阅读说明：本技术 太赫兹超材料生物传感器及其制备方法和检测方法 (Terahertz metamaterial biosensor and preparation method and detection method thereof ) 是由 崔宁 关敏 徐梦轲 张杨 曾一平 于 2019-09-26 设计创作，主要内容包括：一种太赫兹超材料生物传感器及其制备方法和检测方法,该生物传感器包括：衬底；设置在所述衬底上用于响应太赫兹波的超材料结构,其上设有若干检测区；以及识别单元,修饰在所述检测区上,用于识别不同标志物。本发明提供的太赫兹超材料生物传感器,采用的超材料是一种非对称的高Q值结构,对周围环境介质的变化响应灵敏度非常高,癌症标志物与超材料结构表面结合,导致其太赫兹透射光谱的红移,且癌症标志物浓度与峰位移动呈函数相关性。通过该标志物的检测浓度与太赫兹透射光谱对比曲线,可以实现对病人血清中的标志物浓度的检测。(A terahertz metamaterial biosensor, a preparation method and a detection method thereof are provided, the biosensor comprises: a substrate; the metamaterial structure is arranged on the substrate and used for responding to terahertz waves, and a plurality of detection areas are arranged on the metamaterial structure; and a recognition unit, which is modified on the detection area and is used for recognizing different markers. According to the terahertz metamaterial biosensor provided by the invention, the adopted metamaterial is an asymmetric high-Q-value structure, the response sensitivity to the change of a surrounding environment medium is very high, the cancer marker is combined with the surface of the metamaterial structure, so that the red shift of a terahertz transmission spectrum is caused, and the concentration of the cancer marker is in functional correlation with the peak position movement. The detection of the concentration of the marker in the serum of the patient can be realized through the contrast curve of the detection concentration of the marker and the terahertz transmission spectrum.)

1. A terahertz metamaterial biosensor comprising:

a substrate;

the metamaterial structure is arranged on the substrate and used for responding to terahertz waves, and a plurality of detection areas are arranged on the metamaterial structure; and

and the recognition unit is modified on the detection area and used for recognizing different markers.

2. The biosensor of claim 1,

the metamaterial structure is a periodic unit of resonant rings arranged in an array of four square rings;

each square ring in the square ring array is an open square ring or a closed square ring;

each square ring in the array of square rings comprises a single ring, a double ring, or a tricyclic ring;

the rotation angle of each square ring in the square ring array is 0-90 degrees;

the square ring array further comprises a grid, and the grid is arranged between the square ring and the square ring in the square ring array or arranged inside the square ring.

3. The biosensor of claim 1,

the metamaterial structure comprises a bottom metal layer and a surface metal layer;

the bottom metal layer is made of any one or a combination of gold, silver, copper, titanium and nickel;

the surface metal layer of the metamaterial structure is made of gold;

the thickness of the surface metal layer is 100-200 nm;

the total thickness of the bottom metal layer and the surface metal layer of the metamaterial structure is 0.1-2 μm;

the bottom metal layer is obtained by an electroplating or magnetron sputtering method;

the surface metal layer is obtained by an electron beam evaporation method.

4. The biosensor of claim 1,

each of said test zones modifies a recognition element;

the test zone is square or circular in shape.

5. The biosensor of claim 1,

the recognition unit comprises any one of carcinoembryonic antigen, glycoprotein antigen 125, alpha fetoprotein, glycoprotein antigen 15-3, glutamyl transpeptidase isozyme II, glycoprotein antigen 19-9, glycoprotein antigen 42, microRNA4484, microRNA3646 and microRNA probe.

6. The biosensor of claim 1,

the substrate comprises any one of quartz, silicon, gallium arsenide, polyimide or polymethylpentene;

the substrate thickness is less than or equal to 500 μm;

when the substrate is quartz, silicon or gallium arsenide, the thickness of the substrate is not more than 200 μm.

7. The biosensor of claim 1,

the response waveband of the biosensor is a terahertz waveband;

the response wave band of the biosensor is 0.5-3 THz.

8. A method of making the biosensor of any one of claims 1-7, comprising:

preparing a metamaterial structure on a substrate; and

and modifying the recognition unit on the metamaterial structure.

