Isotropic metasurface for ultrasensitive detection of extracellular vesicles

Abstract

The invention discloses an isotropic metasurface for ultrasensitive detection of extracellular vesicles. A square unit cell in the isotropic metasurface is composed of a pair of orthogonal I-shaped protruding structures and four C-shaped split ring resonators. The optical metasurface adopts an isotropic metasurface structure design, and the structure shows the same response to x and y polarization incident light and generates isotropic resonance response. An annular dipole with characteristic end-to-end configuration is generated through interaction between the induction magnetic dipoles, and the annular mode is remarkably enhanced. The optical metasurface can be used for carrying out ultra-sensitive detection on extracellular vesicles, the correlation R2 is 0.962, and the LoD of the extracellular vesicles is 5 / mu L.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to an isotropic metasurface for ultrasensitive detection of extracellular vesicles. Background Art

[0002] Liquid biopsy is an accompanying technology in the field of tumor detection. Extracellular vesicles (EVs), characterized by appearing in the early stage of tumors and carrying tumor-related markers, have become circulating markers with great clinical diagnostic value. Extracellular vesicles (EVs) are a general term for nanoparticles with a phospholipid bilayer structure and a size ranging from nanometers to micrometers actively secreted by cells. EVs carry substances such as proteins, lipids, and nucleic acids from the parent cells to the recipient cells and cause corresponding functional responses, realizing information interaction with the external microenvironment and directly affecting processes such as tumor cell invasion and distant metastasis. Traditional EV separation methods include density gradient centrifugation, ultracentrifugation, size exclusion chromatography, etc., and detection methods include Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy, etc. However, due to the small diameter of EVs and the complexity of the plasma system, the separation and detection of EVs often rely on large instruments, which are time-consuming and cannot be adapted to clinical application scenarios, limiting the practical application of EVs in clinical scenarios.

[0003] Optical metasurface is a highly sensitive, small-sized, and fast-responsive optical sensing technology. Based on the absorption characteristics of the analyte for light of different wavelengths, the spectrum can be analyzed to determine the absorption of light of different wavelengths in the sample.

[0004] Currently, there is no report on the research of using optical metasurface to detect low-concentration extracellular vesicles. Summary of the Invention

[0005] In order to solve the above technical problems and achieve ultrasensitive detection of extracellular vesicles, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides an isotropic metasurface for ultrasensitive detection of extracellular vesicles. The isotropic metasurface includes one or more than two metasurface units. The metasurface unit includes a substrate and a square unit cell. The square unit cell is composed of a pair of orthogonal I-shaped protruding structures on the substrate surface and four protruding C-shaped split-ring resonators.

[0007] Preferably, the four C-shaped split-ring resonators are symmetrically arranged around the intersection point of the I-shaped protruding structures.

[0008] Preferably, the distance between the outer side lines of two opposite C-shaped split ring resonators is 2250 to 3400 nm, for example: 2250 nm, 2350 nm, 2450 nm, 2550 nm, 2675 nm, 2775 nm, 2875 nm, 2900 nm, 3000 nm, 3150 nm, 3275 nm, 3400 nm.

[0009] Furthermore, the distance between the top of the C-shaped split ring resonator and the top of the I-shaped protruding structure is 300 to 450 nm, for example: 300 nm, 325 nm, 350 nm, 368 nm, 400 nm, 450 nm.

[0010] Preferably, the length of the I-shaped protruding structure is 1000 to 1500 nm, for example: 1000 nm, 1050 nm, 1200 nm, 1265 nm, 1300 nm, 1350 nm, 1400 nm, 1465 nm, 1500 nm.

[0011] Preferably, the end width of the I-shaped protruding structure is 500 to 750 nm, for example: 500 nm, 525 nm, 550 nm, 600 nm, 625 nm, 655.5 nm, 700 nm, 725 nm, 750 nm.

[0012] Preferably, the width of the I-shaped protruding structure is 100 to 150 nm, for example: 100 nm, 110 nm, 120 nm, 130 nm, 138 nm, 140 nm, 145 nm, 150 nm.

[0013] Preferably, the outer width of the C-shaped split ring resonator is 1000 to 1500 nm, for example: 1000 nm, 1050 nm, 1200 nm, 1265 nm, 1300 nm, 1350 nm, 1400 nm, 1465 nm, 1500 nm.

