluminum layer. MIR ;

[0019] In the second subunit, the radius R of the large ring NIR1 The radius R of the small ring is 0.31 μm. NIR2 The width W2 of each ring is 0.25 μm, the opening width A2 of each ring is 0.13 μm, the coupling spacing G2 between the two rings is 0.08 μm, and the period length L of the second subunit is 0.25 μm. NIR The diameter is 2.33 μm, and the period width is W. NIR It is 1.2μm.

[0020] In one embodiment of the present invention, the amyloid protein solution to be tested is formed by dissolving and diluting the amyloid protein to be tested using Tris buffer.

[0021] In one embodiment of the present invention, the incident light is directed in the Z direction, and the electric field is polarized in the X direction.

[0022] In one embodiment of the present invention, the step of detecting the refractive index and layer thickness of the protein-forming layer within the groove of a coupled complementary open-circuit resonator operating in the near-infrared band, and obtaining one-dimensional structural information of the amyloid protein-forming layer to be tested based on the obtained refractive index and layer thickness of the protein layer, includes:

[0023] The electromagnetic field generated by the dual-resonance wavelength characteristics of the incident light after being modulated by the optical metasurface unit is applied to the protein layer in a number of periodically arranged second subunits to detect the change in the dual-resonance wavelength.

[0024] Based on the change in the dual resonant wavelength, the layer refractive index sensitivity and layer thickness sensitivity measured in advance based on each resonant wavelength, the refractive index and layer thickness of the protein layer in the periodically arranged multiple second subunits are calculated.

[0025] Based on the calculated refractive index and thickness of the protein layer, the density of the protein layer is calculated using the De Feijter formula;

[0026] The one-dimensional structural information of the amyloid-forming layer to be tested is characterized using the layer thickness and density.

[0027] In one embodiment of the present invention, the dual resonant wavelengths are 1.56 μm and 1.589 μm, respectively.

[0028] In one embodiment of the present invention, the method of identifying the molecular configuration of the constituent molecules within the groove by utilizing the characteristic absorption peaks of molecules operating in the mid-infrared band to obtain the structural information of the amyloid protein molecule to be tested includes:

[0029] The incident light is used to excite the protein molecules in the first subunit, causing the component molecules of the protein layer in the first subunit to undergo vibrational and rotational energy level transitions, and the corresponding absorption spectrum is obtained.

[0030] Based on the position of the characteristic absorption peak in the absorption spectrum, the molecular configuration of the protein in the first subunit is identified to obtain the molecular structural information of the amyloid protein to be tested; wherein, the characteristic absorption peak appears in the amide I band and the amide II band; the molecular structural information of the amyloid protein to be tested includes α-helical amyloid protein molecules and β-sheet folded amyloid protein molecules.

[0031] In one embodiment of the present invention, the optical detection method for amyloid protein conformation based on infrared multi-frequency resonance further includes:

[0032] The microenvironmental parameters of the amyloid protein solution to be tested are changed, and the changes in the conformation of the amyloid protein to be tested under the change of microenvironment are detected; wherein, the microenvironmental parameters include microenvironmental temperature, pH, and metal ion concentration.

[0033] The beneficial effects of this invention are:

[0034] The present invention provides an optical metasurface unit that enables both refractive index measurement and molecular absorption spectroscopy measurement on the same sensing surface. In the near-infrared band, a dual-wavelength interrogation mechanism can be used to detect the structure of the amyloid cambium layer; in the mid-infrared band, the conformation of the protein molecule can be determined based on the infrared absorption spectrum generated by specific molecular vibrational rotational energy level transitions. Based on the difference in the interaction characteristics between near-infrared / mid-infrared light waves and matter, the present invention combines optical refractive index measurement and optical absorption measurement to acquire information on the structure of the protein cambium layer and the conformation of its constituent molecules on the same sensing surface. This allows for convenient and efficient measurement of the conformation of protein molecules with a miniaturized measurement structure, achieving high sensitivity and high specificity. It organically links the one-dimensional structure of the biological cambium layer with the conformation of its constituent molecules, laying the foundation for elucidating protein misfolding caused by conformational changes in amyloid protein. Attached Figure Description

[0035] Figure 1 A schematic flowchart of an optical detection method for amyloid protein conformation based on infrared multi-frequency resonance provided in an embodiment of the present invention;

[0036] Figure 2 This is an electric field distribution diagram of the near-infrared and mid-infrared bands of ccSRR in an embodiment of the present invention;

[0037] Figure 3 A three-dimensional structural diagram of an optical metasurface unit provided in an embodiment of the present invention;

[0038] Figure 4 A top view of an optical metasurface unit provided in an embodiment of the present invention;

[0039] Figure 5 This is a diagram showing the electric field mode distribution of the optical metasurface unit at multiple frequencies in an embodiment of the present invention.

[0040] Figure 6 This is a schematic diagram illustrating the principle of the dual-wavelength interrogation mechanism in an embodiment of the present invention;

[0041] Figure 7 This is the measurement result of the field enhancement factor within a 100nm range of the distance sensing surface in an embodiment of the present invention;

[0042] Figure 8 This is a dual-wavelength reflectance spectrum based on volume refractive index measurement in an embodiment of the present invention;

[0043] Figure 9 This is the reflection spectrum at the 1560nm resonant wavelength in an embodiment of the present invention;

[0044] Figure 10 This is the reflection spectrum at the 1589nm resonant wavelength in an embodiment of the present invention;

[0045] Figure 11 The following are output spectra at different resonant wavelengths in embodiments of the present invention;

[0046] Figure 12 These are the optical geometric parameters of amyloid protein layers with different component molecules obtained by the dual-wavelength interrogation mechanism in this embodiment of the invention.