9. The method of claim 8,

the method also comprises the following steps before the metamaterial structure is prepared on the substrate: sequentially cleaning the substrate with acetone, absolute ethyl alcohol and deionized water;

the preparation of the metamaterial structure on the substrate comprises the following steps:

spin-coating photoresist on a substrate and then exposing to obtain a metamaterial structure pattern;

growing a metal layer on the exposed metamaterial structure pattern to obtain a metamaterial structure;

the step of obtaining the metamaterial structure pattern is realized by adopting a micro-nano processing photoetching technology;

wherein the step of obtaining the metamaterial structure further comprises the following steps: the substrate is thinned to a thickness of less than or equal to 500 μm.

10. A method for detecting a marker using the biosensor of any one of claims 1-7 or the biosensor prepared by the method of any one of claims 8-9, comprising:

adding the serum to be tested into a test area of the biosensor, and testing the biosensor after reaction to obtain a terahertz transmission spectrum of the serum to be tested;

comparing the terahertz transmission spectrum of the serum to be detected with a standard concentration comparison card to obtain the concentration of the cancer marker in the serum to be detected;

wherein the reaction time in the reaction step is 30-60 minutes;

wherein, the step of obtaining the standard concentration contrast card comprises:

testing terahertz transmission spectra of markers with different concentrations;

obtaining a curve which is the standard concentration comparison card according to the relation between the peak position displacement of the obtained terahertz transmission spectrum and the marker concentration;

wherein at least four different concentrations of the cancer markers at different concentrations are tested, and the concentration of the markers is 1ng/mL-10 mug/mL.

Technical Field

The invention relates to the field of biological detection, in particular to a terahertz metamaterial biosensor and a preparation method and a detection method thereof.

Background

Early stages of cancer development often have no obvious signs of disease and are often overlooked by patients, and when symptomatic, the cancer develops to a high degree or has metastasized. Research shows that cancer cells can generate different cancer marker proteins after being formed, and the cancer marker proteins can help people to diagnose whether corresponding cancer cells exist in vivo. Therefore, the concentration of cancer marker protein plays a key role in early cancer diagnosis.

The terahertz spectrum technology has the advantages of instantaneity, no label, nondestructive detection and the like, and with the development of the micro-nano processing technology, the terahertz sensor based on the metamaterial receives great attention. By changing the structure and the size of the metamaterial resonance unit, the resonance characteristic of the metamaterial resonance unit to the terahertz waves can be artificially manipulated, and the metamaterial resonance unit can have high response rate to surface medium changes. Currently, widely used structures include SRR (split ring resonator) type, cross type, and coupling type. Among them, Fano resonance caused by asymmetric metamaterial has sharp spectral shape and high Q factor (quality factor), and is especially suitable for detecting tiny dielectric change on surface. A cancer marker antibody capable of being specifically recognized is modified on a Fano resonance metamaterial, then a corresponding antigen is detected on the cancer marker antibody, and after the antigen antibody is combined, the surface dielectric medium parameter of the metamaterial can be changed and is reflected on a terahertz transmission spectrum, namely the shift of the transmission peak frequency.

Because the dielectric constants of different cancer markers are different, the frequency change rule of different cancer markers combined on the super surface is also different, and a marker concentration comparison card is obtained by measuring the concentration of the cancer markers and the relation curve of the transmission peak displacement in advance. When measuring a certain cancer marker in serum, the concentration of the marker can be obtained only by corresponding the transmission peak displacement to the previously obtained concentration comparison card.

Early clinical diagnosis methods for a certain protein concentration in a serum sample mainly utilized biochemical methods, such as enzyme-linked immunosorbent assay (ELISA), western blot assay (WB), immunofluorescence assay (IFA), etc., however, these methods are complicated and time-consuming to operate, and cannot meet the current demand for rapid detection. Therefore, it is highly desirable to develop new biosensors that are more sensitive, easy to handle, label-free and in particular fast-mode.

Disclosure of Invention

In view of the above, one of the main objectives of the present invention is to provide a terahertz metamaterial biosensor, and a method for manufacturing the same and a method for detecting the same, so as to at least partially solve at least one of the above technical problems.

In order to achieve the above object, as one aspect of the present invention, there is provided a terahertz metamaterial biosensor comprising:

a substrate;

the metamaterial structure is arranged on the substrate and used for responding to terahertz waves, and a plurality of detection areas are arranged on the metamaterial structure; and

and the recognition unit is modified on the detection area and used for recognizing different markers.

