ERalpha functionalized HEMT biosensor and construction method and application thereof

Abstract

The invention provides an ERalpha functionalized HEMT (High Electron Mobility Transistor) biosensor as well as a construction method and application thereof. The ERalpha functionalized HEMT biosensor comprises (1) an ERalpha functionalized HEMT biosensor; (2) a construction method of the ERalpha functionalized HEMT biosensor; and (3) application of the ERalpha functionalized HEMT biosensor in detection of an ERalpha potential antagonist. The biosensor provided by the invention takes an HEMT device as a carrier, is formed by functionally modifying ERalpha through chemical reaction, has the advantages of high sensitivity (the detection limit is as low as 1 fM), strong specificity, wide linear range (1 fM-100 nM) and the like, can efficiently and accurately identify the ERalpha potential antagonist, and provides a method or technical support for screening of ERalpha potential antagonist drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection of receptor antagonists, and in particular to an ERα-functionalized HEMT biosensor, a construction method thereof and an application thereof. Background Art

[0002] ERα plays an important role in various tissues and cells, and is involved in regulating the development and function of the reproductive system, such as promoting the development of mammary alveoli and maintaining the periodic changes of the endometrium. ERα is related to the occurrence and development of various gynecological tumors such as breast cancer, endometrial cancer and ovarian cancer. Research shows that after estrogen binds to ERα, it induces the transcription of target genes by acting on the estrogen response element in the promoter region of the target gene, and then promotes the proliferation, differentiation, invasion and metastasis of breast cancer cells. ERα is one of the key targets for the treatment of diseases such as breast cancer and mammary hyperplasia, and has important therapeutic significance especially in the treatment of estrogen receptor-positive breast cancer. ERα antagonists bind to ERα competitively with estrogen, block the growth-promoting effect of estrogen on cancer cells, and thus inhibit the proliferation of tumor cells. The detection of ERα receptor antagonists is of great significance for designing and developing candidate drugs with novel anti-estrogen receptor potential.

[0003] However, the existing detection technologies for ERα receptor antagonists have many limitations: firstly, the proliferation inhibition experiment based on cell lines requires high-concentration compounds (usually in the μM level) to observe significant activity, resulting in easy omission of low-abundance active molecules (such as metabolites, natural products or novel chemical entities); secondly, the existing detection methods are often complex in operation, high in technical requirements, long in experimental period, and expensive in the instrument equipment used and high in cost, which is not convenient for batch sample detection. Summary of the Invention

[0004] The purpose of the present invention is to provide an ERα-functionalized HEMT biosensor, a construction method thereof and an application thereof, aiming to develop a detection technology with high sensitivity (the detection limit is as low as 1 fM), strong specificity and wide linear range (1 fM - 100 nM), which can efficiently and accurately identify potential ERα antagonists, so as to provide a more efficient tool for drug screening and accelerate the discovery and development of novel ERα antagonist drugs.

[0005] In the first aspect, the present invention provides a construction method of an ERα-functionalized HEMT biosensor, and the method includes: Step S01: Obtain a quartz glass tube with both ends open, and use a HEMT device as a carrier to bond one open end of the quartz glass tube to its gate region to form a sample pool; Step S02: Add an aqueous solution of 3-mercaptopropionic acid to the sample cell to react with the gate region, generating Au-S bonds on the surface to form a self-assembled monolayer. Step S03: Add a mixed aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide for activation to generate a stable amine-activated product. Step S04: After the activation is completed, add the pre-prepared aqueous solution of ERα protein to the sample cell for reaction, and the ERα-functionalized HEMT biosensor is obtained upon completion.

[0006] In some embodiments, the HEMT biosensor is a semiconductor device based on an AlGaAs / InGaAs heterostructure, having advantages such as high sensitivity, high specificity, fast response, and easy integration, and showing great application prospects in biochemical detection. By modifying an active drug target protein (such as a receptor) on the surface of the HEMT device, when an analyte drug (such as a ligand) is added and interacts, it will cause a change in the two-dimensional electron gas density (2DEG) in the HEMT channel, and the detection of the target drug can be achieved through the change in the electrochemical signal. In the present invention, by modifying ERα on the HEMT device, an ERα-functionalized HEMT biosensor is constructed for the detection of potential ERα antagonists, providing technical support for high-throughput drug screening and new drug research and development.