[0047] Figure 13 This is a schematic diagram of the absorption peaks of the α-helix and β-sheet folded components in the embodiments of the present invention;

[0048] Figure 14 The images show the infrared absorption spectra of different molecular components (including two configurations of protein molecules and fat molecules) in the embodiments of the present invention. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0050] This invention provides an optical detection method for amyloid protein conformation based on infrared multi-frequency resonance, belonging to a label-free optical detection method for amyloid protein structural characterization, such as... Figure 1 As shown, the optical detection method for amyloid protein conformation based on infrared multi-frequency resonance may include the following steps:

[0051] S1, Obtain the pre-constructed optical metasurface unit;

[0052] As will be understood by those skilled in the art, a complementary split ring resonator (cSRR) is a complementary structure to a split ring resonator (SRR), i.e., an split ring resonator structure etched onto a metal layer. A cSRR can be considered a metasurface structure. cSRRs possess negative permeability, generating a resonant magnetic dipole moment. Therefore, time-varying electromagnetic fields can excite them to produce a resonant circuit effect, achieving a filtering effect. Currently, cSRRs are widely used in microwave communication, primarily as filters.

[0053] This invention improves upon cSRR and proposes for the first time a ccSRR (coupling complementary splitring resonator) structure.

[0054] Specifically, the ccSRR structure consists of two ccSRRs of different sizes placed side by side with their openings facing each other, meaning the openings of the ccSRRs are both on the outside of each other. The two ccSRRs have a certain coupling distance. Each ccSRR has its own natural resonant frequency. By adjusting the size of the ccSRRs and the coupling distance between the two ccSRRs, a pair of ccSRRs (i.e., the ccSRR structure) can reach the critical coupling condition.

[0055] The inventors discovered that the capacitive effect of cSRRs, with the electric field confined to the sensing surface, enhances the interaction between light and matter, resulting in high sensitivity. When a pair of cSRRs couple and resonate, the filtering effect is significantly enhanced, the output spectrum exhibits a sharp resonance peak, and the detection limit is reduced.

[0056] Specifically, this invention enables infrared light modulation in different wavelength bands by adjusting the size of the cCSRR. Figure 2 Figures (a) and (b) show the electric field distribution in the near-infrared and mid-infrared bands, respectively. Figure 2 Figure (a) corresponds to the electric field mode distribution in the 1.56 μm band. Figure 2 Figure (b) shows the electric field mode distribution in the 6.5μm band.

[0057] Based on ccSRR, this invention proposes an optical metasurface unit. The optical metasurface unit comprises, from top to bottom, an aluminum layer, an aluminum oxide layer, a gold layer, and a silicon substrate layer. Multiple pairs of coupled complementary open-ring resonators are formed on the surface of the aluminum layer through etched grooves. These multiple pairs of coupled complementary open-ring resonators operate in the near-infrared and mid-infrared bands, respectively. Specifically, the operating wavelength range for near-infrared light is 1.52 μm to 1.63 μm, and the operating wavelength range for mid-infrared light is 6.0 μm to 6.6 μm.

[0058] In one optional embodiment, the plurality of coupled complementary open-ring resonators includes a first subunit operating in the mid-infrared band and a plurality of second subunits operating in the near-infrared band; the operating resonant frequencies of the first subunit and the second subunits do not interfere with each other; each subunit includes a pair of coupled complementary open-ring resonators of unequal size, spaced apart and arranged side by side along the length direction on the surface of the aluminum layer, with the opening direction of the two rings on the side away from the center of the two rings, and the two rings having their own inherent resonant frequencies and reaching the critical coupling condition; the length direction of the surface of the aluminum layer corresponds to the X direction, and the width direction corresponds to the Y direction;

[0059] The first subunit is located in the central region of the surface of the aluminum metal layer; the plurality of second subunits are divided into two groups, located on one side of the surface of the aluminum metal layer corresponding to the wide side, and the plurality of second subunits on the same side are arranged periodically along the Y direction; the plurality of second subunits have the same size, and the size of each second subunit is smaller than the size of the first subunit.

[0060] Please see Figure 3 The three-dimensional structure diagram of the optical metasurface unit shown and Figure 4 The top view of the optical metasurface unit shown is provided for illustrative understanding. It can be seen that this embodiment of the invention constructs periodically arranged coupled complementary open-ended resonant rings on a layer of aluminum metal. The XYZ coordinate directions follow the right-hand screw rule. In practical use, the optical metasurface unit of this embodiment can also be periodically repeated to form a larger optical metasurface, which can then be used as a sensing surface for measurement.

[0061] The optical metasurface unit proposed in this embodiment of the invention consists of two subunits. The first subunit is a pair of larger cCSRRs located at the center, which can precisely control mid-infrared light waves with wavelengths of 6.025 μm, 6.2 μm, and 6.5 μm. The second subunit is a pair of smaller cCSRRs located on the wide side of the optical metasurface, which can precisely control near-infrared light waves with wavelengths of 1.56 μm and 1.589 μm. There are four sets of second subunits on both sides of the optical metasurface, together with the first subunit at the center, forming a complete optical metasurface unit. The two subunits operate at their respective resonant frequencies and do not interfere with each other (see [link to relevant documentation]). Figure 5 As shown in the figure, this is the key to achieving multi-frequency resonance on the same sensing surface. Figure 5 The figures show the electric field mode distribution of the optical metasurface unit at multiple frequencies in the embodiments of the present invention, wherein (a) represents the electric field mode distribution in the 1.565μm band, (b) represents the electric field mode distribution in the 1.589μm band, (c) represents the electric field mode distribution in the 6.2μm band, and (d) represents the electric field mode distribution in the 6.5μm band.