As another aspect of the present invention, there is also provided a method for preparing the biosensor as described above, comprising the steps of:

preparing a metamaterial structure on a substrate; and

and modifying the recognition unit on the metamaterial structure.

As still another aspect of the present invention, there is also provided a method of detecting a marker using the biosensor as described above or the biosensor prepared by the method as described above, comprising:

adding the serum to be tested into a test area of the biosensor, and testing the biosensor after reaction to obtain a terahertz transmission spectrum of the serum to be tested;

and comparing the terahertz transmission spectrum of the serum to be detected with a standard concentration comparison card to obtain the concentration of the cancer marker in the serum to be detected.

Based on the technical scheme, compared with the prior art, the terahertz metamaterial biosensor, the preparation method and the detection method thereof have at least one of the following advantages:

1. according to the terahertz metamaterial biosensor provided by the invention, the adopted metamaterial is an asymmetric high-Q-value structure, the response sensitivity to the change of a surrounding environment medium is very high, the cancer marker is combined with the surface of the metamaterial structure, so that the red shift of a terahertz transmission spectrum is caused, and the concentration of the cancer marker is in functional correlation with the peak position movement. The detection of the concentration of the marker in the serum of the patient can be realized through the comparison curve of the detection concentration of the marker and the terahertz transmission spectrum;

2. the detection method provided by the invention has strong reliability and high sensitivity, can quickly detect the concentrations of different cancer markers, and is suitable for detecting most cancer markers and biospecific recognition.

Drawings

FIG. 1 is a schematic diagram illustrating a terahertz metamaterial biosensor testing principle according to an embodiment of the present invention;

FIG. 2 is an electron microscope image of a metamaterial structure according to an embodiment of the present invention, wherein a is a single ring plus gate structure, b is a basic double ring structure, c is a rotating double ring structure, d is a double ring plus outer gate structure, e is a double ring plus inner gate structure, and f is a three ring nested structure;

FIG. 3 is a graph showing the shift of terahertz peaks of carcinoembryonic antigen proteins at different concentrations in a rotating double-ring structure test according to an embodiment of the present invention;

fig. 4 is a graph showing the relationship between the peak shift of the terahertz of the rotating double-ring structure and the concentration of the carcinoembryonic antigen protein according to the embodiment of the present invention.

Description of reference numerals:

1-a substrate; 2-metamaterial structure; 3-an identification unit; 4-a marker.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The invention discloses a terahertz metamaterial biosensor, which comprises:

a substrate;

the metamaterial structure is arranged on the substrate and used for responding to terahertz waves, and a plurality of detection areas are arranged on the metamaterial structure; and

and the recognition unit is modified on the detection area and used for recognizing different markers.

The metamaterial structure is a periodic unit of resonant rings arranged in an array of four square rings;

each square ring in the square ring array is an open square ring or a closed square ring;

wherein each square ring in the array of square rings comprises a single ring, a double ring, or a tricyclic ring;

wherein the rotation angle of each square ring in the square ring array is 0-90 degrees;

the square ring array further comprises a grid, and the grid is arranged between the square ring and the square ring in the square ring array or arranged inside the square ring.

The metamaterial structure comprises a bottom metal layer and a surface metal layer;

the bottom metal layer is made of any one or a combination of more of gold, silver, copper, titanium and nickel;

the surface metal layer of the metamaterial structure is made of gold;

wherein the thickness of the surface metal layer is 100-200 nm;

wherein the total thickness of the bottom metal layer and the surface metal layer of the metamaterial structure is 0.1-2 μm;

wherein the bottom metal layer is obtained by an electroplating or magnetron sputtering method;

wherein the surface metal layer is obtained by an electron beam evaporation method.

Wherein each of said test zones modifies a recognition element;

wherein the test zone is square or circular in shape.

Wherein, the recognition unit comprises any one of carcinoembryonic antigen, glycoprotein antigen 125, alpha fetoprotein, glycoprotein antigen 15-3, glutamyl transpeptidase isozyme II, glycoprotein antigen 19-9, glycoprotein antigen 42, microRNA4484, microRNA3646 and microRNA probe.

Wherein the substrate comprises any one of quartz, silicon, gallium arsenide, polyimide, or polymethylpentene;

wherein the substrate thickness is less than or equal to 500 μm;

wherein, when the substrate is quartz, silicon or gallium arsenide, the thickness of the substrate is not more than 200 μm.