[0014] Preferably, the inner width of the C-shaped split ring resonator is 800 to 1200 nm, for example: 800 nm, 825 nm, 850 nm, 900 nm, 950 nm, 989 nm, 1000 nm, 1050 nm, 1100 nm, 1150 nm, 1200 nm.

[0015] Preferably, the thickness of the I-shaped protruding structure and the C-shaped split ring resonator is 90 to 120 nm, for example: 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm.

[0016] Preferably, the substrate material is CaF 2A substrate, wherein the surfaces of the I-shaped protruding structure and the C-shaped split-ring resonator are gold films.

[0017] Furthermore, between the substrate and the gold film is a titanium (Ti) adhesion layer.

[0018] Preferably, the preparation process of the isotropic annular optical metasurface is as follows: The I-shaped protruding structure and the C-shaped split-ring resonator are lithographed on a CaF 2 substrate by electron beam lithography. Then, a Ti adhesion layer is deposited by magnetron sputtering, and then a gold film (Au) is deposited.

[0019] In a second aspect, the present invention provides a detection device for extracellular vesicles, which includes the isotropic metasurface described in the first aspect.

[0020] Preferably, the extracellular vesicles are derived from any one of cell supernatant, gastric juice, blood, urine, breast milk, saliva or cerebrospinal fluid, and more preferably cell supernatant.

[0021] Furthermore, the cell supernatant is the supernatant of AGS cells.

[0022] In a third aspect, the present invention provides the application of the isotropic metasurface described in the first aspect in detecting extracellular vesicles.

[0023] Preferably, the extracellular vesicles are derived from any one of cell supernatant, gastric juice, blood, urine, breast milk, saliva or cerebrospinal fluid, and more preferably cell supernatant.

[0024] Furthermore, the cell supernatant is the supernatant of AGS cells.

[0025] Furthermore, the isotropic metasurface is used to detect the resonance frequency shift data of the transmittance of AGS extracellular vesicles.

[0026] Advantages of the present invention:

[0027] 1. The optical metasurface of the present invention adopts an isotropic metasurface structure design. This structure shows the same response to x- and y-polarized incident light, generating an isotropic resonance response. The interaction between induced magnetic dipoles generates an annular dipole with a characteristic head-to-tail configuration, significantly enhancing the annular mode.

[0028] 2. The metasurface of the present invention is used to detect the resonance frequency shift data of the transmittance of AGS EVs, achieving ultrasensitive detection of extracellular vesicles, with an R 2 of 0.962 and a LoD of extracellular vesicles of 5 particles / μL. Description of the Drawings

[0029] Figure 1The square unit cell structure in the isotropic metasurface is shown. W represents the length outside the I-shaped protruding structure and the C-shaped split-ring resonator. a represents the end width of the I-shaped protruding structure. s represents the width of the I-shaped protruding structure and the C-shaped split-ring resonator. L represents the distance between the outer side lines of two opposite C-shaped split-ring resonators. g represents the distance between the top of the C-shaped split-ring resonator and the top of the I-shaped protruding structure.

[0030] Figure 2 The characterization of the isotropic metasurface is shown. A is the current distribution generated by the toroidal dipole. B is the transmittance of the metasurface with only internal (red) and external (blue) structures. C is the transmittance of the simulated and measured isotropic toroidal metasurface. D is the radiation power of the induced electric dipole moment (blue) and toroidal dipole moment (red) in the isotropic toroidal metasurface. E is the simulated transmittance of the isotropic toroidal metasurface coated with a cladding layer. F is the dependence of the toroidal resonance splitting Δλ on the Δε perturbation.

[0031] Figure 3 The performance of the isotropic metasurface in EVs detection is shown. A is a schematic diagram of the metasurface detection process. B is the transmittance spectral shift of the air, PBS, MB, and EVs samples detected. C is the transmittance spectral shift detected for different concentrations of EVs samples. D is the LoD curve fitted according to the concentration gradient of EVs in the transmittance spectral shift. Detailed implementation mode

[0032] The technical solution of the present invention will be further described below in conjunction with the embodiments and the drawings. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention.

