Terahertz metasurface sensor for multiple detection of tumor markers and application of terahertz metasurface sensor

Abstract

The invention belongs to the field of terahertz, and particularly relates to a terahertz metasurface sensor for multiple detection of tumor markers and application of the terahertz metasurface sensor. The terahertz metasurface sensor comprises a microstructure unit array and a substrate layer, and the microstructure unit array is located on the substrate layer. Gold nanoparticles with different particle sizes are fixed on the surface of the terahertz metasurface sensor, and the gold nanoparticles with different particle sizes are respectively coated with binding reagents for specifically recognizing different tumor markers.

Description

Technical Field

[0001] The present invention belongs to the field of terahertz, and in particular relates to a terahertz metasurface sensor for multiple detection of tumor markers and its application. Background Art

[0002] In recent years, with the continued rise in cancer morbidity and mortality, early diagnosis and accurate detection of cancer have become critical issues in medical research and clinical applications. Numerous studies have demonstrated that serum tumor markers (such as AFP, CEA, CA125, and CA199) have important monitoring and diagnostic value in the development and progression of tumors. Therefore, developing a highly sensitive and specific detection technology platform capable of simultaneously detecting multiple tumor markers is crucial for early screening and personalized treatment of cancer.

[0003] Traditional tumor marker detection methods mainly include ELISA, electrochemical detection, fluorescence immunoassay, and mass spectrometry. Although these methods have certain sensitivity and reliability, they generally suffer from complex operation, long detection cycles, high costs, and difficulty in achieving simultaneous multi-marker detection, which limits their application in large-scale screening and point-of-care diagnosis.

[0004] Terahertz (THz) electromagnetic waves, located between microwaves and infrared, possess advantages such as non-ionization, strong penetration into biological tissue, and distinct spectral response characteristics. They have been widely studied for bio-component identification and disease diagnosis. Furthermore, terahertz metasurfaces, due to their highly designable structures and significant resonance enhancement effects, can effectively enhance the interaction between THz waves and trace substances, making them a key development direction in the field of THz sensing.

[0005] However, terahertz metasurface sensors have difficulty in simultaneously detecting multiple markers, which may require complex structural design and limit their applications. Therefore, there is an urgent need to develop a new type of terahertz metasurface structure sensor that can quickly and simultaneously detect multiple tumor markers in the same sample with high sensitivity, high specificity and good stability. Summary of the Invention

[0006] In order to solve the above problems, in a first aspect, the present invention provides a terahertz metasurface sensor for multiple detection of tumor markers, characterized in that the terahertz metasurface sensor comprises a microstructure unit array and a substrate layer, wherein the microstructure unit array is located on the substrate layer; and gold nanoparticles of different particle sizes are fixed on the surface of the terahertz metasurface sensor, and the gold nanoparticles of different particle sizes are respectively coated with binding reagents that specifically recognize different tumor markers.

[0007] Furthermore, the gold nanoparticles of different particle sizes include gold nanoparticles with particle sizes of 10 nm and 40 nm.

[0008] Furthermore, the gold nanoparticles with a particle size of 10 nm are coated with a binding reagent that specifically recognizes a first tumor marker, and the gold nanoparticles with a particle size of 40 nm are coated with a binding reagent that specifically recognizes a second tumor marker.

[0009] Furthermore, those skilled in the art will understand that the gold nanoparticles of different particle sizes may also include gold nanoparticles of other particle sizes, such as gold nanoparticles of 60 nm, 80 nm, 100 nm, etc., and the gold nanoparticles of each particle size are coated with a binding reagent that specifically recognizes different tumor markers.

[0010] Furthermore, the material of the microstructure unit is one of gold, aluminum, copper and aluminum, and the thickness is 120nm to 250nm.

[0011] Furthermore, those skilled in the art will understand that the present invention is applicable to microstructure unit arrays having resonance units of any structure.

[0012] As an example, the resonant unit of the microstructure unit array is in the shape of a hollow square ring structure, with dimensions of outer length / width L=20μm~100μm, square ring line width w=2μm~30μm, and period Px=Py=40μm~200μm.

[0013] Furthermore, the base layer is silicon dioxide, high-resistance silicon or polyimide.

[0014] Furthermore, the thickness of the microstructure unit array is in the range of 100 nm to 300 nm, and the thickness of the base layer is in the range of 100 μm to 2 mm.

[0015] In a second aspect, the present invention provides a method for preparing a terahertz metasurface sensor as described herein, comprising:

[0016] (1) forming a microstructure unit array on the substrate layer to obtain a terahertz metasurface sensor;

[0017] (2) Surface modification of terahertz metasurface sensors using mercaptosilane coupling agents;

[0018] (3) A suspension of gold nanoparticles of different sizes coated with binding reagents that specifically recognize different tumor markers is added dropwise onto the terahertz metasurface sensor to obtain a terahertz metasurface sensor for multiple detection of tumor markers.