[0007] Further, in the step S01: The quartz glass tube has an inner diameter of 5 mm and a height of 15 mm. The gate region includes a gate, and the surface of the gate is coated with a gold film.

[0008] Further, in the step S02: The concentration of the aqueous solution of 3-mercaptopropionic acid is 15 - 25 mM. The reaction time is 10 - 14 h, and the reaction temperature is 10 - 30 °C.

[0009] Further, in the step S03: The concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 15 - 25 mM, the concentration of N-hydroxysuccinimide is 40 - 60 mM, and the volume ratio is 1:1. The activation time is 15 - 30 min, and the activation temperature is 10 - 30 °C.

[0010] Further, in the step S04: The concentration of the aqueous solution of ERα protein is 8 - 12 μg / mL. The reaction time is 10 - 14 h, and the reaction temperature is 2 - 8 °C.

[0011] In a second aspect, the present invention provides an ERα-functionalized HEMT biosensor constructed according to the above method.

[0012] In a third aspect, the present invention provides an application of the HEMT biosensor constructed according to the above method in detecting potential ERα antagonists, and the application includes: Loading a solution of the compound to be tested onto the constructed ERα-functionalized HEMT biosensor; Using an electrochemical workstation to scan and record the current intensity between the source and drain of the ERα-functionalized HEMT biosensor loaded with the solution of the compound to be tested, repeating the measurement three times to obtain three current intensity values I1, I2, and I3; Using the solvent used to prepare the solution of the compound to be tested as a blank control, using an electrochemical workstation to scan and record the current intensity between the source and drain of the ERα-functionalized HEMT biosensor loaded with the blank control, repeating the measurement three times to obtain three current intensity values I 01 、I 02 and I 03 ; Taking the three current intensity values I1, I2, I3 as the first group of data, and taking the three current intensity values I 01 、I 02 、I 03 as the second group of data, and using the t-test method to determine whether there is a significant difference between the first group of data and the second group of data; If there is a significant difference, then the compound to be tested is a potential ERα antagonist.

[0013] Furthermore, the detection method is linear sweep voltammetry; Setting the constant voltage range of the linear sweep voltammetry to 0 - 2 V, the current limit to 0.1 A, and the step value to 2 mV.

[0014] In a fourth aspect, the present invention provides an application of the above ERα-functionalized HEMT biosensor in detecting the strength of the interaction between ERα protein and potential ERα antagonists, and the application includes: Weighing a certain weight of potential ERα antagonist and preparing a stock solution with a concentration of 10 mM; Taking the stock solution and performing serial dilution by a factor of 10 to obtain a set of ERα potential antagonist solutions with gradient concentrations; On the constructed ERα-functionalized HEMT biosensor, loading different concentrations of ERα potential antagonist solutions in ascending order of concentration, using an electrochemical workstation to scan and record the current intensity I between the source and drain, repeating the measurement three times, calculating the average value, and obtaining I; Using the solvent used to prepare the ERα potential antagonist solution as a blank control, an electrochemical workstation was used to scan and record the source-drain current intensity of the ERα-functionalized HEMT biosensor loaded with the blank control. The measurement was repeated three times, and the average value was calculated to obtain I0; With the logarithm of the concentration of the ERα potential antagonist solution, Lg[A g as the abscissa and the relative value of the current change (I - I0) / I0 as the ordinate, at least five consecutive data points were selected for a linear fitting to obtain at least one linear fitting function, and the target linear fitting function with R 2 > 0.9 was selected from the at least one linear fitting function; Two data points belonging to the endpoints in the target linear fitting function were obtained, and a target linear range was generated with the concentrations of the ERα potential antagonist solution corresponding to the two data points belonging to the endpoints. Within the target linear range, with the concentration of the ERα potential antagonist solution [A g as the abscissa and the ratio of the concentration [A g to the current change value I - I0 as the ordinate, a quadratic linear fitting was performed to obtain a quadratic linear fitting function, and the dissociation equilibrium constant K D value of the interaction between the ERα protein and the ERα potential antagonist was calculated according to the quadratic linear fitting function; According to the magnitude of the dissociation equilibrium constant K D value, the strength of the interaction between the ERα protein and the ERα potential antagonist was judged. The smaller this value, the stronger the interaction. Description of the Drawings