[0033] Example 1 Preparation of the isotropic metasurface

[0034] According to Figure 1 the design, an optimized mid-infrared toroidal metasurface was constructed by electron beam lithography and lift-off on a CaF 2 substrate. A 5-nm Ti adhesion layer was deposited by magnetron sputtering technology, and then 100 nm of gold (Au) was deposited to prepare the metal film. The lift-off process was carried out in an acetone solution. The relevant size data of the optical metasurface are as follows: the length of the I-shaped protruding structure is 1265 nm, the end width of the I-shaped protruding structure is 655.5 nm, and the width of the I-shaped protruding structure is 138 nm. The outer width of the C-shaped split-ring resonator is 1265 nm, and the inner width of the C-shaped split-ring resonator is 989 nm. The distance between the outer side lines of two opposite C-shaped split-ring resonators is 2875 nm, and the distance between the top of the C-shaped split-ring resonator and the top of the I-shaped protruding structure is 368 nm.

[0035] As Figure 2As shown in A, under y - polarized illumination, the currents in the two C - shaped metal rings oscillate in opposite directions, indicating that the magnetic dipoles are out of phase.

[0036] As Figure 2 Figure B shows the spectral responses of the metasurfaces with only orthogonal I - shaped CWs or only four C - shaped SRRs. The metasurface with only orthogonal I - shaped CWs shows a symmetric Lorentz - type resonance dip at 5.62 μm, while the metasurface with only C - shaped SRRs shows another dip at 6.91 μm. The corresponding current distributions reveal the electric - dipole - dominant mode in the I - shaped CWs and two mirror - symmetric magnetic - dominant modes in the C - shaped SRRs.

[0037] As Figure 2 As shown in C, when the orthogonal I - shaped protruding structure and four C - shaped open resonators are combined, this coupled structure generates a sharp Fano - type resonance at 7.78 μm. The simulated curve shape of the isotropic annular metasurface transmittance is similar to the actual detection result, indicating that the isotropic annular metasurface meets the design requirements.

[0038] As Figure 2 As shown in D, the electric dipole (Py) shows strong scattering over the entire wavelength range, indicating that the y - polarized electric field excites the metasurface. Near the Fano resonance wavelength, the toroidal dipole (Ty) shows a significant enhancement, reflecting the strong excitation of the toroidal mode within the metasurface. At 7.67 μm, Ty becomes the main contributor, even exceeding Py. The interference between Py and Ty results in an asymmetric Fano - type spectrum, causing the resonance to drop sharply.

[0039] As Figure 2 As shown in E and Figure 2 F, by calculating the transmission spectra of the metasurfaces with different dielectric - constant coatings, it can be seen that as the dielectric constant increases from 1 to 2, the toroidal resonance dip shifts from 7.67 μm to 8.69 μm, indicating that the metasurface has a high sensing ability for the surrounding environment.

[0040] Figure 2 The results show that the isotropic metasurface sets periodic boundary conditions along the x and y directions to simulate an infinite - period array, and a perfectly matched layer is set along the z direction. Therefore, when a y - polarized plane wave is vertically incident on the isotropic annular metasurface, the toroidal mode enhances the sensing performance of the light wave.

[0041] Example 2: Detection of extracellular vesicles by an isotropic metasurface

[0042] 1. Preparation of AGS EVs

[0043] In this experiment, the gastric cancer cell line AGS was prepared by culturing in DMEM F12 medium, antibiotics, and exosome-free serum. After reaching 100% cell density, the cell supernatant was collected and subjected to ultracentrifugation (UC) at 12,000 g for 70 minutes. Then the AGS EVs particles were suspended in PBS solution and stored at -80 °C for subsequent processing.

[0044] 2. Preparation of magnetic beads

[0045] 2.1 Take DSPE-PEG2000-Biotin powder (ruixibio R-0040) and dissolve it in filtered PBS solution to obtain a DSPE-PEG2000-Biotin solution with a concentration of 0.5 mg / mL.

[0046] 2.2 Take magnetite beads with a diameter of 10 nm (MB, Xianfeng Nano, 104090) and prepare a magnetite bead solution with a concentration of 1 mg / mL.

[0047] 2.3 Mix the magnetite bead solution and the DSPE-PEG2000-Biotin solution thoroughly at room temperature for 1 hour, and wash twice with PBS solution to obtain the magnetic bead solution.

[0048] 3. Treatment of EVs detection samples

[0049] Take 1 μL of the EVs sample and dissolve it in 10 μL of PBS solution, then add it to the mixture of DSPE-MB, mix thoroughly at room temperature for 30 minutes to obtain the EVs detection sample, and store it at 4 °C.