[0062] The embodiments of this invention employ an aluminum layer because it has a large window in the infrared band and its processing technology is compatible with CMOS, which helps reduce processing costs and complexity. The aluminum oxide layer used, as a low-loss dielectric layer, can increase optical resonance. Since near-infrared detection uses the reflection spectrum, the use of a gold layer can effectively enhance light reflection.

[0063] The thicknesses of the aluminum layer, the alumina layer, the gold layer, and the silicon substrate can be set as needed in embodiments of the present invention. In one optional embodiment, the thicknesses of the aluminum layer, the alumina layer, the gold layer, and the silicon substrate are 0.1 μm, 0.25 μm, 0.6 μm, and 725 μm, respectively.

[0064] In embodiments of the present invention, the size parameters of the first subunit and the second subunit can be set according to control requirements, etc. Please refer to... Figure 4 As shown, in one optional embodiment, for the optical metasurface unit, the length L of the aluminum layer surface is... MIR It is 14μm in diameter and has a width of W. MIR It is 5μm;

[0065] In the first subunit, the radius R of the large ring MIR1 The diameter is 1.49 μm, and the radius R of the small ring is... MIR2 The diameter is 1.34 μm, the width W1 of both rings is 0.37 μm, the opening width A1 of both rings is 0.24 μm, the coupling spacing G1 between the two rings is 0.4 μm, and the period length L of the first subunit is 1.34 μm. NIR The length L corresponding to the surface of the aluminum metal layerMIR The periodic width of the first subunit corresponds to the width W of the surface of the aluminum layer. MIR That is, the length L of a complete optical metasurface unit. MIR and width W MIR That is, the size of one cycle of the mid-infrared subunit (i.e., the first subunit).

[0066] In the second subunit, the radius R of the large ring NIR1 The radius R of the small ring is 0.31 μm. NIR2 The width W2 of each ring is 0.25 μm, the opening width A2 of each ring is 0.13 μm, the coupling spacing G2 between the two rings is 0.08 μm, and the period length L of the second subunit is 0.25 μm. NIR The diameter is 2.33 μm, and the period width is W. NIR It is 1.2μm.

[0067] This invention provides a novel optical metasurface unit based on coupled complementary open resonant rings. By simply adjusting the geometry, near-infrared and mid-infrared light waves can be manipulated on the same sensing surface to achieve field enhancement and filtering effects. It can also achieve multi-frequency resonance in the infrared band without interference.

[0068] S2, add the amyloid protein solution to be tested to the optical metasurface unit, so that the amyloid protein solution to be tested accumulates in the grooves corresponding to the multiple pairs of coupled complementary open resonant rings;

[0069] The amyloid protein solution to be tested can be added to the optical metasurface unit using a microfluidic chip. Since the ccSRR is formed by etching grooves, the amyloid protein solution to be tested will accumulate in the grooves corresponding to the coupled complementary open resonant rings of each subunit.

[0070] Since the amyloid protein to be tested is a solid substance, in order to perform the detection, a solution of the amyloid protein to be tested is first prepared using a suitable buffer solution, which is selected according to the different solute molecules (i.e., amyloid protein as described here).

[0071] In one optional embodiment, the amyloid protein solution to be tested is prepared by dissolving and diluting the amyloid protein to be tested using Tris buffer.

[0072] Tris represents tris(hydroxymethyl)aminomethane. For details on Tris buffer, please refer to the relevant technical explanations. It will not be described in detail here.

[0073] S3, electromagnetic field excitation is achieved by irradiating incident light onto the optical metasurface unit;

[0074] As mentioned earlier, the surface of the optical metasurface unit corresponds to the XY plane. Therefore, the direction of the incident light is the Z direction, and the electric field is polarized along the X direction. The magnetic field direction is perpendicular to the electric field direction. Specifically, the incident light perpendicularly illuminates the optical metasurface unit. The incident light is a plane wave propagating along the Z direction, the electric field vibration direction is along the X direction, and the magnetic field vibration direction is along the Y direction.

[0075] S4. For a coupled complementary open resonator operating in the near-infrared band, the refractive index and layer thickness of the protein-forming layer in the groove are detected, and the one-dimensional structural information of the amyloid protein-forming layer to be tested is obtained based on the obtained refractive index and layer thickness of the protein layer.

[0076] In one optional implementation, S4 may include:

[0077] S41, using the electromagnetic field generated by the double resonant wavelength characteristics of the incident light after being modulated by the optical metasurface unit, acting on the protein layer in a plurality of periodically arranged second subunits, and detecting the change in the double resonant wavelength;

[0078] Specifically, the optical metasurface unit of this invention can be used for one-dimensional structural analysis of amyloid-forming layers. The etched regions in the ccSRR structure of the plurality of second subunits serve as the sensing surface. On the one hand, the electromagnetic field is confined to the groove surface; on the other hand, the groove structure facilitates the capture of amyloid molecules. In the near-infrared working region, after adding the amyloid solution to be tested to the grooves corresponding to the ccSRR structure of the plurality of second subunits, protein molecules form a protein layer on the surface of the annular grooves. The incident light is modulated by the optical metasurface unit to generate an electromagnetic field with dual-resonant wavelength characteristics. For the structural parameters of the optical metasurface unit described above, the dual-resonant wavelengths are 1.56 μm and 1.589 μm, respectively, and the electric field energy is effectively confined to the sensing surface. The electric field interacts with the protein layer on the surface of the plurality of second subunits, manifesting as a response of the resonant wavelength to the optical parameters of the protein layer (including layer refractive index and layer thickness). Specifically, the interaction between the electric field and the protein layer causes the two sets of resonant wavelengths to change to different degrees. This embodiment of the invention is based on a dual-wavelength interrogation mechanism to measure changes in the photogeometry parameters of the sensing surface medium. Specifically, S41 involves real-time detection of the changes in each of the two resonant wavelengths. The changes in the two resonant wavelengths at 1.56 μm and 1.589 μm can be expressed as Δλ, respectively. 1.56 and Δλ 1.589 .

