Terahertz Metamaterial Biosensor and Its Applications

Abstract

The present invention provides a terahertz metamaterial biosensor and its application. Based on micro-nano processing technology, the present invention attaches a subwavelength metal resonant ring array on a dielectric substrate to form a metamaterial sensor. The subwavelength metal resonant ring is composed of two fan-shaped resonant rings with an angle of 60° to form a structure similar to the shape of an "8", and two gaps are formed at the upper and lower ends of the structure to generate annular dipole resonance, which can achieve high quality factor and high sensitivity sensing in the terahertz band. Colloidal gold nanoparticles functionalized by biomodification technology not only enhance the sensing performance of the metamaterial biosensor and significantly reduce the detection limit, but also achieve specific recognition of the cancer marker carcinoembryonic antigen. The metamaterial sensor combined with functionalized gold nanoparticles proposed by the present invention has excellent high quality factor sensing performance, can achieve high sensitivity and specific detection of carcinoembryonic antigen, the method is simple and effective, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of metamaterial structure design and biological detection, and in particular relates to a terahertz metamaterial biosensor and its application. Background Art

[0002] Carcinoembryonic antigen (CEA) is a broad-spectrum cancer marker that is highly expressed in cancer patients (colon cancer, rectal cancer, breast cancer, etc.) compared to healthy people. It has been identified as one of the essential markers for cancer diagnosis, monitoring, and efficacy evaluation. In the past few decades, enzyme-linked immunosorbent assay (ELISA), impedance immunobiosensors, fluorescence-based methods, and spectroscopic techniques such as localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS) have been used in the diagnosis of cancer markers and have made great progress. However, since the test requires strict test methods, skilled technicians, or a high-quality testing environment, it often has disadvantages such as being time-consuming, costly, and complex to operate. Therefore, it is of great significance to develop innovative detection methods with high sensitivity, specificity, and label-free to achieve trace CEA detection.

[0003] Terahertz time-domain spectroscopy (THz-TDS), an emerging coherent detection spectroscopy technique, can reveal structural information about intramolecular and inter-molecular vibrational modes, enabling precise molecular-level detection, thanks to its high signal-to-noise ratio (SNR), low photon energy, and wide bandwidth. It has been rapidly developing in biological detection. Recently, metamaterials with subwavelength artificial electromagnetic composite structures have been combined with terahertz biosensors. By utilizing the surface plasmon resonance generated by these metamaterials under electromagnetic wave excitation to form localized electromagnetic field enhancement, they are highly sensitive to the surrounding medium and enable highly sensitive analysis. These sensors offer the advantages of low cost, minimal energy consumption, real-time, and non-destructive testing.

[0004] The sensing characteristics of terahertz metamaterial biosensors are generally characterized by sensitivity (S), while the quality factor (Q) describes the sharpness of the resonance peak, reflecting the strength of the resonance peak and playing an important role in characterizing the sensor's ability to identify analytes. To date, research has been conducted on the use of quality factors to characterize the sensing performance of designed terahertz metamaterial biosensors. For example, CN111766218A designed a terahertz metamaterial biosensor using a periodic structure of asymmetric split resonant rings, with a theoretical quality factor of up to 83; CN108572162A designed a terahertz metamaterial sensor based on the electromagnetically induced transparency effect using a periodic structure consisting of split circular resonant rings and split square resonant rings, with a theoretical transmission peak quality factor of up to 85. However, due to the accuracy currently available from terahertz time-domain spectrometers, the realization of these two types of terahertz metamaterial biosensors is currently difficult. Furthermore, as terahertz metamaterial biosensors continue to advance in the field of precise detection of extremely small amounts of samples, simply changing metamaterial sensor parameters is no longer sufficient to achieve the required detection sensitivity, and their ability to accurately identify target samples needs to be improved. Therefore, in practical applications, it is not only necessary to design a terahertz metamaterial biosensor with high sensitivity and a high quality factor that can be realized to meet the practical needs of large-scale production; it is also extremely important to find a method that can enhance the sensing performance of terahertz metamaterial biosensors and achieve specific detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a terahertz metamaterial biosensor with a high quality factor and applications thereof.

[0006] Another object of the present invention is to provide a method for detecting biomaterial-specificity with simple operation and high sensitivity.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a terahertz metamaterial biosensor, comprising a dielectric substrate and a subwavelength metal double-sector resonant ring array attached to the dielectric substrate.

[0008] The subwavelength metal double-fan resonant ring array is composed of at least 100×100 metal structural units of the same size and uniform arrangement.

[0009] The size of the metal structure unit is 50 μm×50 μm.

[0010] The metal structure unit is composed of two sector-shaped resonant rings of the same size with an included angle of 45-75 degrees, forming a structure similar to an "8" shape, and two notches of the same size are formed above and below the "8" structure.