[0015] Figure 1 are the electrochemical signals and X-ray photoelectron spectroscopy (XPS) results of the HEMT biosensor before and after modification with 3-mercaptopropionic acid and ERα protein. Among them, (a) is the characterization result of the electrochemical signal, (b) is the total XPS scan spectrum, (c) is the XPS carbon fine scan spectrum, and (d) is the XPS nitrogen fine scan spectrum; Figure 2 is the response of the ERα-functionalized HEMT sensor to the test compound solution. Among them, (a) to (e) are the responses of the ERα-functionalized HEMT sensor of the present invention to tamoxifen, toremifene, raloxifene, sucrose, and sodium chloride at a concentration of 1 pM; Figure 3 are the results of detecting tamoxifen using the ERα-functionalized HEMT biosensor. Among them, (a) is the I DS -V DS signal change with the concentration of tamoxifen, and (b) is the relative current change (I - I0) / I0 and the logarithm concentration of tamoxifen Lg[A gThe linear relationship between (c) the ratio of tamoxifen concentration to current change [A g / ΔI and the tamoxifen concentration [A g ; Figure 4 Shows the results of detecting toremifene using an ERα-functionalized HEMT biosensor. Among them, (a) is the change of I DS -V DS signal with the change of toremifene concentration, (b) is the linear relationship between the relative current change (I - I0) / I0 and the logarithm concentration Lg[A g of toremifene, and (c) is the ratio of toremifene concentration to current change [A g / ΔI and the toremifene concentration ([A g ); Figure 5 Shows the results of detecting raloxifene using an ERα-functionalized HEMT biosensor. Among them, (a) is the change of I DS -V DS signal with the change of raloxifene concentration, (b) is the linear relationship between the relative current change (I - I0) / I0 and the logarithm concentration Lg[A g of raloxifene, and (c) is the ratio of raloxifene concentration to current change [A g / ΔI and the toremifene concentration ([A g );

[0016] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meaning as understood by those of ordinary skill in the art in the field to which the present invention belongs. The words such as "including" used herein mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects.

[0018] Example 1 (1) Obtain a quartz glass tube with both ends open (inner diameter 5 mm × height 15 mm), and use the HEMT device as a carrier. Stick one open end of the quartz glass tube on its gate region to form a sample cell. The gate region includes a gate, the surface of which is coated with a gold film. Then, add 50 μL of 20 mM 3-mercaptopropionic acid aqueous solution to the sample cell and soak and react at room temperature for 12 h to form a self-assembled monolayer. After the formation of the Au-S bond, wash the chip surface three times with water to remove physically adsorbed 3-mercaptopropionic acid.

[0019] (2) Add 50 μL of a mixed aqueous solution of 20 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and an equal volume of 50 mM N-hydroxysuccinimide to activate the carboxyl group. After reacting at room temperature for 30 min, wash three times with water. After the activation reaction is completed, a stable amine activation product is formed on the chip surface.

[0020] (3) After diluting the ERα protein with water to a concentration of 10 μg / mL, take 50 μL and add it to the sample cell, and react at 4 °C for 12 h. After the reaction is completed, the ERα-functionalized HEMT biosensor is obtained.