[0079] S42, based on the change in the dual resonant wavelength, the layer refractive index sensitivity and layer thickness sensitivity measured in advance based on each resonant wavelength, the refractive index and layer thickness of the protein layer in the multiple periodically arranged second subunits are calculated.

[0080] Please see Figure 6 Understand the principle of dual-wavelength query mechanism; Figure 6 Figure (a) shows the response of two independent sets of dual-resonance wavelengths (1.56 μm and 1.589 μm) to the same protein layer, and the curves showing the change of resonant wavelengths over time (t). Real-time measurement and recording of the changes in the dual-resonance wavelengths (1.56 μm and 1.589 μm) can yield... Figure 6 The results in Figure (a) show that the responses of the same protein layer at resonant wavelengths of 1.56 μm and 1.589 μm are independent, allowing for the analysis of two separate curves corresponding to different resonant wavelengths at the same time (t1). Each curve represents an infinite number of protein layer refractive indices and thicknesses; each set of refractive indices and thicknesses corresponds to a protein layer structure, and each set serves as the optical response of its corresponding protein layer. Therefore, for each curve, the possible protein layers are infinite. Only the intersection of two curves corresponds to a protein layer that is the solution for both resonant wavelengths. In other words, the intersection of the curves represents the solution that simultaneously satisfies the change in resonant wavelength, i.e., the true protein layer thickness and refractive index values, such as... Figure 6 As shown in Figure (b), Figure 6 Figure (b) shows two curves representing the refractive index and thickness of an infinite number of layers at time t1, derived from the change in resonant wavelength. The intersection of these curves represents the true refractive index and thickness of the protein layer. Therefore, near-infrared subunits (i.e., multiple second subunits) can achieve highly sensitive measurement of the one-dimensional structure of protein layers.

[0081] The above Figure 6 As shown, the process of calculating the refractive index and layer thickness of the protein layer in the plurality of second subunits can be expressed by the following formula:

[0082]

[0083] Where, Δλ 1.56 and Δλ 1.589 This represents the change in wavelength between the two resonants at 1.56 μm and 1.589 μm; This indicates the layer refractive index sensitivity corresponding to a resonant wavelength of 1.56 μm; This indicates the layer thickness sensitivity corresponding to a resonant wavelength of 1.56 μm; This indicates the layer refractive index sensitivity corresponding to a resonant wavelength of 1.589 μm;

[0084] Δn1 represents the layer thickness sensitivity corresponding to a resonant wavelength of 1.589 μm; Δn1 represents the refractive index of the protein layer; Δa represents the layer thickness of the protein layer; Δn1 and Δa are quantities to be determined. It was determined in advance through experiments.

[0085] The ccSRR structure proposed in this invention exhibits both field enhancement and filtering effects. The following analysis and verification will focus on these two characteristics, and will also address... The determination process will be explained. The field enhancement effect can be measured using the field enhancement factor and sensitivity index. The filtering effect can be measured using the detection limit index.

[0086] First, the field enhancement factor of the electric field in the etched portion of the cCSRR structure was calculated, starting from the etched groove surface (sensing surface) up to a distance of 100 nm from the groove surface. Figure 7 The results are shown. The results indicate that the electric field is effectively confined to the sensing surface, where the enhancement factor is strongest. The enhancement factor decreases exponentially with distance from the sensing surface. The strongest electric field at the sensing surface is beneficial for the detection of analyte molecules because the thickness of the monolayer homogeneous biocluster is in the range of 4 nm to 12 nm, and a single protein molecule is only a few nanometers thick. The strongest electric field at the sensing surface facilitates the interaction between the field and the analyte molecules, thereby effectively enhancing sensitivity.

[0087] Secondly, the sensitivity of the volume solution refractive index (volume solution refers to the buffer solution within the sensing unit without a protein layer) was tested using an optical metasurface unit, and the results are as follows: Figure 8 As shown.

[0088] Figure 8 It is a dual-wavelength reflectance spectrum based on volume refractive index measurement. Figure 8 Figure (a) corresponds to the 1560nm resonant wavelength. Figure 8 Figure (b) corresponds to a resonant wavelength of 1589 nm; Figure 8 In the two figures, 1.331, 1.332, 1.333, 1.334, 1.335, and 1.336 represent refractive indices, with units of RIU. Testing the refractive index of a bulk solution using an optical metasurface unit refers to applying a solution without a biological forming layer on the sensing surface (i.e., without applying a solution containing biological substances such as amyloid protein), and measuring the refractive index of the liquid on the sensing surface. This allows for the measurement of bulk refractive index sensitivity, which characterizes the sensor's sensitivity and verifies its performance. Since sensitivity is an inherent parameter of the sensor and is independent of the analyte, a bulk solution is used for the test.

[0089] Since Tris buffer is commonly used to dissolve and dilute protein molecules, it can be used as a bulk solution for measuring bulk refractive index sensitivity. Bulk solution sensitivity (bulk refractive index sensitivity) is defined as the change in resonant wavelength caused by a change in refractive index (bulk refractive index), denoted as . Where λ represents the resonant wavelength, n s Indicates the volume refractive index, Indicates the sensitivity of the bulk solution.