[0011] The dielectric substrate may be a rigid substrate or a flexible substrate.

[0012] Preferably, the material of the hard substrate is silicon, quartz, etc., and the thickness is 0.5-3 mm; more preferably, the material of the hard substrate is quartz, and the thickness is 1 mm.

[0013] Preferably, the flexible substrate is made of polyimide, PDMS, etc., with a thickness of 5-100 μm; more preferably, the flexible substrate is made of polyimide, with a thickness of 25 μm.

[0014] The metal is made of gold, silver, copper or aluminum, preferably gold.

[0015] Preferably, the metal wire width used to make the metal structure unit is 2-4 μm, and the thickness is 150-450 nm; the gap width is 1-10 μm, the straight-line distance from the center of the metal structure unit to the inner edge end point of the gap is 19-21 μm, and the fan-shaped resonant ring width is 20-23 μm.

[0016] More preferably, the metal wire used to make the metal structure unit has a width of 3 μm and a thickness of 200 nm; the gap width is 3 μm, the straight-line distance from the center of the metal structure unit to the inner edge end point of the gap is 20 μm, and the fan-shaped resonant ring width is 22 μm; the metal structure unit is composed of two fan-shaped resonant rings of the same size with an angle of 60° to form a structure similar to the shape of an "8".

[0017] Furthermore, the subwavelength metal double open ring resonator array is 8 mm×8 mm.

[0018] In the present invention, the metal structural unit can be attached to the dielectric substrate by photolithography technology and / or metallization process.

[0019] In a second aspect, the present invention provides applications of the biosensor in biomaterial detection (the application includes non-disease diagnosis and treatment purposes).

[0020] The biological material includes but is not limited to proteins, DNA or RNA, such as proteins such as biomarkers.

[0021] Preferably, the protein is carcinoembryonic antigen.

[0022] In a third aspect, the present invention provides a method for detecting a biological material (the method has non-disease diagnosis and treatment purposes). When the biological material is a carcinoembryonic antigen, the method comprises the following steps:

[0023] (1) Preparation of functionalized gold nanoparticles: a colloidal gold nanoparticle solution and 3-mercaptopropionic acid are mixed and incubated at a certain volume ratio to obtain 3-mercaptopropionic acid-modified colloidal gold nanoparticles; then, a carcinoembryonic antigen antibody is reacted with the 3-mercaptopropionic acid-modified colloidal gold nanoparticles through a dehydration condensation reaction to obtain functionalized gold nanoparticles;

[0024] (2) forming a ring dipole resonance in the biosensor under the excitation of a terahertz wave (the direction of the electric field of the terahertz wave is parallel to the direction of the notch);

[0025] (3) Detection of carcinoembryonic antigen: The functionalized gold nanoparticles are mixed and incubated with carcinoembryonic antigens of different concentrations, and the reaction product is dripped onto the surface of the biosensor to obtain terahertz spectral information of the carcinoembryonic antigen. Based on the terahertz spectral information, the offset of the ring dipole resonant frequency with different concentrations of carcinoembryonic antigen is further obtained, and a model between the carcinoembryonic antigen concentration and the offset is established to achieve qualitative and quantitative analysis of the carcinoembryonic antigen.

[0026] Furthermore, step (1) includes: mixing 0.45-0.9 mL of a 0.6-1 nM colloidal gold nanoparticle solution with 0.15-0.3 mL of 3-mercaptopropionic acid, and reacting at 22° C. for 70 minutes to obtain 3-mercaptopropionic acid-modified colloidal gold nanoparticles; then, mixing an anti-carcinoembryonic antigen antibody with the 3-mercaptopropionic acid-modified colloidal gold nanoparticles in a solution volume ratio of 4-1:1-1, and reacting at 22° C. for 90 minutes to obtain functionalized gold nanoparticles.

[0027] The particle size of the colloidal gold nanoparticles in the colloidal gold nanoparticle solution is 3-100 nm.

[0028] Furthermore, the concentration of the carcinoembryonic antigen in step (3) is 100 ng / ml-2 μg / ml.

[0029] Furthermore, the minimum detection limit of the carcinoembryonic antigen is 34.83 ng / ml.

[0030] In a specific embodiment of the present invention, the present invention provides a terahertz band metamaterial biosensor with a high quality factor, the sensor comprising a dielectric substrate and a subwavelength metal double-fan-shaped resonant ring array attached to the dielectric substrate layer; wherein the subwavelength metal double-fan-shaped resonant ring array is composed of at least 100×100 metal structural units of the same size and uniform arrangement; the size of the metal structural unit is 50μm×50μm; the metal structural unit is composed of two fan-shaped resonant rings with an angle of 60° to form a structure similar to an "8" shape, and two notches are formed above and below the structure, and the direction of the terahertz wave electric field is parallel to the direction of the notch, thereby exciting the formation of a ring dipole resonance.