[0021] Example 2 Use an electrochemical workstation and an X-ray photoelectron spectrometer (XPS) to characterize the electrochemical signals and elemental changes of the HEMT biosensor before and after modification with 3-mercaptopropionic acid and ERα protein during the construction process of Example 1. Figure 1 The electrochemical signals and XPS measurement results of the HEMT biosensor before and after modification with 3-mercaptopropionic acid and ERα protein are shown. Specifically, as can be seen from (a) in Figure 1 , compared with the bare chip, the current changes significantly after modification with 3-mercaptopropionic acid and ERα protein. As can be seen from (b) in Figure 1 to (d) in Figure 1 , after modification with 3-mercaptopropionic acid, an Au-S (S 2p) peak is formed at 162.5 eV, and a new carbonyl peak (O-C=O) appears at 289.0 eV, indicating the formation of SAM; while after modification with ERα protein, two new N1s peaks appear, located at 401.4 eV and 403.5 eV respectively, corresponding to the neutral amino group (NH2) and protonated amino group (NH3 + ) of the protein, and a C1s peptide bond peak (-CONH-) appears at 286.5 eV. The above results all indicate that 3-mercaptopropionic acid and ERα protein are successfully modified on the HEMT device, and the ERα-functionalized HEMT biosensor is successfully constructed.

[0022] Example 3 (1) It is known that sucrose and sodium chloride are not ERα antagonists (ligands), and tamoxifen, toremifene and raloxifene are all ERα antagonists. Tamoxifen, toremifene and raloxifene are prepared into a test solution with a concentration of 1 pM using 5% DMSO, and sucrose and sodium chloride are prepared into a test solution with a concentration of 1 pM using purified water. 50 μL of each of the above test solutions were loaded on the ERα functionalized HEMT biosensor prepared in Example 1, and reacted at room temperature for 1 h. The current intensity between the source and drain of the ERα functionalized HEMT biosensor loaded with the above test solution was scanned and recorded using an electrochemical workstation. Each sample was measured three times to obtain I1, I2 and I3; (2) Use an electrochemical workstation to scan and record the current intensity between the source and drain of each ERα-functionalized HEMT biosensor loaded with 50 μL of blank control. Repeat the measurement three times to obtain I 01 ,I 02 and I 03 ; (3) Take the three current intensity values ​​I1, I2, and I3 as the first set of data, and the three current intensity values ​​I 01 ,I 02 ,I 03 The second set of data was used to determine whether there was a significant difference between the first and second sets of data. Specifically, the current intensities I1, I2, and I3 measured after the ERα-functionalized HEMT biosensor was loaded with tamoxifen, toremifene, raloxifene, sucrose, and sodium chloride solutions were compared with I 01 ,I 02 and I 03 Conduct a t-test.

[0023] The results are as follows Figure 2 As shown in (a) to (e), tamoxifen, toremifene and raloxifene solutions produce strong responses on the ERα-functionalized HEMT biosensor, and there is a significant difference in the current intensity compared with the blank control (p < 0.01); while sucrose and sodium chloride solutions produce weak responses or no responses on the ERα-functionalized HEMT biosensor, and there is no significant difference in the current intensity compared with the blank control. This indicates that the ERα-functionalized HEMT biosensor in the present application can detect tamoxifen, toremifene and raloxifene as potential ERα antagonists, while sucrose and sodium chloride are not potential ERα antagonists, which is consistent with the actual situation.

[0024] Embodiment 4 (1) Preparation of the test solution: Weigh 3.87 mg of tamoxifen and prepare a stock solution with a concentration of 10 mM in a 5% DMSO aqueous solution. Then take the stock solution and dilute it successively by 10-fold to obtain a set of tamoxifen solutions with gradient concentrations in the range of 1 fM - 1 nM.

