An isotropic metasurface for ultrasensitive detection of extracellular vesicles

Abstract

This invention discloses an isotropic metasurface for ultrasensitive detection of extracellular vesicles. The square unit cell of the isotropic metasurface consists of a pair of orthogonal I-shaped protrusions and four C-shaped open-ring resonators. The optical metasurface of this invention employs an isotropic metasurface structure design, which exhibits the same response to x- and y-polarized incident light, generating an isotropic resonance response. The interaction between induced magnetic dipoles generates a ring dipole with a characteristic head-and-tail configuration, significantly enhancing the ring mode. This optical metasurface enables ultrasensitive detection of extracellular vesicles, with a correlation R... 2 The value was 0.962, and the LoD of extracellular vesicles was 5 / μL.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an isotropic metasurface for ultrasensitive detection of extracellular vesicles. Background Technology

[0002] Liquid biopsy is an accompanying technology in the field of tumor detection. Extracellular vesicles, characterized by their early appearance in tumors and carrying tumor-related biomarkers, have become circulating biomarkers with significant clinical diagnostic value. Extracellular vesicles (EVs) are a collective term for particles ranging in size from nanometers to micrometers, actively secreted by cells and possessing a phospholipid bilayer structure. EVs carry proteins, lipids, nucleic acids, and other substances derived from the mother cell to recipient cells, triggering corresponding functional responses and facilitating information exchange with the external microenvironment. This directly influences processes such as tumor cell invasion and distant metastasis. Traditional methods for EV separation include density gradient centrifugation, ultracentrifugation, and size exclusion chromatography. Detection methods include Western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy. However, the small diameter of EVs and the complexity of the plasma system often lead to EV separation and detection relying on large instruments, which are time-consuming and unsuitable for clinical applications, limiting the practical application of EVs in clinical settings.

[0003] Optical metasurfaces are a highly sensitive, small-sized, and fast-response optical sensing technology. Based on the absorption characteristics of the analyte for light of different wavelengths, the absorption of light of different wavelengths in the sample can be determined by analyzing the spectrum.

[0004] Currently, there are no reports on the use of optical metasurfaces to detect low concentrations of extracellular vesicles. Summary of the Invention

[0005] To address the aforementioned technical problems and achieve ultrasensitive detection of extracellular vesicles, this invention provides the following technical solution.

[0006] In a first aspect, the present invention provides an isotropic metasurface for ultrasensitive detection of extracellular vesicles, the isotropic metasurface comprising one or more metasurface units, the metasurface unit comprising a substrate and a square cell, the square cell comprising a pair of orthogonal I-shaped protrusions on the surface of the substrate and four protruding C-shaped open-ring resonators.

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

[0008] Preferably, the distance between the outer edges of the two opposite C-shaped open-ring resonators is 2250–3400 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 open ring resonator and the top of the I-shaped protruding structure is 300-450nm, for example: 300nm, 325nm, 350nm, 368nm, 400nm, 450nm.

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

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

[0012] Preferably, the width of the I-shaped protrusion is 100-150 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 open-ring resonator is 1000-1500nm, for example: 1000nm, 1050nm, 1200nm, 1265nm, 1300nm, 1350nm, 1400nm, 1465nm, 1500nm.

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

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

[0016] Preferably, the substrate material is a CaF2 substrate, and the surfaces of the I-type protrusion structure and the C-shaped open-ring resonator are gold films.

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

[0018] Preferably, the fabrication process of the isotropic ring optical metasurface is as follows: the I-type protrusion structure and the C-shaped open ring resonator are etched on a CaF2 substrate using electron beam photolithography. Then, a Ti adhesion layer is deposited using magnetron sputtering, followed by the deposition of a gold film (Au).

[0019] In a second aspect, the present invention provides a device for detecting extracellular vesicles, the device comprising 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 from cell supernatant.

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

[0022] Thirdly, the present invention provides the application of the isotropic metasurface described in the first aspect in the detection of 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 from cell supernatant.

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

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

[0026] The beneficial effects of this invention are:

[0027] 1. The optical metasurface of this invention employs an isotropic metasurface structure design, which exhibits the same response to x- and y-polarized incident light, generating an isotropic resonant response. The interaction between the induced magnetic dipoles produces a ring dipole with a characteristic head-to-tail configuration, significantly enhancing the ring mode.

[0028] 2. The metasurface of this invention is used to detect the resonant frequency shift data of the transmittance of AGS EVs, achieving ultrasensitive detection of extracellular vesicles. 2 The value was 0.962, and the LoD of extracellular vesicles was 5 / μL. Attached Figure Description

[0029] Figure 1The diagram shows a square cell structure in an isotropic metasurface. W represents the length of the outer side of the I-shaped protrusion and the C-shaped open-ring resonator, a represents the end width of the I-shaped protrusion, s represents the width of the I-shaped protrusion and the C-shaped open-ring resonator, L represents the distance between the outer edges of the two opposite C-shaped open-ring resonators, and g represents the distance between the top of the C-shaped open-ring resonator and the top of the I-shaped protrusion.

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

[0031] Figure 3 The diagram shows the performance of isotropic metasurfaces in EV detection; A is a schematic diagram of the metasurface detection process; B is the transmission spectrum shift of air, PBS, MB and EV samples; C is the transmission spectrum shift of EV samples with different concentrations; D is the LoD curve fitted based on the concentration gradient of EVs in the transmission spectrum shift. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0033] Example 1: Preparation of Isotropic Metasurfaces

[0034] according to Figure 1 An optimized mid-infrared toroidal metasurface was constructed using electron beam lithography and lift-off from a CaF2 substrate. A 5 nm Ti adhesion layer was deposited via magnetron sputtering, followed by a 100 nm gold (Au) film. The lift-off process was performed in acetone solution. The relevant dimensional data for the optical metasurface are as follows: the length of the I-shaped protrusion is 1265 nm, the end width of the I-shaped protrusion is 655.5 nm, and the width of the I-shaped protrusion is 138 nm. The outer width of the C-shaped open-ring resonator is 1265 nm, and the inner width of the C-shaped open-ring resonator is 989 nm. The distance between the outer edges of two opposing 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 protrusion is 368 nm.