[0031] The dielectric substrate is a 0.5-3 mm hard substrate such as silicon or quartz or a 5-100 μm flexible substrate such as polyimide or PDMS, preferably 1 mm quartz or 25 μm polyimide.

[0032] Furthermore, the metal wire width of the metamaterial structure is 2-4 μm, preferably 3 μm; the width of the gap is 1-10 μm, preferably 3 μm; the straight-line distance from the center of the metal structure to the inner edge endpoint of the gap is 19-21 μm, preferably 20 μm; the angle between the two metal wires is 45-75°, preferably 60°; the structure width is 20-23 μm, preferably 22 μm; the thickness of the metal structure is 150-450 nm, preferably 200 nm; the material of the double open resonant ring is gold, silver, copper or aluminum, preferably gold.

[0033] Furthermore, the subwavelength metal double open ring resonator array is 8 mm×8 mm.

[0034] Furthermore, the metamaterial sensor can generate a high-quality-factor annular dipole resonance valley under the excitation of a terahertz wave in a horizontal electric field.

[0035] Furthermore, in a specific embodiment of the present invention, the metamaterial sensor uses a 1 mm quartz substrate whose dielectric constant in the terahertz band is approximately 3.69 as determined by experimental testing.

[0036] In simulation experiments, the quality factor of the resonance valley of the metamaterial sensor is 15.04; when the thickness of the analyte is 4 μm, the theoretical sensitivity of the transmission valley in the obtained terahertz metamaterial biosensor is 280 GHz / RIU.

[0037] Furthermore, the metamaterial sensor is prepared by photolithography technology and / or metallization process.

[0038] Secondly, the terahertz metamaterial biosensor proposed in the present invention is applied to biomarkers such as proteins, as well as DNA and RNA detection (including non-disease diagnosis and treatment purposes).

[0039] Among them, biomarker proteins can come from cancer cells, such as carcinoembryonic antigen CEA.

[0040] In a third aspect, the present invention provides a method for simple operation and high sensitivity and specific detection, the method comprising: (1) preparation of functionalized gold nanoparticles: using biomodification technology, the prepared colloidal gold nanoparticles are covalently bound to 3-mercaptopropionic acid via gold-sulfur bonds, and the reaction is carried out at room temperature of 22°C for 70 minutes; then, a carcinoembryonic antigen antibody with a concentration of 750 ng / ml is bound to the carboxyl group of 3-mercaptopropionic acid via a dehydration condensation reaction, and the reaction is carried out at room temperature of 22°C for 90 minutes, thereby obtaining functionalized gold nanoparticles; (2) detection process: the functionalized gold nanoparticles are reacted with carcinoembryonic antigens of different concentrations at room temperature of 22°C for 2 hours, 5 μl is taken and dripped onto the surface of the metamaterial sensor, the terahertz spectrum information of the carcinoembryonic antigen is obtained, the offset of the ring dipole resonant frequency with different concentrations of carcinoembryonic antigen is obtained, a model between the carcinoembryonic antigen concentration and the offset is established, and qualitative and quantitative analysis of the carcinoembryonic antigen is achieved.

[0041] Specifically, the method comprises the following steps:

[0042] (1) Colloidal gold nanoparticles were prepared according to conventional methods and stored at 4°C until use;

[0043] (2) The prepared colloidal gold nanoparticles were mixed with 3-mercaptopropionic acid in a certain volume ratio and incubated at room temperature (22°C) for 70 minutes;

[0044] (3) combining the anti-CEA antibody with the 3-mercaptopropionic acid-modified colloidal gold nanoparticles obtained in (2) through a dehydration condensation reaction, and reacting at room temperature (22°C) for 90 minutes to obtain functionalized gold nanoparticles;

[0045] (4) Dilute carcinoembryonic antigen with 18.2 MΩ ultrapure water, add it to (3) in a certain proportion, and incubate at room temperature (22°C) for 2 hours;

[0046] (5) dropping the mixed solution in (4) onto the surface of the metamaterial sensor and drying it to serve as a sample to be tested;

[0047] (6) The metamaterial sensor with or without the sample to be tested is used to obtain the corresponding terahertz spectrum information using the terahertz time-domain spectroscopy system as the sample time-domain signal E sam (t), the terahertz spectrum information obtained from the substrate test of the metamaterial sensor is used as the reference time domain signal E ref (t);

[0048] (7) Using fast Fourier transform, we can obtain the sample time domain signal E sam (t) and the reference time domain signal E ref (t) the frequency domain signal E sam (ω) and E ref(ω), and through T=E sam (ω) / E ref (ω), obtain the transmittance of the sample relative to the dielectric substrate;

[0049] (8) The resonance frequencies corresponding to the resonance valleys of the samples with different concentrations are obtained according to the frequency-transmittance curve in (7), and a standard equation between the resonance frequencies and the resonance valleys of the samples with different concentrations is established based on the frequency shift of the resonance frequencies.