[0090] according to Figure 8 The calculated volume refractive index sensitivity is 1162 nm / RIU, derived from... Figure 7 and Figure 8 Experimental results confirm that the ccSRR structure proposed in this embodiment of the invention has a good field enhancement effect.

[0091] Furthermore, the sensitivity of amyloid-forming layers composed of molecules with different configurations was tested using optical metasurface units, and the results are as follows: Figure 9 , Figure 10 As shown. Figure 9 This is the reflection spectrum at the 1560nm resonant wavelength. Figure 9 In Figures (a) and (b), the reflection spectra of the α-helical configuration components are shown. In Figure (a), the refractive index of the layer changes while the layer thickness remains constant, and each legend represents a different refractive index. In Figure (b), the layer thickness changes while the refractive index remains constant, and each legend represents a different thickness. Figure 9 In the diagrams (c) and (d), we see the reflection spectra of the β-sheet folded component molecules. In (c), the layer refractive index changes while the layer thickness remains constant, and in (d), the layer thickness changes while the layer refractive index remains constant.

[0092] Figure 10 This is the reflection spectrum at the 1589nm resonant wavelength. Figure 10 In the diagrams (a) and (b), we see the reflection spectra of the α-helical configuration component molecules. In (a), the layer refractive index changes while the layer thickness remains constant, and in (b), the layer thickness changes while the layer refractive index remains constant. Figure 10 In the diagrams (c) and (d), we see the reflection spectra of the β-sheet folded component molecules. In (c), the layer refractive index changes while the layer thickness remains constant, and in (d), the layer thickness changes while the layer refractive index remains constant.

[0093] Because wavelength interrogation is used, the sensitivity of the cambium is defined as the change in resonant wavelength caused by each change in refractive index (layer refractive index n1), denoted as... This is called the layer refractive index sensitivity, and the change in resonant wavelength caused by a change in the thickness of each biological layer (denoted as a) is denoted as... This is called layer thickness sensitivity. Because the biocambium sensitivity is independent of the biocambium being measured, the sensitivity of the optical metasurface to the biocambium can be defined using the cambium composed of bovine serum albumin molecules.

[0094] according to Figure 9 , 10 The calculated layer refractive index sensitivity is 0.5 nm / RIU, and the layer thickness sensitivity is 0.2 nm / nm. Therefore, following the above method, by calculating for resonant wavelengths of 1.56 μm and 1.589 μm respectively, we can obtain... For S42, the variation Δλ between the dual resonant wavelengths of 1.56 μm and 1.589 μm is... 1.56 and Δλ 1.589 and the results obtained from preliminary experiments Substituting into the above formula (1), the refractive index Δn1 and layer thickness Δa of the protein layer in the plurality of second subunits can be calculated.

[0095] Furthermore, the detection limit is determined by the resonance peak profile, spectrometer resolution, and noise during the detection process. Laboratory-grade infrared spectrometers can achieve a resolution of 1 pm. Noise primarily originates from temperature disturbances in the testing environment, typically around 10 fm. The calculated detection limits for amyloid molecules with α-helical and β-sheet fold configurations are 155 pg / mm². 2 and 88pg / mm 2 This confirms that the optical metasurface unit exhibits good filtering effect. For an explanation of the concept of detection limit, please refer to the relevant technical documentation; it will not be elaborated upon here.

[0096] S43. Based on the calculated refractive index and thickness of the protein layer, the density of the protein layer is calculated using the De Feijter formula.

[0097] The De Feijter formula is the existing calculation formula, expressed as follows: Where Γ is the layer density, a is the layer thickness, n1 is the layer refractive index, and n s Where dn is the bulk refractive index, dn / dc is the refractive index growth rate, and dn / dc is a constant that varies for different types of substances; for proteins, it is 0.186 cm⁻¹. 3 For details on the De Feijter formula, please refer to the prior art understanding.

[0098] S44, using the layer thickness and the density, characterize the one-dimensional structural information of the amyloid-forming layer to be tested.

[0099] The layer thickness and density can be used to characterize the one-dimensional structure of the amyloid layer formed by the amyloid molecules to be tested.

[0100] To facilitate understanding of the S4 processing procedure, the following exemplary description is provided.

[0101] Measuring the one-dimensional structure of amyloid layers typically involves dissolving and diluting amyloid proteins using Tris buffer. Therefore, before testing, Tris buffer is first added to the sensing region, specifically the etched region of the second subunit, to act as a bulk solution. Then, solutions of amyloid proteins with different molecular conformations are added sequentially. Amyloid standard reagents can be used for this experimental step. The reflectance spectrum is shown below. Figure 11 As shown. Figure 11 Figure (a) corresponds to a resonant wavelength of 1.56 μm, and Figure (b) corresponds to a resonant wavelength of 1.589 μm.

[0102] Figure 11 The spectrum clearly shows that the different resonant wavelength shifts caused by protein layers composed of molecules with different configurations (α-helices and β-sheet folds) are significant, indicating a strong interaction between the light waves and the protein layers, and different responses to protein layers composed of different components. This demonstrates high sensitivity and specificity for protein layer detection. Based on the dual-wavelength interrogation mechanism, the photogeometry parameters of the protein layers were obtained; the results are shown below. Figure 12 , Figure 12 Figure (a) shows the α-helical component molecules, and Figure (b) shows the β-sheet folded component molecules. The calculated values ​​are in high agreement with the published experimental results. The calculated values ​​for the α-helical protein layer are: refractive index 1.406 RIU, layer thickness 3.08 nm; the calculated values ​​for the β-sheet folded protein layer are: refractive index 1.383 RIU, layer thickness 9.53 nm. According to the De Feijter formula, the density of the protein layer composed of α-helical components is calculated to be 1200 pg / mm². 2 The density of the protein layer composed of β-sheet folding components is 2500 pg / mm². 2 .