Wherein the response waveband of the biosensor is a terahertz waveband;

wherein the response band of the biosensor is 0.5-3 THz.

The invention also discloses a method for preparing the biosensor, which comprises the following steps:

preparing a metamaterial structure on a substrate; and

and modifying the recognition unit on the metamaterial structure.

Wherein, the method also comprises the following steps before the metamaterial structure is prepared on the substrate: sequentially cleaning the substrate with acetone, absolute ethyl alcohol and deionized water;

wherein, the preparation of the metamaterial structure on the substrate comprises the following steps:

spin-coating photoresist on a substrate and then exposing to obtain a metamaterial structure pattern;

growing a metal layer on the exposed metamaterial structure pattern to obtain a metamaterial structure;

the step of obtaining the metamaterial structure pattern is realized by adopting a micro-nano processing photoetching technology;

wherein the step of obtaining the metamaterial structure further comprises the following steps: the substrate is thinned to a thickness of less than or equal to 500 μm.

The present invention also discloses a method for detecting a marker using the biosensor according to any one of claims 1 to 7 or the biosensor prepared by the method according to any one of claims 8 to 9, comprising:

adding the serum to be tested into a test area of the biosensor, and testing the biosensor after reaction to obtain a terahertz transmission spectrum of the serum to be tested;

comparing the terahertz transmission spectrum of the serum to be detected with a standard concentration comparison card to obtain the concentration of the cancer marker in the serum to be detected;

wherein the reaction time in the reaction step is 30-60 minutes;

wherein, the step of obtaining the standard concentration contrast card comprises:

testing terahertz transmission spectra of markers with different concentrations;

obtaining a curve which is the standard concentration comparison card according to the relation between the peak position displacement of the obtained terahertz transmission spectrum and the marker concentration;

wherein at least four different concentrations of the cancer markers at different concentrations are tested, and the concentration of the markers is 1ng/mL-10 mug/mL.

In an exemplary embodiment of the invention, a negative refractive index terahertz metamaterial biosensor applied to early warning of cancer comprises a substrate 1, a negative refractive index metamaterial structure 2 grown on the substrate, and antibodies or probes (i.e. identification units) 3 which are modified on the metamaterial structure and used for identifying different cancer markers.

In some embodiments of the present invention, the substrate may be quartz, high resistivity silicon, gallium arsenide, polyimide, polymethylpentene, or the like. After the growth of the metamaterial metal layer is finished, the substrate needs to be thinned, and the thickness is controlled to be not higher than 500 mu m.

In some embodiments of the present invention, the metamaterial structure is a periodic unit of metal open resonant rings (SRRs) arranged in an array, where each square ring in the square ring array may be an open square ring or a closed square ring;

each square ring in the array of square rings can be monocyclic, bicyclic, or tricyclic;

each square ring in the square ring array can rotate for a certain angle, and the rotation angle range is 0-90 degrees;

the square ring array further comprises a grid; the grid can be added between the square rings in the square ring array or inside the square rings.

The rotation and grid-adding function is to improve the Q factor of the metamaterial structure and improve the sensitivity.

In the following, several structures are specifically exemplified by taking fig. 2 as an example:

the periodic unit of the resonance ring (SRR) can be a metamaterial structure such as a double ring, a rotary double ring, a double ring grid, a three-ring nested ring grid, a single ring grid and the like based on an open square ring.

As shown in FIG. 2a, it is a single ring with gate structure;

as shown in fig. 2b, the inner and outer nested structure is adopted, the distance between the two rings is consistent with the line width, and the openings can be in the same direction or opposite directions;

as shown in fig. 2c, the rotating angle of the double-ring rotating structure is 0-90 °;

as shown in fig. 2e, the double-ring gate structure can add a horizontal or vertical gate in the middle of four square ring groups, and the gate length is 85-130 μm; the grid can also be added in the inner part of each ring, as shown in the d diagram in FIG. 2, the grid length is 8-48 μm;

as shown in FIG. 2f, the three-ring nested structure is a three-ring nested structure, the side length of the three-ring nested structure ring is 80-150 μm, the line width of the ring is 2-6 μm, and the ring distance is consistent with the line width.

In some embodiments of the present invention, the metal in the metamaterial structure may be one or more of gold, silver, copper, titanium, and nickel, wherein gold is a surface layer of the metamaterial structure.