[0050] Furthermore, the size of the colloidal gold nanoparticles can be controlled between 3-100 nm. In a specific implementation method of the present invention, the average diameter of the gold nanoparticles is 27 nm.

[0051] Furthermore, the concentration of 3-mercaptopropionic acid is 1 μg / ml-20 μg / ml. In a specific implementation method of the present invention, the concentration of 3-mercaptopropionic acid is 1.22 μg / ml.

[0052] Furthermore, the volume ratio of the colloidal gold nanoparticles and 3-mercaptopropionic acid for incubation is 3:1.

[0053] Furthermore, in a specific embodiment of the present invention, the volume ratio of the carcinoembryonic antigen to the functionalized gold nanoparticles is 1:1.

[0054] Furthermore, in a specific embodiment of the present invention, the concentration of carcinoembryonic antigen antibody is 750 ng / ml.

[0055] Furthermore, in one embodiment of the present invention, the frequency shift changes of pure carcinoembryonic antigen solution, pure colloidal gold nanoparticle solution and a solution obtained by incubating a mixture of colloidal gold nanoparticles and carcinoembryonic antigen at the same concentration and different dosages were compared.

[0056] Furthermore, the measurement conditions of the terahertz time-domain spectroscopy system are: 20-25° C. (preferably 22° C.), relative humidity <15%, and a frequency range of 0-3.0 THz.

[0057] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0058] (1) The present invention provides a metamaterial sensor with a high quality factor in the terahertz band, with a quality factor of 15.04.

[0059] (2) The metamaterial structure provided by the present invention can excite ring dipole resonance under the excitation of terahertz waves and has high sensitivity. In simulations, when the analyte thickness is 4μm, the sensitivity of the transmission valley can reach 280GHz / RIU, which has excellent sensing performance in biological detection.

[0060] (3) The terahertz metamaterial biosensor provided by the present invention can adjust the resonance valley frequency within a certain frequency range by changing the opening size.

[0061] (4) Compared with existing methods for detecting biological samples, the method provided by the present invention for detecting the performance of highly sensitive terahertz-band metamaterial sensors requires only the addition of the required colloidal gold nanoparticles and incubation. This method offers advantages such as simplicity, effectiveness, low cost, and a significant reduction in the detection limit. In one embodiment of the present invention, the detection limit was reduced by 3 times.

[0062] (5) The method provided by the present invention for achieving specific detection of terahertz-band metamaterial sensors can accurately identify the cancer marker carcinoembryonic antigen. It has great room for expansion by optimizing the biological modification process and has great application potential in early cancer screening in the future.

[0063] (6) The method for high sensitivity enhancement and specific detection of terahertz-band metamaterial sensors provided by the present invention can be applied to the identification and detection of proteins such as DNA, RNA, and biomarkers (including non-disease diagnosis and treatment purposes), and has a broad application market. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram and optical microscope image of the terahertz metamaterial sensor designed in the preferred embodiment of the present invention.

[0065] Figure 2 This is the simulated transmission spectrum of the terahertz metamaterial sensor designed in the preferred embodiment of the present invention.

[0066] Figure 3 1 and 2. The transmission spectra (a) corresponding to analytes with different refractive indices and the linear relationship between the frequency shift and the refractive index (b) when the thickness of the analyte is 4 μm in a preferred embodiment of the present invention.

[0067] Figure 4 This is the transmission spectrum corresponding to changing the size of the upper and lower opening gaps from 1 μm to 9 μm in a preferred embodiment of the present invention.

[0068] Figure 5 This is the UV-visible absorption spectrum of the colloidal gold nanoparticles prepared in the preferred embodiment of the present invention.

[0069] Figure 6 Schematic diagram of transmission electron microscopy of colloidal gold nanoparticles prepared in a preferred embodiment of the present invention.

[0070] Figure 7Graphs showing different dose-transmission curves of a solution (a) incubated with a mixture of colloidal gold nanoparticles and carcinoembryonic antigen, a pure colloidal gold nanoparticle solution (b), and a pure carcinoembryonic antigen solution (c) in a preferred embodiment of the present invention.

[0071] Figure 8 Schematic diagram of different dose-frequency offsets of a solution (a) after mixed incubation of colloidal gold nanoparticles and carcinoembryonic antigen, a pure colloidal gold nanoparticle solution (b), and a pure carcinoembryonic antigen solution (c) in a preferred embodiment of the present invention.

[0072] Figure 9 Schematic diagram of carcinoembryonic antigen CEA concentration-resonance frequency in a preferred embodiment of the present invention.