[0103] S5, targeting a coupled complementary open-ended resonant ring operating in the mid-infrared band, uses the characteristic absorption peaks of molecules to identify the molecular configuration of the constituent molecules in the groove, thereby obtaining the structural information of the amyloid protein molecule to be tested.

[0104] S5 measures the infrared absorption peak of the constituent molecules within the groove corresponding to the first subunit, thereby obtaining molecular configuration information.

[0105] In one optional implementation, S5 may include:

[0106] S51, the incident light is used to excite the protein molecules in the first subunit, causing the component molecules of the protein layer in the first subunit to undergo vibrational rotational energy level transitions, and the corresponding absorption spectrum is obtained.

[0107] S52, Based on the position of the characteristic absorption peak in the absorption spectrum, identify the protein molecular configuration in the first subunit to obtain the molecular structure information of the amyloid protein to be tested.

[0108] The characteristic absorption peaks appear in the amide I and amide II bands; the molecular structure information of the amyloid protein to be tested includes α-helical amyloid protein molecules and β-sheet folded amyloid protein molecules.

[0109] α-helical amyloid molecules and β-lamellar folded amyloid molecules correspond to α-helical component molecules and β-lamellar folded component molecules, respectively.

[0110] Specifically, in the mid-infrared working region, light waves excite the component molecules on the sensing surface to undergo vibrational and rotational energy level transitions. The configuration of the component molecules can be identified by using the characteristic absorption peaks of characteristic functional groups in the absorption spectrum.

[0111] Amide band I (characteristic absorption peak at 6 μm) and amide band II (characteristic absorption peak at 6.5 μm) are characteristic absorption peaks for protein identification.

[0112] Figure 13 The absorption peaks of different component molecules are shown in Figure (a) for α-helical amyloid molecules and Figure (b) for β-lamellar amyloid molecules. It can be seen that both amide band I and amide band II are clearly visible in the spectra. For α-helical amyloid molecules, amide band I is at 6.02 μm and amide band II is at 6.5 μm; for β-lamellar amyloid molecules, amide band I is at 6.25 μm and amide band II is at 6.5 μm. It can be seen that the characteristic absorption peak values ​​are greater than 44% and significantly higher than surrounding peaks. To further verify the specificity of this optical metasurface unit, the absorption spectra of lipid molecules in the wavelength range of 6.0 μm to 6.5 μm were measured. The results are as follows... Figure 14 As shown, because the characteristic absorption peaks of fat molecules are not within the wavelength range mentioned above, neither amide band I nor amide band II appears in the spectrum. This indicates that the optical metasurface unit can specifically recognize protein molecules.

[0113] As can be seen, the molecular structure information of the amyloid protein to be tested can be obtained through S5, such as whether it is an α-helical amyloid protein molecule or a β-sheet folded amyloid protein molecule.

[0114] S6. Using the one-dimensional structural information of the amyloid-forming layer to be tested and the structural information of the amyloid molecule to be tested, determine the conformational analysis results of the amyloid protein to be tested and the relationship between the conformation of the protein forming layer and the protein component molecules.

[0115] This invention utilizes the one-dimensional structural information of the amyloid-forming layer and the structural information of the amyloid molecule to jointly characterize the conformational analysis results of the amyloid protein. The relationship between the protein-forming layer and the conformation of the protein component molecules refers to the unique mapping relationship between the protein-forming layer and the conformation of the protein component molecules of the same amyloid protein.

[0116] Existing label-free optical detection methods, particularly those based on refractive index measurement, suffer from low specificity, limiting their application to protein molecular formation layers. Spectroscopic detection methods also exhibit low sensitivity due to the weak interaction between mid-infrared light and constituent molecules. This invention utilizes an optical metasurface to combine refractive index and absorption light measurements, achieving both refractive index-based and molecular absorption spectroscopy-based measurements on the same sensing surface. This provides more comprehensive measurement information and enables more accurate protein conformation analysis. Furthermore, the optical metasurface units in this invention exhibit field enhancement effects, strengthening the interaction between the field and matter and improving sensitivity. Therefore, it achieves highly sensitive and specific protein molecular conformation measurements. Simultaneously, this invention determines the relationship between the one-dimensional structure of the amyloid formation layer and the protein molecular conformation, organically linking the layer structure with the molecular conformation. This lays the foundation for elucidating protein misfolding caused by conformational changes in amyloid.

[0117] In one optional embodiment, the optical detection method for amyloid protein conformation based on infrared multi-frequency resonance further includes:

[0118] The microenvironmental parameters of the amyloid protein solution to be tested are changed, and the changes in the conformation of the amyloid protein to be tested under the change of microenvironment are detected; wherein, the microenvironmental parameters include microenvironmental temperature, pH, and metal ion concentration; however, the microenvironmental parameters in the embodiments of the present invention are not limited to these.

[0119] Specifically, changes in the conformation of a certain amyloid protein can be determined by altering microenvironmental parameters such as pH, for example, a change from an α-helical conformation to a β-sheet conformation. Changes in the microenvironment can lead to conformational changes in amyloid proteins. Since measuring protein layer structure is relatively simple, this embodiment of the invention can obtain information on changes in the one-dimensional structure of the amyloid protein layer by measuring the drift of the resonant wavelength in the output spectrum. Based on this, it can be determined whether the protein molecule conformation has changed. If it has changed, the specific conformation can be further identified by the characteristic absorption peaks in the mid-infrared spectrum. This simplifies the measurement process and achieves more efficient and accurate measurements.