[0025] (2) Sample determination: On the ERα-functionalized HEMT biosensor constructed in Example 1, add 50 μL of tamoxifen solution in ascending order of concentration, react at room temperature for 1 h, use an electrochemical workstation to scan and record the source-drain current intensity of the ERα-functionalized HEMT biosensor loaded with tamoxifen solutions at different concentrations, repeat the determination three times, calculate the average value of the three current intensities to obtain I; (3) Using 50 μL of 5% DMSO aqueous solution as a blank control, use an electrochemical workstation to scan and record the source-drain current intensity of the ERα-functionalized HEMT biosensor loaded with 5% DMSO aqueous solution, repeat the determination three times, calculate the average value to obtain I0; (4) Using the logarithm of the tamoxifen concentration (Lg[A g ) as the abscissa and the relative value of the current change (I - I0) / I0 as the ordinate, select at least five consecutive data points for a linear fitting to obtain at least one linear fitting function, and select the target linear fitting function with R 2 > 0.9 from the at least one linear fitting function; (5) Obtain two data points that belong to the endpoints in the target linear fitting function, and generate a target linear range with the concentrations of the ERα potential antagonist solutions corresponding to the two data points that belong to the endpoints. Within the target linear range, use the tamoxifen concentration ([A g ) as the abscissa and the ratio of the concentration ([A g ) to the current change value (ΔI = I - I0) as the ordinate for a quadratic linear fitting to obtain a linear equation of = a + b, and calculate the dissociation equilibrium constant K D = b / a.

[0026] Measurement results: The I DS - V DS signals of ERα interacting with tamoxifen solutions at different concentrations are as shown in Figure 3 (a). When the concentration range is from 1 fM to 1 nM, the I DS signal shows a regular downward trend as the concentration of tamoxifen increases. As shown in Figure 3 (b), using the logarithm of the tamoxifen concentration (Lg[A gUsing the concentration ([A]]) as the abscissa and the relative value of current change ((I - I0) / I0) as the ordinate for linear fitting, when the concentration range is from 10 fM to 100 pM, a good linear relationship is presented, and the linear equation is =-0.0192 -0.1617 (R 2 =0.9477). As shown in (c) of Figure 3 using the concentration of tamoxifen ([A g ) as the abscissa and the concentration ([A g ) / current change (I - I0, ΔI) as the ordinate for linear fitting, the obtained linear equation is =1726.7577 -1.0824×10 -9 (R 2 =0.9996), with a good linear relationship. The dissociation equilibrium constant K D for the interaction between tamoxifen and ERα protein is calculated to be 6.2684×10 -13 M.

[0027] Derivation of the calculation process for the dissociation equilibrium constant K D : ; In the formula, [A b is the concentration of ERα protein, [A g is the concentration of tamoxifen solution [C], K A is the binding constant, K D is the dissociation constant, is the value of current change, is the maximum value of current change.

[0028] Example 5 This example is basically the same as Example 4, except that: (1) Preparation of the test solution: Weigh 4.06 mg of toremifene and prepare a stock solution with a concentration of 10 mM using a 5% DMSO aqueous solution. Then take the stock solution and perform serial 10-fold dilutions to obtain a set of toremifene solutions with gradient concentrations in the range of 10 fM - 100 nM.

[0029] Measurement results: The I DS -V DS signals for the interaction between ERα and toremifene solutions with different concentrations are as shown in (a) of Figure 4 . When the concentration range is from 10 fM to 100 nM, the I DS signals show a regular downward trend as the concentration of toremifene increases. As shown in (b) of Figure 4 using the logarithm of the toremifene concentration (Lg[A gUsing ) as the abscissa and the relative value of current change (I - I0) / I0 as the ordinate for linear fitting, when the concentration range is 1 pM - 100 nM, a good linear relationship is presented, and the linear equation is = -0.0058 - 0.1449 (R 2 = 0.9114). As shown in Figure 4 (c) of, using the concentration of toremifene ([A g ) as the abscissa and the concentration ([A g ) / current change (I - I0, ΔI) as the ordinate for linear fitting, the obtained linear equation is = -430.7821 - 7.1412×10 -8 (R 2 = 0.9999), and the linear relationship is good. Finally, the dissociation equilibrium constant K of the interaction between toremifene and ERα protein is calculated to be D 1.6577×10 -10 M.