[0019] In a third aspect, the present invention provides the use of the terahertz metasurface sensor as described herein in the multiplex detection of tumor markers.

[0020] Beneficial effects of the present invention

[0021] The present inventors have discovered that depositing gold nanoparticles of varying sizes on a terahertz metasurface sensor designed in this invention can produce distinct detection signals, with the shift in the resonance peak position positively correlated with the gold nanoparticle size. Leveraging this discovery, multiplexed detection of different tumor markers can be achieved by coating gold nanoparticles of varying sizes with binding reagents that recognize different targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic structural diagram of the terahertz metasurface sensor of the present invention is shown.

[0023] Figure 2 The results of detecting EGFR samples with different concentrations using a terahertz metasurface sensor containing only EGFR antibody-coated gold nanoparticles are shown.

[0024] Figure 3 The results of detecting TGFB2 samples with different concentrations using a terahertz metasurface sensor containing only TGFB2 antibody-coated gold nanoparticles are shown.

[0025] Figure 4 The results of detecting samples containing EGFR and TGFB2 using a terahertz metasurface sensor containing EGFR antibody-coated gold nanoparticles and TGFB2 antibody-coated gold nanoparticles are shown. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0027] Example 1

[0028] This embodiment provides a terahertz metasurface sensor for multiplex detection of tumor markers. Figure 1 The schematic diagram of the structure of the terahertz metasurface sensor designed by the present invention is shown in FIG. Figure 1 As shown, the terahertz metasurface sensor comprises a base layer 100, a microstructure unit array layer 101, and a gold nanoparticle layer 102, all fabricated in a sequential, bottom-up, integrated manner. The square lattice 103 in base layer 100 has an x-axis period px of 70 μm and a y-axis period py of 70 μm. Made of SiO2, it has a thickness of 200 μm. The metal layer 101 consists of hollow square ring resonant unit structures arranged within the square lattice 103. Its dimensions are 70 μm in length / width (L) and 5 μm in width (w).

[0029] Its preparation method is as follows:

[0030] (1) Clean the surface of the silicon substrate, spin-coat a layer of silicon dioxide film on the silicon substrate, spin-coat a layer of photoresist on the silicon dioxide surface using a coating machine, then bake it, and expose it using a UV exposure system. It can then be developed in a developer to form a pattern. After the sample is dried, metal is deposited using an electron beam evaporation deposition system. Finally, the photoresist is removed with acetone, and then sliced ​​with a microtome to obtain a terahertz metasurface sensor;

[0031] (2) The terahertz metasurface sensor was surface modified with mercaptosilane coupling agent KH580 to introduce mercapto groups on the sensor surface;

[0032] (3) Gold nanoparticles with a particle size of 10 nm were mixed with 1.22 μg / ml 3-mercaptopropionic acid at a volume ratio of 3:1 and incubated at room temperature (22°C) for 70 minutes. EGFR antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was combined with 3-mercaptopropionic acid-modified gold nanoparticles via a dehydration condensation reaction. The final concentration of EGFR antibody was 750 ng / ml. The reaction was carried out at room temperature (22°C) for 90 minutes to obtain EGFR antibody-coated gold nanoparticles, which were then resuspended in PBS containing 1% BSA at a concentration of 1 mg / ml.

[0033] Gold nanoparticles with a particle size of 40 nm were mixed with 1.22 μg / ml 3-mercaptopropionic acid at a volume ratio of 3:1 and incubated at room temperature (22°C) for 70 minutes. TGFB2 antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was conjugated to 3-mercaptopropionic acid-modified gold nanoparticles via a dehydration condensation reaction at a final concentration of 750 ng / ml. The TGFB2 antibody was reacted at room temperature (22°C) for 90 minutes to obtain TGFB2 antibody-coated gold nanoparticles, which were then resuspended at 1 mg / ml in PBS containing 1% BSA.

[0034] (4) Take 10 μl of EGFR antibody-coated gold nanoparticle suspension or 10 μl of TGFB2 antibody-coated gold nanoparticle suspension or an equal volume mixture thereof, and add it dropwise to the terahertz metasurface sensor treated in step (2), so that the gold nanoparticles are fixed on the sensor surface by reacting with the thiol groups on the sensor surface. After washing, a terahertz metasurface sensor for multiple detection of tumor markers is obtained.

[0035] Example 2: Detection of tumor markers by the terahertz metasurface sensor of the present invention

[0036] Test solutions containing 0 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL of EGFR were prepared and added dropwise to the terahertz metasurface sensor containing only EGFR antibody-coated gold nanoparticles prepared in Example 1. The relationship between the analyte content and the resonant frequency shift of the resonance peak was evaluated. The transmission spectra obtained were as follows: Figure 2 shown.