[0035] like Figure 2As shown in Figure 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] like Figure 2 B shows the spectral responses of metasurfaces with only orthogonal I-shaped CWs or only four C-shaped SRRs. The metasurface with only orthogonal I-shaped CWs exhibits a symmetrical Lorentz-type resonance tilt at 5.62 μm, while the metasurface with only C-shaped SRRs exhibits another tilt at 6.91 μm. The corresponding current distributions reveal an electric dipole-dominated mode in the I-shaped CWs and two mirror-symmetric magnetic-dominated modes in the C-shaped SRRs.

[0037] like Figure 2 As shown in Figure C, when the orthogonal I-shaped protrusion structure is combined with four C-shaped open resonators, this coupling structure produces a sharp Fano-type resonance at 7.78 μm. The simulated curve of the transmittance of the isotropic toroidal metasurface has a shape similar to the actual test results, indicating that the isotropic toroidal metasurface meets the design requirements.

[0038] like Figure 2 As shown in Figure D, the electric dipole (Py) exhibits strong scattering across 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 dominant contributor, even surpassing Py. The interference between Py and Ty results in an asymmetric Fano-type spectrum, causing a sharp drop in resonance.

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

[0040] Figure 2 The results show that the isotropic metasurface, with periodic boundary conditions along the x and y directions to simulate an infinite periodic array, and a perfectly matched layer along the z direction, exhibits enhanced sensing performance of the light wave when a y-polarized plane wave is incident perpendicularly on the isotropic toroidal metasurface, with the toroidal mode enhancing the light wave's sensing performance.

[0041] Example 2: Detection of extracellular vesicles using isotropic metasurfaces

[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 ultracentrifuged at 12000g (UC) for 70 minutes. The AGS EVs particles were then resuspended in PBS solution and stored at -80°C for subsequent processing.

[0044] 2. Preparation of magnetic beads

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

[0046] 2.2 Take 10 nm diameter iron oxide magnetic beads (MB, Xianfeng Nano, 104090) and prepare a 1 mg / mL iron oxide magnetic bead solution.

[0047] 2.3 Mix the trioxide magnetic bead solution with 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. Sample processing for EVs detection

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

[0050] 4. Sample testing and analysis

[0051] 4.1 Apply 3 μL of PBS solution, 3 μL of magnetic bead solution, and 3 μL of EVs sample to the metasurface device, respectively, and allow for magnetic adsorption for approximately 2 minutes. Remove excess water and allow to air dry naturally, using air as a blank control. Perform FTIR detection on the samples using a LUMOS II spectrometer equipped with a transmission (TR) spectrometer (Bruker, Germany), recording spectra in the range of 600–4000 cm⁻¹. -1 Within a range of 4cm -1 The resolution was measured in 32 scans.

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

[0053] 4.3 Plotting the detection limit curve

[0054] The concentration of the EVs samples was adjusted 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, samples of EVs at different concentrations were taken and analyzed according to method 4.1 to obtain the transmittance resonant frequency shift value. A curve was plotted with the frequency shift value on the ordinate and the EVs concentration on the abscissa. Figure 3 D).

[0055] The limit of detection (LoD) is calculated as follows: 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] like Figure 3 As shown, specific detection of EV samples was achieved by incorporating DSPE into the phospholipid bilayer of EVs using MB. The captured EVs were concentrated in the metasurface response region by magnetic separation, and analyzed after removing excess moisture. Figure 3 A). Fourier transform infrared spectroscopy is used to analyze samples in the mid-infrared range, measuring their absorbance and transmittance at specific wavelengths.

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

[0058] like Figure 3 As shown in C, substances with different refractive indices will cause a significant shift in the resonance peak in the spectrum.

[0059] like Figure 3 As shown in D, the resonant wavelength shift is strongly linearly correlated with different EV concentration gradients, with a correlation coefficient R. 2 = 0.962. The limit of detection (LoD) is calculated as 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 scope of the technical concept of the present invention, various simple modifications can be made to the technical solution 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, each metasurface unit including a substrate and a square cell. The square cell consists of a pair of orthogonal I-shaped protrusions on the surface of the substrate and four protruding C-shaped open-ring resonators. The C-shaped open-ring resonators are symmetrically arranged around the intersection of the I-shaped protrusions. The length of the I-shaped protrusion is 1000~1500 nm, the end width of the I-shaped protrusion is 500~750 nm, and the width of the I-shaped protrusion is 100~150 nm. 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.

2. The isotropic metasurface according to claim 1, characterized in that, The distance between the outer edges of the two opposing 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.

3. The isotropic metasurface according to any one of claims 1-2, characterized in that, The thickness of the I-shaped protruding structure and the C-shaped open-ring resonator is 90~120 nm.

4. The isotropic metasurface according to any one of claims 1-2, characterized in that, The substrate material is a CaF2 substrate, and the surfaces of the I-shaped protrusion structure and the C-shaped open ring resonator material are gold films.

5. The isotropic metasurface according to claim 4, characterized in that, A titanium adhesion layer is formed between the substrate and the gold film.

6. A device for detecting extracellular vesicles, characterized in that, The device includes the isotropic metasurface as described in any one of claims 1-5.

7. The use of the isotropic metasurface according to any one of claims 1-5 in the preparation of products for detecting extracellular vesicles.

Images