[0073] Figure 10 FIG. 4 is a histogram of carcinoembryonic antigen (CEA) concentration versus resonance frequency offset in a preferred embodiment of the present invention.

[0074] Figure 11 Comparison of the frequency shift histograms of the metamaterial sensor designed in the preferred embodiment of the present invention sensing four different proteins. DETAILED DESCRIPTION

[0075] The present invention provides a terahertz band metamaterial sensor having a double-fan-shaped metamaterial structure to form a high quality factor, and a method for achieving high sensitivity enhancement and specific sensing in biological detection.

[0076] The present invention adopts the following technical solutions:

[0077] First, a terahertz-band metamaterial sensor with a dual-sector metamaterial structure to form a high-quality factor is provided. The sensor comprises a dielectric substrate and a subwavelength metal dual-sector resonant ring array attached to the dielectric substrate. The subwavelength metal dual-sector resonant ring array comprises at least 100×100 uniformly arranged metal structural units of equal size. The metal structural units are formed by two sector-shaped resonant rings at an angle of 60°, forming a figure-8 structure with two notches formed above and below the structure. The terahertz wave electric field is parallel to the direction of the notches, exciting a toroidal dipole resonance. The dielectric substrate is a quartz substrate with a dielectric constant of 3.69 and a thickness of 1 mm.

[0078] Among them, the metal wire width of the metamaterial structure is 2-4μm; the width of the gap is 1-10μm; the straight-line distance from the center of the metal structure to the inner edge endpoint of the gap is 19-21μm; the angle between the two metal wires is 45-75°; the structure width is 20-23μm; and the thickness of the metal structure is 150-450nm.

[0079] The double open resonant ring is made of gold, silver, copper or aluminum, preferably gold.

[0080] The size of the metal structure unit is 50 μm×50 μm.

[0081] The sub-wavelength metal double-open resonant ring array is 8mm×8mm.

[0082] Secondly, the designed metamaterial sensor was modeled and simulated using commercial CST simulation software. Under the excitation of terahertz waves in a horizontal electric field, a high-quality ring dipole resonance valley with a quality factor of 15.04 was generated. When the thickness of the analyte was 4μm, the theoretical sensitivity of the transmission valley in the obtained terahertz metamaterial biosensor was 280GHz / RIU, which can achieve high-performance sensing in biological detection.

[0083] Then, the metamaterial sensor is fabricated by conventional photolithography and metallization processes.

[0084] Finally, the present invention provides a method for achieving high sensitivity and specificity sensing in biological detection, which can significantly reduce the detection limit. The method comprises:

[0085] (1) Prepare the required colloidal gold nanoparticles according to conventional methods and store them at 4°C until use;

[0086] (2) The prepared colloidal gold nanoparticles were mixed with 3-mercaptopropionic acid in a certain volume ratio and incubated at room temperature (22°C) for 70 minutes;

[0087] (3) Carcinoembryonic antigen antibody was combined with 3-mercaptopropionic acid-modified colloidal gold nanoparticles via a dehydration condensation reaction at room temperature (22°C) for 90 minutes to obtain functionalized gold nanoparticles;

[0088] (4) Dilute carcinoembryonic antigen with 18.2 MΩ ultrapure water, add it to (3) in a certain proportion, and incubate at room temperature (22°C) for 2 hours;

[0089] (5) adding the solution incubated in (4) dropwise onto the surface of the metamaterial sensor and drying it to serve as a sample to be tested;

[0090] (6) The metamaterial sensor with or without the sample to be tested is used to obtain the corresponding terahertz spectrum information using the terahertz time-domain spectroscopy system as the sample time-domain signal E sam (t) The terahertz spectrum information obtained by testing the dielectric substrate used in the metamaterial sensor is used as the reference time domain signal E ref (t);

[0091] (7) Using fast Fourier transform, we can obtain the sample time domain signal E sam (t) and the reference time domain signal E ref The frequency domain signal E of (t)sam (ω) and E ref (ω), and through T=E sam (ω) / E ref (ω), obtain the transmittance of the sample relative to the dielectric substrate;

[0092] (8) The resonance frequencies corresponding to the resonance valleys of the samples with different concentrations are obtained based on the frequency-transmittance curve in (7), and a standard equation between the resonance frequencies and the frequency shift of the samples with different concentrations is established.

[0093] The measurement conditions of the terahertz time-domain spectroscopy system are: 20-25° C. (preferably 22° C.), frequency range 0-3.0 THz, nitrogen gas filled into the terahertz optical path during the test, and humidity <5%.

[0094] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0095] In the description of the present invention, unless otherwise specified, "multiple" means two or more. Terms such as "upper," "lower," "inner," and "outer" indicating orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They do not indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and are therefore not to be construed as limiting the present invention.