In some embodiments of the present invention, the total thickness of the metamaterial metal layer is 100nm-2 μm, and the thickness of the surface layer gold is 100-200 nm.

The preparation method of the metal comprises the following steps: the growth method of the bottom metal layer is electroplating or magnetron sputtering, and the growth method of the surface gold layer is electron beam evaporation.

In some embodiments of the invention, the response band of the biosensor is in the terahertz range, which is 0.5-3 THz.

In some embodiments of the invention, the antibodies or probes recognizing different cancer markers modified on the metamaterial are: CEA (carcinoembryonic antigen), CA125 (glycoprotein antigen 125), AFP (alpha fetoprotein), CA15-3 (glycoprotein antigen 15-3), GGT-II (glutamyl transpeptidase isozyme II), CA19-9 (glycoprotein antigen 19-9), CA42 (glycoprotein antigen 42), microRNA4484, microRNA3646 and other conventional cancer marker antibodies and microRNA probes.

The detection method of the negative refractive index terahertz metamaterial biosensor for early warning of cancer, which is described above, has the principle shown in fig. 1, and comprises the following steps:

(1) firstly, cancer markers with different concentrations are sequentially added into a metamaterial test area, and the area of the test area is 0.04-1cm²；

According to the difference of the markers, the reaction time is 30-60 minutes, and then the terahertz transmission spectrum of the sensor is tested;

(2) obtaining a marker standard concentration comparison card through a curve of terahertz peak position displacement delta f changing along with concentration; the metamaterial structure can respond to a terahertz wave band, has a tuning effect on a terahertz transmission peak, and can generate displacement when a medium around the metamaterial is changed.

(3) The THz transmission spectrum of the patient serum is analyzed by a comparison card of the standard concentration, so that the concentration of the cancer marker in the patient serum can be obtained.

According to the curve of the transmission peak position migration quantity delta f and the change of the marker concentration, the concentration comparison card of various cancer markers can be obtained.

In another exemplary embodiment of the invention, a negative refractive index terahertz metamaterial biosensor applied to early warning of cancer, as shown in fig. 1, comprises a substrate 1, a negative refractive index metamaterial structure 2 grown on the substrate, and a recognition unit 3 modified on the metamaterial and identifying different cancer markers 4, wherein the recognition unit is an antibody or a probe 3. The preparation method comprises the following specific steps:

(1) substrate preparation:

the substrate of the negative refractive index terahertz metamaterial biosensor applied to early warning of cancers is quartz, silicon, gallium arsenide, polyimide or polymethylpentene and the like.

Before the metamaterial processing technology is carried out, the substrate material is placed in an acetone solution to be boiled for 5-15 minutes to remove organic pollutants on the surface of the substrate, then the substrate material is boiled in an absolute ethyl alcohol solution for 5-15 minutes to remove acetone, finally the substrate material is washed with deionized water for three times and dried with nitrogen.

(2) Constructing a metamaterial structure:

the metamaterial with the negative refractive index is a metal open resonance ring (SRR) periodic unit which is arranged in an array mode, and the resonant ring (SRR) periodic unit can be a metamaterial structure which is based on an open square ring and has a double ring structure, a rotating double ring structure, a double ring grid structure, a three-ring nested structure, a single ring grid structure and the like.

And transferring the metamaterial pattern on the substrate by adopting a micro-nano processing photoetching technology. First, a photoresist (AR-N4340) is coated on the previously processed substrate by spin coating, and exposed to light to build those metamaterial structures on the substrate.

In some embodiments of the invention, the metamaterial structures are arranged periodically in groups of four square ring structures; the side length of the square ring with the opening is 40-60 mu m, the distance between the two square rings is 5-10 mu m, the line width of the ring is 2-6 mu m, and the width of the opening of the ring is consistent with that of the ring;

in some embodiments of the present invention, as shown in fig. 2a, the metamaterial structure is a single-ring gate structure;

in some embodiments of the present invention, as shown in fig. 2b, the metamaterial structure is a dual-opening ring, which is an inside-outside nested structure, the distance between two rings is consistent with the line width, and the openings may be in the same direction or in opposite directions;

in some embodiments of the present invention, as shown in fig. 2c, the metamaterial structure is a double-ring rotating structure, and the rotating angle of the double-ring rotating structure is 0-90 °;