[0120] This approach not only allows for the simple and accurate acquisition of conformational changes in the amyloid protein molecules being tested, but also enables the identification of the correlation between the one-dimensional structure of the protein molecular layer and the conformation of the protein components, laying the foundation for elucidating the influence of changes in microenvironmental factors on the folding behavior of amyloid proteins.

[0121] Specifically, since changes in the one-dimensional structure of the amyloid-forming layer are easier to measure and identify, the process first involves detecting the one-dimensional structural information of the amyloid-forming layer to determine whether the molecular conformation has changed; then, the molecular conformation is identified using mid-infrared spectroscopy. Combining this with the obtained molecular conformations of the amyloid-forming layer and protein components, the changes in the structural information of the amyloid molecule are determined.

[0122] Because the embodiments of this invention can determine the conformational analysis results of the amyloid protein to be tested, the relationship between the conformational structure of the protein forming layer and the molecular conformation of the protein components, and the changes in the conformational structure of the protein molecules, it can not only be used for amyloid protein conformational measurement and analysis in the experimental environment, but also applied to the early clinical diagnosis of Alzheimer's disease. For example, amyloid protein can be extracted from the blood and cerebrospinal fluid of patients, and the optical detection of the amyloid protein conformation can be performed using the methods of the embodiments of this invention. Specifically, it can identify whether it is an α-helical conformational amyloid protein molecule or a β-sheet conformational amyloid protein molecule, and determine the mechanism of change from an α-helical conformational amyloid protein molecule to a β-sheet conformational amyloid protein molecule, thereby assisting in the diagnosis and treatment of neurodegenerative diseases.

[0123] Compared to traditional methods for measuring and analyzing amyloid protein conformation, the test structure employed in this invention primarily comprises periodically arranged optical metasurface units, enabling highly integrated and miniaturized on-chip experimental devices. Furthermore, based on the characteristics of optical signals, rapid measurements unaffected by external electromagnetic interference are possible. Moreover, this invention not only obtains static and steady-state structural information but is also suitable for monitoring dynamic changes in protein structure.

[0124] Compared to existing label-free optical detection methods, this invention utilizes a cCSRR metasurface to modulate the incident light field. On one hand, it greatly confines the light field to the sensing surface, effectively improving measurement sensitivity; on the other hand, it generates sharp resonance peaks based on the "filtering effect," effectively enhancing detection performance. Furthermore, it acquires rich information on biomolecular structures, including the one-dimensional structure of the biomolecular forming layer and the biomolecular configuration. In addition, the sensing structure is miniaturized, highly integrated, and simple to operate.

[0125] Both optical detection methods based on refractive index measurement and spectroscopic detection belong to label-free optical detection methods, but they measure different light signals. One converts biological reactions into optical refractive index, while the other converts them into optical absorbance. Therefore, the measured light signals are optical refractive index and optical absorbance, respectively. The inherent sensing structures of SPR and DPI technologies can only operate in the near-infrared wavelength range and can only support refractive index measurement. FTIR and SERS, based on their inherent sensing structures, can only operate in the mid-infrared band and can only detect optical absorbance. Because optical metasurfaces have a high degree of control over the light field, only by using optical metasurfaces can the two measurement methods be combined.

[0126] Achieving both near-infrared refractive index measurements and mid-infrared absorption spectroscopy measurements on the same sensing surface yields better results than using separate methods. This is because measuring on the same sensing surface ensures the consistency of the sample and eliminates measurement errors caused by variations in the sample itself. For example, when the sample is added to the sensing surface, all cCSRR structures aggregate at the same time the sample is added. However, if two techniques are used, i.e., adding the sample to different sensing surfaces, the unique correspondence between the biological layer and the constituent molecules cannot be guaranteed, leading to measurement errors. More importantly, it is difficult to accurately correlate the biological layer structure with the molecular structure, which is detrimental to monitoring the influence of the microenvironment on configurational changes.