[0096] Example 1 Design and preparation of metamaterial structure and terahertz metamaterial biosensor

[0097] This embodiment provides a terahertz-band metamaterial sensor with a high-quality factor and a dual-sector metamaterial structure. The designed metamaterial sensor was modeled using commercial CST simulation software. The sensor includes a dielectric substrate and a subwavelength metal dual-sector resonant ring array attached to the dielectric substrate, measuring 8 mm x 8 mm. The subwavelength metal dual-sector resonant ring array is composed of at least 100 x 100 uniformly arranged metal structural units. The metal structural units have dimensions a of 50 μm x 50 μm. The metal resonant ring units are formed by two sector-shaped resonant rings at a 60° angle, forming a figure-8-shaped structure with two equal-sized notches formed above and below the structure. The metal wire width of the metamaterial structure is w = 3 μm; the notch width is d = 3 μm; the straight-line distance from the center of the metal structure to the inner edge of the notch is r = 20 μm; the angle θ between the two metal wires is 60°; the structure width is D = 22 μm; and the metal structure is Au with a thickness of H = 200 nm. The dielectric substrate is a quartz substrate with a dielectric constant of 3.69 and a thickness of h = 1 mm. Figure 1 The metamaterial sensor is fabricated by conventional photolithography and metallization processes.

[0098] Example 2 Performance Analysis of Terahertz Metamaterial Biosensor

[0099] This embodiment uses quality factor and sensitivity as the performance evaluation criteria for terahertz metamaterial sensors, and the sensitivity formula is defined as: S = Δf / Δn, where Δf represents the frequency offset of the resonance valley corresponding to the object to be measured with different refractive indices, and Δn represents the change in the refractive index of the object to be measured; the quality factor formula is defined as: Q = f0 / Δν, where f0 represents the resonance frequency of the resonance valley, and Δν represents the half-height width of the resonance valley. The present invention sets the boundary conditions in the CST simulation software as follows: the x direction is the electric field direction, the y direction is the magnetic field direction, and the z direction is the terahertz wave incident direction, and the terahertz wave is vertically incident from this end of the metamaterial structure. In the simulation, under the excitation of the terahertz wave in the horizontal electric field, a high-quality annular dipole resonance valley appears in the transmission spectrum, with a resonance frequency of 1.76THz and a quality factor of 15.04, as shown in FIG. Figure 2 To analyze the sensing performance of the designed terahertz metamaterial biosensor, the thickness of the analyte is set to 4 μm, and the refractive index of the analyte is increased from 1.1 to 1.9 in intervals of 0.2 and simulation is performed. The transmission spectrum obtained is shown in Figure 3 As shown in (a), it can be seen that with the increase of the refractive index of the analyte, the frequency of the resonance valley has a significant red shift. According to the sensitivity formula, the theoretical sensitivity of the transmission valley of the terahertz metamaterial biosensor is 280GHz / RIU, as shown in Figure 3(b) In order to adjust the resonant peak frequency of the generated transmission spectrum to meet the needs of practical applications, the sizes of the upper and lower openings in the structure were selected for simulation analysis. The opening gap was increased from 1μm to 9μm at intervals of 2, and the transmission spectrum obtained was as shown in Figure 4 As shown in the figure, it can be seen that with the increase of the opening gap, the resonant frequency has a red shift phenomenon, showing a certain resonant frequency selection characteristic.

[0100] Example 3 Preparation and Characterization of Colloidal Gold Nanoparticles

[0101] This embodiment illustrates the preparation and characterization of colloidal gold nanoparticles. The preparation of colloidal gold nanoparticles is to heat 100 ml of 0.01% by mass HAuCl4 aqueous solution to boiling, then add 1.5 ml of 1% by mass sodium citrate aqueous solution, and then stop the reaction after continuous stirring for 20 minutes. The reaction is cooled to room temperature in a water bath to obtain the required colloidal gold nanoparticles, and then stored at 4°C. In addition, the size of the colloidal gold nanoparticles can be adjusted by adjusting the amount or concentration of sodium citrate. Using ultraviolet-visible absorption spectrum test analysis (the prepared colloidal gold nanoparticle solution is diluted by half during the test), it can be seen that the prepared colloidal gold nanoparticles have an absorption peak at 529 nm, as shown in FIG. Figure 5 As shown, it shows that colloidal gold nanoparticles have been successfully prepared with a concentration of about 0.8nM. The morphology of colloidal gold nanoparticles was characterized by transmission electron microscopy, and it was found that the average diameter of the colloidal gold nanoparticles was about 27nm, and they had a certain aspect ratio, as shown in Figure 2. Figure 6 shown.