[0030] Example Six This example is basically the same as Example Four, except that: (1) Preparation of the test solution: Weigh 4.06 mg of raloxifene and prepare a stock solution with a concentration of 10 mM in 5% DMSO aqueous solution. Then take the stock solution and dilute it successively by 10 times to obtain a set of raloxifene solutions with gradient concentrations, and the concentration range is 1 fM - 10 nM.

[0031] Measurement result: The I DS -V DS signal of ERα interacting with raloxifene solutions with different concentrations is as shown in Figure 5 (a) of. When the concentration range is 1 fM to 1 nM, the I DS signal shows a regular upward trend as the concentration of raloxifene increases. As shown in Figure 5 (b) of, using the logarithm of raloxifene concentration (Lg[A g ) as the abscissa and the relative value of current change (I - I0) / I0 as the ordinate for linear fitting, when the concentration range is 1 fM - 100 pM, a good linear relationship is presented, and the linear equation is = 0.0152 + 0.2341 (R 2 = 0.9688). As shown in Figure 5 (c) of, using the concentration of raloxifene ([A g ) as the abscissa and the concentration ([A gTaking the change in fluorescence intensity (F - F0, ΔF) / current change (I - I0, ΔI) as the ordinate, a linear fit is performed, and the linear equation obtained is = 2719.8192 + 2.6400×10 -10 (R 2 = 0.9993), indicating a good linear relationship. Finally, the dissociation equilibrium constant K D for the interaction between raloxifene and ERα protein is calculated to be 9.7065×10 -14 M.

[0032] It should also be noted that for Examples 2 to 6, the detection method for the ERα-functionalized HEMT biosensor is linear sweep voltammetry, with a constant voltage range of 0 - 2V, a current limit of 0.1 A, and a step value of 2 mV. During the test, the biosensor is placed on a probe station, the probe is pressed on the source and drain electrodes, and it is placed in an electromagnetic shielding box. The current intensity (I DS ) between the source and drain and the corresponding voltage (V DS ) are scanned and recorded.

[0033] As can be seen from Examples 4 to 6, the ERα-functionalized HEMT biosensor prepared by the present invention can effectively detect the dissociation equilibrium constant K D reflecting the interaction between ERα protein and ERα antagonist. The smaller the dissociation equilibrium constant, the stronger the affinity between ERα protein and ERα antagonist, and vice versa.

[0034] In summary, as can be seen from Examples 1 to 6, the ERα-functionalized HEMT biosensor constructed by the present invention has advantages such as high sensitivity (the lowest detection limit is as low as 1 fM), strong specificity, and a wide linear detection range (1 fM - 100 nM), thus enabling the efficient and accurate detection of potential ERα antagonists and providing technical support for drug screening and research.

[0035] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A method for constructing an ERα-functionalized HEMT biosensor, characterized in that, The method includes: Step S01: Obtain a quartz glass tube with both ends open, and using a HEMT device as a carrier, stick one open end of the quartz glass tube onto its gate region to form a sample cell. Step S02: Add an aqueous solution of 3-mercaptopropionic acid into the sample cell, react with the gate region, and form Au-S bonds on the surface to form a self-assembled monolayer. Step S03: Add a mixed aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide for activation to generate an amine-activated product. Step S04: After the activation is completed, add the pre-prepared aqueous solution of ERα protein into the sample cell for reaction, and the ERα-functionalized HEMT biosensor can be obtained upon completion.

2. The construction method of the ERα-functionalized HEMT biosensor according to claim 1, characterized in that, In the said Step S01: The quartz glass tube has an inner diameter of 5 mm and a height of 15 mm. The gate region includes a gate, and the surface of the gate is coated with a gold film.

3. The construction method of the ERα-functionalized HEMT biosensor according to claim 1, characterized in that, In the said Step S02: The concentration of the aqueous solution of 3-mercaptopropionic acid is 15 - 25 mM. The reaction time is 10 - 14 h, and the reaction temperature is 10 - 30 °C.