[0127] The present invention provides an optical metasurface unit that enables both refractive index measurement and molecular absorption spectroscopy measurement on the same sensing surface. In the near-infrared band, a dual-wavelength interrogation mechanism can be used to detect the structure of the amyloid cambium layer; in the mid-infrared band, the conformation of the protein molecule can be determined based on the infrared absorption spectrum generated by specific molecular vibrational rotational energy level transitions. Based on the difference in the interaction characteristics between near-infrared / mid-infrared light waves and matter, the present invention combines optical refractive index measurement and optical absorption measurement to acquire information on the structure of the protein cambium layer and the conformation of its constituent molecules on the same sensing surface. This allows for convenient and efficient measurement of the conformation of protein molecules with a miniaturized measurement structure, achieving high sensitivity and high specificity. It organically links the one-dimensional structure of the biological cambium layer with the conformation of its constituent molecules, laying the foundation for elucidating protein misfolding caused by conformational changes in amyloid protein.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. An optical detection method for amyloid protein conformation based on infrared multi-frequency resonance, characterized in that, include: Obtain a pre-constructed optical metasurface unit; wherein, the optical metasurface unit comprises, from top to bottom, a metal aluminum layer, an aluminum oxide layer, a metal gold layer and a silicon substrate layer; the surface of the metal aluminum layer is formed with multiple pairs of coupled complementary open resonant rings by etching grooves, and the multiple pairs of coupled complementary open resonant rings operate in the near-infrared band and the mid-infrared band respectively; An amyloid protein solution to be tested is added to the optical metasurface unit, causing the amyloid protein solution to be tested to accumulate in the grooves corresponding to the multiple pairs of coupled complementary open resonant rings; Electromagnetic field excitation is achieved by irradiating the optical metasurface unit with incident light. For a coupled complementary open resonator operating in the near-infrared band, the refractive index and layer thickness of the protein-forming layer in the groove are detected, and the one-dimensional structural information of the amyloid protein-forming layer to be tested is obtained based on the obtained refractive index and layer thickness of the protein layer. For coupled complementary open resonant rings operating in the mid-infrared band, the characteristic absorption peaks of molecules are used to identify the molecular configuration of the components in the groove, thereby obtaining the structural information of the amyloid protein molecule to be tested. Using the one-dimensional structural information of the amyloid-forming layer and the structural information of the amyloid molecule, the conformational analysis results of the amyloid protein and the relationship between the conformation of the protein-forming layer and the protein component molecules are determined. The plurality of coupled complementary open-ended resonant rings include a first subunit operating in the mid-infrared band and a plurality of second subunits operating in the near-infrared band; the operating resonant frequencies of the first subunit and the second subunits do not interfere with each other; each subunit includes a pair of coupled complementary open-ended resonant rings of unequal size, spaced apart and arranged side by side along the length direction on the surface of the aluminum layer, with the opening direction of the two rings on the side away from the center of the two rings, and the two rings having their own inherent resonant frequencies and reaching the critical coupling condition; the length direction of the surface of the aluminum layer corresponds to the X direction, and the width direction corresponds to the Y direction; The first subunit is located in the central region of the surface of the aluminum metal layer; the plurality of second subunits are divided into two groups, located on one side of the surface of the aluminum metal layer corresponding to the wide side, and the plurality of second subunits on the same side are arranged periodically along the Y direction; the plurality of second subunits have the same size, and the size of each second subunit is smaller than the size of the first subunit.

2. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 1, characterized in that, The thicknesses of the aluminum layer, the aluminum oxide layer, the gold layer, and the silicon substrate are all 0.1 mm. 0.25 0.6 and 725 .

3. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 2, characterized in that, For the aforementioned optical metasurface unit, the length of the aluminum layer surface 14 ,width 5 ; In the first subunit, the radius of the large ring It is 1.49 The radius of the small ring It is 1.34 The width of the two rings Both are 0.37 The opening width of the two rings Both are 0.24 The coupling spacing between the two rings It is 0.4 The period length of the first subunit Corresponding to the length of the surface of the aluminum metal layer The periodic width of the first subunit corresponds to the width of the surface of the aluminum layer. ; In the second subunit, the radius of the large ring It is 0.31 The radius of the small ring It is 0.25 The width of the two rings Both are 0.13 The opening width of the two rings Both are 0.1 The coupling spacing between the two rings It is 0.08 The period length of the second subunit It is 2.33 Period width It is 1.2 .

4. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 1, characterized in that, The amyloid protein solution to be tested is prepared by dissolving and diluting the amyloid protein to be tested using Tris buffer.

5. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 1, characterized in that, The incident light is directed in the Z direction, and the electric field is polarized in the X direction.

6. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to any one of claims 1 to 5, characterized in that, The method involves detecting the refractive index and thickness of the protein-forming layer within the groove of a coupled complementary open-loop resonator operating in the near-infrared band. Based on the obtained refractive index and thickness of the protein layer, one-dimensional structural information of the amyloid protein-forming layer to be tested is obtained, including: The electromagnetic field generated by the dual-resonance wavelength characteristics of the incident light after being modulated by the optical metasurface unit is applied to the protein layer in a number of periodically arranged second subunits to detect the change in the dual-resonance wavelength. Based on the change in the dual resonant wavelength, the layer refractive index sensitivity and layer thickness sensitivity measured in advance based on each resonant wavelength, the refractive index and layer thickness of the protein layer in the periodically arranged multiple second subunits are calculated. Based on the calculated refractive index and thickness of the protein layer, the density of the protein layer is calculated using the De Feijter formula. The layer thickness and layer density are used to characterize the one-dimensional structural information of the amyloid protein forming layer to be tested.

7. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 6, characterized in that, The dual resonant wavelengths are 1.56 nm and 1.56 nm respectively. and 1.589 .

8. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 7, characterized in that, The aforementioned coupled complementary open-loop resonator operating in the mid-infrared band utilizes characteristic absorption peaks to identify the molecular configuration within the groove, thereby obtaining structural information of the amyloid protein molecule to be tested, including: The incident light is used to excite the protein molecules in the first subunit, causing the component molecules of the protein layer in the first subunit to undergo vibrational and rotational energy level transitions, and the corresponding absorption spectrum is obtained. Based on the positions of characteristic absorption peaks in the absorption spectrum, the protein molecular conformation in the first subunit is identified, and the molecular structural information of the amyloid protein to be tested is obtained; wherein, the characteristic absorption peaks appear in the amide I band and the amide II band; the molecular structural information of the amyloid protein to be tested includes Helical amyloid protein molecules and Laminar folded configuration amyloid protein molecule.

9. The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance according to claim 8, characterized in that, The optical detection method for amyloid protein conformation based on infrared multi-frequency resonance also includes: The microenvironmental parameters of the amyloid protein solution to be tested are changed, and the changes in the conformation of the amyloid protein to be tested under the change of microenvironment are detected; wherein, the microenvironmental parameters include microenvironmental temperature, pH, and metal ion concentration.

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