[0102] Example 4 Method for Realizing Highly Sensitive Terahertz Metamaterial Biosensor Performance

[0103] This example provides a method for enhancing the performance of a highly sensitive terahertz metamaterial biosensor. Carcinoembryonic antigen (CEA) was selected as the target protein analyte, and three sets of experiments were set up for comparison: a mixture of CEA and colloidal gold nanoparticles, a pure CEA solution, and pure colloidal gold nanoparticles. The specific steps are as follows:

[0104] (1) Preparation of three sample solutions: ① Dilute the 1 mg / ml standard CEA solution with ultrapure water to 1 μg / ml, take 1 ml of colloidal gold nanoparticles and 1 ml of 1 μg / ml CEA solution and mix them. After incubation, the concentration of CEA solution is 500 ng / ml and is used as the sample solution to be tested, marked as solution 1; ② Mix 1 ml of colloidal gold nanoparticles with 1 ml of ultrapure water and shake well, marked as solution 2; ③ Mix 1 ml of 1 μg / ml CEA solution with 1 ml of ultrapure water and shake well, marked as solution 3. All three solutions were incubated at room temperature (22°C) for 2 hours before testing.

[0105] (2) Test process: ① The terahertz time-domain spectroscopy system is used to collect the terahertz time-domain information of the quartz medium substrate used as a reference signal; ② The three sample solutions are dripped onto the surface of the designed metamaterial sensor at intervals of 5 μl, and the corresponding terahertz time-domain information is obtained after drying as the sample signal; ③ The obtained reference time-domain signal and sample time-domain signal are Fourier transformed to obtain the reference frequency domain amplitude E ref (ω) and the sample frequency domain amplitude E sam (ω); ④ Using T=E sam (ω) / E ref (ω), the transmittance of the three sample solutions relative to the dielectric substrate is obtained, as shown in Figure 7 (a-c) ⑤ According to the frequency-transmittance curve, the resonance frequency corresponding to the resonance peak of different sample amounts is obtained. By comparing the offset of the resonance frequency with different sample amounts, the frequency-transmittance curve of different dose samples and the standard equation between the dose and the frequency offset (relative to the resonance frequency generated by adding 0μl sample to the metamaterial sensor) are obtained. Figure 8 (a-c) The relationships between the three sample solution contents and the resulting frequency shift can be expressed as: y = 0.733 × x, y = 0.46 × x, and y = 0.488 × (x-10), respectively. The THz time-domain spectroscopy system measurement conditions were: temperature 22°C, frequency range 0-3.0 THz, nitrogen gas in the THz optical path, and humidity <5%.

[0106] (3) Result analysis: By comparing the standard equations between the sample content and the frequency shift obtained for the three sample solutions, it can be found that for sample solution 1, the transmission valley can produce a larger frequency shift at the same dose, achieving higher sensitivity detection; for sample solution 3, the transmission valley cannot produce a frequency shift under 10μl CEA solution, that is, the detection function cannot be achieved, reaching the detection limit. At the same time, it shows that the addition of gold nanoparticles reduces the detection limit by more than 3 times, and further proves that the addition of colloidal gold nanoparticles can achieve high sensitivity terahertz metamaterial biosensor sensing performance.

[0107] Example 5 Method for realizing specific detection of carcinoembryonic antigen using terahertz metamaterial biosensor

[0108] This embodiment provides a method for implementing specific detection of a terahertz metamaterial biosensor, and the specific steps are as follows:

[0109] (1) Preparation of functionalized gold nanoparticles: Colloidal gold nanoparticles with an average particle size of 27 nm were mixed with 1.22 μg / ml of 3-mercaptopropionic acid at a volume ratio of 3:1 and incubated at room temperature (22°C) for 70 minutes. Carcinoembryonic antigen antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was combined with 3-mercaptopropionic acid-modified colloidal gold nanoparticles via a dehydration condensation reaction, with the final concentration of carcinoembryonic antigen antibody being 750 ng / ml. Functionalized gold nanoparticles were obtained by reacting at room temperature (22°C) for 90 minutes.

[0110] (2) Test process: First, functionalized gold nanoparticles were mixed with carcinoembryonic antigens of different concentrations in a volume ratio of 1:1 and incubated for 2 hours. Secondly, the terahertz time-domain spectroscopy system was used to collect the terahertz time-domain information of the quartz medium substrate used as a reference signal. The three sample solutions were dripped onto the surface of the designed metamaterial sensor at intervals of 5 μl. After drying, the corresponding terahertz time-domain information was obtained as the sample signal. Finally, based on Fourier transform, the reference frequency domain amplitude E was obtained. ref (ω) and the sample frequency domain amplitude E sam (ω), using T=E sam (ω) / E ref (ω), the frequency-transmittance curves of carcinoembryonic antigen samples with different concentrations (0ng / ml, 100ng / ml, 300ng / ml, 500ng / ml, 750ng / ml) and the standard equation between their concentration and frequency shift (relative to the resonant frequency generated by adding 0μl sample to the metamaterial sensor) were obtained. In order to characterize the specific detection of carcinoembryonic antigen by the method, the resonant frequency shift generated by 15μl of carcinoembryonic antigen, carbohydrate antigen 125, SARS-CoV-2S1 protein and S-RBD protein on the terahertz metamaterial sensor was obtained. The measurement conditions of the terahertz time-domain spectroscopy system are: temperature of 22°C, frequency range of 0-3.0THz, nitrogen gas filled in the terahertz optical path during the test, and humidity <5%.