4. The preparation method of the ERα-functionalized HEMT biosensor according to claim 1, characterized in that, In the said Step S03: The concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 15 - 25 mM, the concentration of N-hydroxysuccinimide is 40 - 60 mM, and the volume ratio is 1:

1. The activation time is 15 - 30 min, and the temperature is 10 - 30 °C.

5. The construction method of the ERα-functionalized HEMT biosensor according to claim 4, characterized in that, In the said Step S04: The concentration of the aqueous solution of ERα protein is 8 - 12 μg / mL. The reaction time is 10 - 14 h, and the reaction temperature is 2 - 8 °C.

6. An ERα-functionalized HEMT biosensor constructed by the method according to any one of claims 1 - 5.

7. Use of the ERα-functionalized HEMT biosensor according to claim 6 in detecting potential antagonists of ERα, characterized in that, The application includes: Loading a solution of the compound to be measured onto the constructed ERα-functionalized HEMT biosensor. Using an electrochemical workstation to scan and record the current intensity between the source and drain of the ERα-functionalized HEMT biosensor loaded with the solution of the compound to be measured, repeat the measurement three times to obtain three current intensity values I1, I2, and I3. The solvent used to prepare the test compound solution was used as a blank control. The electrochemical workstation was used to scan and record the current intensity between the source and drain of the ERα-functionalized HEMT biosensor loaded with the blank control. The measurement was repeated three times to obtain three current intensity values ​​I 01 ,I 02 and I 03 ; Taking three current intensity values I1, I2, and I3 as the first group of data, and taking three current intensity values I 01 , I 02 , I 03 as the second group of data, the t-test method is used to determine whether there is a significant difference between the first group of data and the second group of data; If there is a significant difference, then the compound to be measured is a potential ERα antagonist.

8. Use of the ERα-functionalized HEMT biosensor according to claim 7 in the detection of potential ERα antagonists, characterized in that, The detection method is linear sweep voltammetry. Set the constant voltage range of linear sweep voltammetry to 0 – 2 V, the current limit to 0.1 A, and the step value to 2 mV.

9. Use of the ERα-functionalized HEMT biosensor according to claim 6 in detecting the strength of the interaction between the ERα protein and an ERα potential antagonist, characterized in that, The application includes: Weigh a certain weight of the potential ERα antagonist, and prepare a stock solution with a concentration of 10 mM. Take the stock solution and perform gradient dilution by a factor of 10 to obtain a set of ERα potential antagonist solutions with gradient concentrations. On the constructed ERα-functionalized HEMT biosensor, load different concentrations of the ERα potential antagonist solutions in ascending order of concentration, use an electrochemical workstation to scan and record the current intensity I between the source and drain, repeat the measurement three times, calculate the average value, and obtain I. Using the solvent used to prepare the ERα potential antagonist solution as a blank control, an electrochemical workstation was used to scan and record the source-drain current intensity of the ERα-functionalized HEMT biosensor loaded with the blank control. The measurement was repeated three times, and the average value was calculated to obtain I0; Taking the logarithm Lg[A g of the concentration of the potential ERα antagonist solution as the abscissa and the relative value of the current change (I - I0) / I0 as the ordinate, at least five consecutive data points are selected for a linear fitting to obtain at least one linear fitting function, and an objective linear fitting function with R 2 > 0.9 is selected from the at least one linear fitting function; Obtain two data points that are endpoints in the target linear fitting function, and generate a target linear range with the concentrations of the ERα potential antagonist solutions corresponding to the two data points that are endpoints. Within the target linear range, use the concentration [A g as the abscissa, and use the ratio of the concentration [A g to the current change value I-I0 as the ordinate to perform quadratic linear fitting to obtain a quadratic linear fitting function, and calculate the dissociation equilibrium constant K D value of the interaction between the ERα protein and the ERα potential antagonist; Based on the dissociation equilibrium constant K D to determine the strength of the interaction between ERα protein and potential ERα antagonists.

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