[0037] Test solutions containing 0 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL of TGFB2 were prepared and added dropwise to the terahertz metasurface sensor containing only TGFB2 antibody-coated gold nanoparticles prepared in Example 1. The relationship between the analyte content and the resonant frequency shift of the resonance peak was evaluated. The transmission spectra obtained were as follows: Figure 3 shown.

[0038] It can be seen that as the analyte content increases, the resonance peak resonant frequency moves toward a low frequency, and there is a significant difference between the resonance peak resonant frequency of EGFR (the frequency of 0 ng / mL is between 1.21 and 1.22 THz) and the resonance peak resonant frequency of TGFB2 (the frequency of 0 ng / mL is between 1.23 and 1.24 THz), which indicates that different particle size gold nanoparticles coated with antibodies can produce different frequency peaks on the terahertz metasurface sensor of the present invention. This makes it possible to judge which protein is present by the appearance of different frequency peaks when different particle size gold nanoparticles coated with different antibodies are simultaneously present on the terahertz metasurface sensor of the present invention. When the sample to be tested is added to the terahertz metasurface sensor containing EGFR antibody-coated gold nanoparticles (10 nm) and TGFB2 antibody-coated gold nanoparticles (40 nm), if a frequency shift of the frequency peak between 1.21 and 1.23 THz appears, EGFR is present in the sample, and if a frequency shift of the frequency peak between 1.23 and 1.24 THz appears, TGFB2 is present in the sample.

[0039] To verify this design, a test solution containing 200 ng / mL EGFR and 100 ng / mL TGFB2 was prepared and added dropwise to the terahertz metasurface sensor containing both EGFR antibody-coated gold nanoparticles (10 nm) and TGFB2 antibody-coated gold nanoparticles (40 nm) prepared in Example 1. The relationship between the analyte content and the resonant frequency shift of the resonance peak was evaluated. The transmission spectrum obtained is shown in FIG. Figure 4As shown, the resonance peak resonance frequency peak of EGFR and the resonance peak resonance frequency peak of TGFB2 can be seen simultaneously in the transmission spectrum, and their respective frequency shifts can be detected, indicating that the terahertz metasurface sensor of the present invention containing both EGFR antibody-coated gold nanoparticles (10 nm) and TGFB2 antibody-coated gold nanoparticles (40 nm) can simultaneously detect EGFR and TGFB2, and can achieve multiple detections of multiple different targets in the same sample.

[0040] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A terahertz metasurface sensor for multiplex detection of tumor markers, characterized in that: The terahertz metasurface sensor comprises a microstructure unit array and a substrate layer, wherein the microstructure unit array is located on the substrate layer; and gold nanoparticles of different particle sizes are fixed on the surface of the terahertz metasurface sensor, and the gold nanoparticles of different particle sizes are respectively coated with binding reagents that specifically recognize different tumor markers.

2. The terahertz metasurface sensor according to claim 1, characterized in that The gold nanoparticles with different particle sizes include gold nanoparticles with particle sizes of 10 nm and 40 nm.

3. The terahertz metasurface sensor according to claim 2, characterized in that: The gold nanoparticles with a particle size of 10 nm are coated with a binding reagent that specifically recognizes a first tumor marker, and the gold nanoparticles with a particle size of 40 nm are coated with a binding reagent that specifically recognizes a second tumor marker.

4. The terahertz metasurface sensor according to claim 1, wherein: The material of the microstructure unit is gold, aluminum, copper or aluminum.

5. The terahertz metasurface sensor according to claim 1, wherein: The shape of the resonant unit of the microstructure unit array is a hollow square ring structure, the size of which is outer length / width L=20μm~100μm, square ring line width w=2μm~30μm, period P x =P y =40μm~200μm.

6. The terahertz metasurface sensor according to claim 1, characterized in that: The base layer is silicon dioxide, high-resistance silicon or polyimide.

7. The terahertz metasurface sensor according to claim 1, characterized in that: The thickness of the microstructure unit array is in the range of 100 nm to 300 nm, and the thickness of the base layer is in the range of 100 μm to 2 mm.

8. A method for preparing a terahertz metasurface sensor according to any one of claims 1 to 7, characterized in that: include: (1) forming a microstructure unit array on the substrate layer to obtain a terahertz metasurface sensor; (2) Surface modification of terahertz metasurface sensors using mercaptosilane coupling agents; (3) A suspension of gold nanoparticles of different sizes coated with binding reagents that specifically recognize different tumor markers is added dropwise onto the terahertz metasurface sensor to obtain a terahertz metasurface sensor for multiple detection of tumor markers.

9. Use of the terahertz metasurface sensor according to any one of claims 1 to 7 in multiplex detection of tumor markers.

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