[0111] (3) Result analysis: Figure 9 The transmittance spectrum generated by dropping 5μl of carcinoembryonic antigen solutions with different concentrations on the surface of the terahertz metamaterial biosensor shows that as the concentration of carcinoembryonic antigen increases, the resonant frequency shift also increases. In order to achieve quantitative detection of carcinoembryonic antigen concentration, Figure 9 The frequency-transmittance curve was used to obtain the frequency offset of carcinoembryonic antigen solutions with different concentrations, and a concentration-frequency offset curve was established. The fitting equation was Δf=2.37×ln(x+11.61)(R 2 =0.991), where x is the concentration of the added carcinoembryonic antigen solution. Figure 10According to the detection limit calculation formula, it can be concluded that the minimum detection limit of CEA achievable by this method is 34.83ng / ml. Figure 11 The resonant frequency shifts of the terahertz metamaterial sensor caused by the four proteins are shown in FIG. 4 , which show obvious differences.

[0112] According to the above method, the terahertz metamaterial sensor can achieve qualitative and quantitative detection of carcinoembryonic antigen with good specificity, and also proves the effectiveness of the sensing test method and analysis of the present invention.

[0113] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for detecting carcinoembryonic antigen, characterized in that: The method is for non-disease diagnosis and treatment purposes and comprises the following steps: (1) Preparation of functionalized gold nanoparticles: 0.45-0.9 mL of a 0.6-1 nM colloidal gold nanoparticle solution was mixed with 0.15-0.3 mL of 3-mercaptopropionic acid, and the mixture was reacted at 22°C for 70 minutes to obtain 3-mercaptopropionic acid-modified colloidal gold nanoparticles; then, carcinoembryonic antigen antibody and the 3-mercaptopropionic acid-modified colloidal gold nanoparticles were mixed at a solution volume ratio of (1-4):1, and the mixture was reacted at 22°C for 90 minutes to obtain functionalized gold nanoparticles; (2) The terahertz metamaterial biosensor forms a ring dipole resonance under the excitation of the terahertz wave; (3) Detection of carcinoembryonic antigen: the functionalized gold nanoparticles are mixed and incubated with carcinoembryonic antigen at different concentrations, and the reaction product is dripped onto the surface of the biosensor to obtain terahertz spectrum information of the carcinoembryonic antigen. Based on the terahertz spectrum information, the offset of the ring dipole resonant frequency with different concentrations of carcinoembryonic antigen is further obtained, and a model between the carcinoembryonic antigen concentration and the offset is established to achieve qualitative and quantitative analysis of the carcinoembryonic antigen; The particle size of the colloidal gold nanoparticles in the colloidal gold nanoparticle solution is 3-100 nm; The terahertz metamaterial biosensor includes a dielectric substrate and a subwavelength metal double-fan-shaped resonant ring array attached to the dielectric substrate; The subwavelength metal double-fan resonant ring array is composed of at least 100×100 metal structural units of the same size and uniform arrangement; Wherein, the size of the metal structure unit is 50 μm×50 μm; The metal wire used to make the metal structural unit has a width of 3μm and a thickness of 200nm; the gap width is 3μm, the straight-line distance from the center of the metal structural unit to the inner edge endpoint of the gap is 20μm, and the fan-shaped resonant ring width is 22μm; the metal structural unit is composed of two identical fan-shaped resonant rings with an angle of 60°, forming a structure similar to an "8" shape; and two identical gaps are formed above and below the "8" structure.

2. The method according to claim 1, characterized in that The dielectric substrate is a rigid substrate or a flexible substrate; The material of the hard substrate is quartz and has a thickness of 1 mm; The flexible substrate is made of polyimide and has a thickness of 25 μm.

3. The method according to claim 1, characterized in that The material of the metal is gold.

4. The method according to claim 1, wherein The metal structure unit is attached to the dielectric substrate through photolithography and / or metallization process.

5. The method according to claim 1, wherein The concentration of the carcinoembryonic antigen in step (3) is 100 ng / ml-2 μg / ml.

6. The method according to any one of claims 1 to 5, characterized in that The minimum detection limit of the carcinoembryonic antigen is 34.83 ng / ml.

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