in some embodiments of the present invention, as shown in fig. 2e, the double-ring gate-added structure may add a horizontal or vertical gate in the middle of the four square ring groups, the gate length is 85-130 μm, or add a gate in the interior of each ring, the gate length is 8-48 μm, as shown in fig. 2 d;

in some embodiments of the present invention, as shown in fig. 2f, a three-ring nested structure is provided, wherein the side length of the outermost ring is 80-150 μm, the line width of the ring is 2-6 μm, and the ring distance is consistent with the line width;

in some embodiments of the invention, the metamaterial coverage area on the surface of the biosensor is a test area, each test area is square or round, and the side length or the round radius of each square is 0.2-1 cm;

in some embodiments of the invention, the metamaterial pattern is a rotating double-opening ring, the rotation angle is 22.5 degrees, and the opening is 2 μm; and the double-ring external grid structure has a grid length of 105 mu m.

(3) Metal growth

The metal in the metamaterial structure can be one or more of gold, silver, copper, titanium and nickel, wherein the gold is a surface layer of the metamaterial structure.

Wherein the total thickness of the metamaterial metal is 100nm-2 μm, and the thickness of the surface gold is 100-200 nm;

the growth method of the metal is electroplating or magnetron sputtering, and the growth method of the surface gold is electron beam evaporation.

(4) Substrate thinning

After the step (3) is finished, the substrate needs to be thinned, and the thickness is controlled to be below 500 mu m.

When the substrate is quartz, high-resistance silicon or gallium arsenide, the thickness is not more than 200 μm.

(5) Modified cancer marker antibodies

The surface of the test area was incubated with 1-1.5. mu.g/mL mercaptopropionic acid (MPA) at room temperature for 15-24h, followed by incubation with 10-60mM N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 1: 1 mixing, as an activating agent, and incubating for 10-30 minutes at room temperature;

antibodies or probes that modify different cancer markers: CEA (carcinoembryonic antigen), CA125 (glycoprotein antigen 125), AFP (alpha fetoprotein), CA15-3 (glycoprotein antigen 15-3), GGT-II (glutamyl transpeptidase isozyme II), CA19-9 (glycoprotein antigen 19-9), CA42 (glycoprotein antigen 42), microRNA4484, microRNA3646 and other conventional cancer marker antibodies and microRNA probes.

Adding the antibody or the probe into the test area, and incubating for 10-24h at 0-10 ℃ according to different markers.

Wherein one test region modifies one antibody or probe;

wherein the concentration of the modified antibody or probe is 1-10 mug/mL.

2. The method for rapidly and accurately detecting the cancer marker protein by using the negative-refractive-index terahertz metamaterial biosensor comprises the following steps:

(1) firstly, cancer markers with different concentrations are sequentially added into a test area of a metamaterial structure, and the area of the test area is 0.04-1cm²；

According to the difference of the markers, the reaction time is 30-60 minutes, and then the terahertz transmission spectrum of the sensor is tested;

(2) obtaining a marker standard concentration comparison card through a curve of terahertz peak position displacement delta f changing along with concentration;

(3) the concentration of the cancer marker in the serum of the patient can be detected by analyzing the terahertz transmission spectrum of the serum of the patient and a standard concentration comparison card.

The different cancer detection markers in the invention are conventional cancer markers such as carcinoembryonic antigen CEA (carcinoembryonic antigen), CA125 (glycoprotein antigen 125), AFP (alpha fetoprotein), CA15-3 (glycoprotein antigen 15-3), GGT-II (glutamyl transpeptidase isozyme II), CA19-9 (glycoprotein antigen 19-9), CA42 (glycoprotein antigen 42), microRNA4484, microRNA3646 and the like, and microRNA.

Wherein, the obtained curve of the peak position displacement delta f changing along with the concentration at least comprises 4 groups of concentration and peak position values;

wherein, the concentration of the marker in the step (2) can be at least four groups of concentrations in the range of 1ng/mL to 10 mu g/mL.

The technical solution of the present invention is further illustrated by the following specific examples. It should be noted that the following specific examples are given by way of illustration only and the scope of the present invention is not limited thereto.

The chemicals and raw materials used in the following examples were either commercially available or self-prepared by a known preparation method.

FIG. 1 is a schematic diagram of a negative refractive index terahertz metamaterial biosensor testing according to an embodiment of the invention.