[0050] 4. Sample detection and analysis

[0051] 4.1 Apply 3 μL of PBS solution, 3 μL of magnetic bead solution, and 3 μL of EVs detection sample to the metasurface device respectively, and magnetically adsorb for about 2 minutes. Remove the excess water and air-dry naturally, with air as the blank control. The samples were subjected to FTIR detection using a spectrometer (LUMOS II, Bruker, Germany) equipped with transmission (TR), and the spectra were recorded in the range of 600 - 4000 cm -1 and scanned 32 times at a resolution of 4 cm -1 .

[0052] 4.2 Adjust the concentration of the EVs detection sample to 10 7 particles / mL, 10 9 particles / mL, 10 11 particles / mL respectively. Take 3 μL of the EVs detection sample with different concentrations and 3 μL of magnetic bead solution (blank control), and perform detection according to the method in 4.1.

[0053] 4.3 Plotting the Detection Limit Curve

[0054] Adjust the concentrations of the EVs detection samples to 10 5 particles / mL, 10 6 particles / mL, 10 7 particles / mL, 10 8 particles / mL, 10 9 particles / mL, 10 10 particles / mL, 10 11 particles / mL respectively. Take the EVs detection samples with different concentrations and perform the detection according to the method in 4.1 to obtain the transmittance resonance frequency shift values. Plot a curve with the frequency shift value as the ordinate and the EVs concentration as the abscissa ( Figure 3 D).

[0055] Calculation formula for the limit of detection (LoD): LoD = 3.3σ / S, where σ represents the standard deviation of the blank response value and S represents the slope of the standard curve.

[0056] As Figure 3 shown, DSPE was incorporated into the EVs phospholipid bilayer through MB, realizing the specific detection of EVs samples. The captured EVs were concentrated in the metasurface response region through magnetic separation and analyzed after removing the excess moisture ( Figure 3 A). Fourier transform infrared spectroscopy microscopy was used to analyze the samples in the mid-infrared range and measure their absorbance and transmittance at specific wavelengths.

[0057] As Figure 3 shown in B, the blank control, PBS solution, magnetic bead solution, and AGS EVs samples were detected separately to evaluate the metasurface EVs detection performance. Different substances will cause refractive index changes, thus affecting the response results.

[0058] As Figure 3 shown in C, substances with different refractive indices will cause obvious resonance peak shifts in the spectrum.

[0059] As Figure 3 shown in D, the resonance wavelength shift has a strong linear correlation with different EVs concentration gradients, and the correlation coefficient R 2 is 0.962. The limit of detection (LoD) is calculated to be 5 particles / μL.

[0060] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An isotropic metasurface for ultrasensitive detection of extracellular vesicles, characterized in that: The isotropic metasurface includes one or more metasurface units, and the metasurface unit includes a substrate and a square unit cell. The square unit cell is composed of a pair of orthogonal I-shaped protruding structures on the surface of the substrate and four protruding C-shaped open ring resonators.

2. The isotropic metasurface according to claim 1, characterized in that: The C-shaped split ring resonator is symmetrically arranged around the intersection of the I-shaped protruding structure.

3. The isotropic metasurface according to claim 2, characterized in that: The distance between the outer side lines of the two opposite C-shaped open ring resonators is 2875 nm, and the distance between the top of the C-shaped open ring resonator and the top of the I-shaped protruding structure is 266-400 nm.

4. The isotropic metasurface according to any one of claims 1 to 3, characterized in that: The length of the I-shaped protruding structure is 1000-1500 nm, the width of the end of the I-shaped protruding structure is 500-750 nm, and the width of the I-shaped protruding structure is 100-150 nm.

5. The isotropic metasurface according to any one of claims 1 to 3, characterized in that: The outer width of the C-shaped open ring resonator is 1000-1500 nm, and the inner width of the C-shaped open ring resonator is 800-1200 nm.

6. The isotropic metasurface according to any one of claims 1 to 3, characterized in that: The thickness of the I-type protrusion structure and the C-shaped open ring resonator is 90-120 nm.

7. The isotropic metasurface according to any one of claims 1 to 3, characterized in that: The substrate material is a CaF2 substrate, and the surfaces of the I-type protruding structure and the C-shaped open ring resonator material are gold films.

8. The isotropic metasurface according to claim 7, characterized in that: A titanium adhesion layer is provided between the substrate and the gold film.

9. A device for detecting extracellular vesicles, characterized in that: The device comprises the isotropic metasurface described in any one of claims 1-8.

10. Use of the isotropic supersurface according to any one of claims 1 to 8 in the preparation of a product for detecting extracellular vesicles.

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