ERα-functionalized HEMT biosensor and its construction method and application

Abstract

The present invention provides an ERα-functionalized HEMT biosensor and its construction method and application, including: (1) an ERα-functionalized HEMT biosensor; (2) a construction method of an ERα-functionalized HEMT biosensor; and (3) the application of the ERα-functionalized HEMT biosensor in the detection of potential ERα antagonists. The biosensor of the present invention uses a HEMT device as a carrier and is functionalized and modified with ERα through a chemical reaction. It has the advantages of high sensitivity (detection limit as low as 1 fM), strong specificity, and a wide linear range (1 fM-100 nM). It can efficiently and accurately identify potential ERα antagonists, providing a method or technical support for the screening of potential ERα antagonist drugs.

Description

Technical Field

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

[0002] ERα plays an important role in various tissues and cells, regulating the development and function of the reproductive system. For example, it promotes the development of mammary alveoli and maintains the cyclical changes of the endometrium. ERα is implicated in the development and progression of various gynecological tumors, including breast cancer, endometrial cancer, and ovarian cancer. Studies have shown that estrogen, upon binding to ERα, acts on estrogen response elements in the promoter regions of target genes, inducing target gene transcription, thereby promoting the proliferation, differentiation, invasion, and metastasis of breast cancer cells. ERα is a key target for the treatment of diseases such as breast cancer and breast hyperplasia, and is particularly important in the treatment of estrogen receptor-positive breast cancer. ERα antagonists competitively bind to ERα, blocking the growth-promoting effects of estrogen on cancer cells and thereby inhibiting tumor cell proliferation. The detection of ERα receptor antagonists is crucial for the design and development of novel drug candidates with anti-estrogen receptor potential.

[0003] However, existing detection technologies for ERα receptor antagonists have many limitations: first, cell line-based proliferation inhibition experiments rely on high concentrations of compounds (usually in the μM range) to observe significant activity, which leads to the easy omission of low-abundance active molecules (such as metabolites, natural products, or new chemical entities); second, existing detection methods are often complex to operate, have high technical requirements, and have long experimental cycles. In addition, the instruments and equipment used are expensive and costly, making them inconvenient for batch sample testing. Summary of the Invention

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

[0005] In a first aspect, the present invention provides a method for constructing an ERα-functionalized HEMT biosensor, the method comprising:

[0006] Step S01: obtaining a quartz glass tube with two open ends, and using a HEMT device as a carrier, gluing one open end of the quartz glass tube to its gate region to form a sample cell;

[0007] Step S02: adding an aqueous solution of 3-mercaptopropionic acid to the sample cell to react with the gate region to generate Au-S bonds on the surface and form a self-assembled monolayer;

[0008] Step S03: adding a mixed aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide for activation to generate a stable amine activated product;

[0009] Step S04: After activation is completed, the pre-prepared ERα protein aqueous solution is added to the sample cell for reaction, thereby obtaining an ERα functionalized HEMT biosensor.

[0010] In some embodiments, HEMT biosensors are semiconductor devices based on an AlGaAs / InGaAs heterostructure. They offer advantages such as high sensitivity, high specificity, rapid response, and ease of integration, showing great promise in biochemical detection. Active drug target proteins (such as receptors) are modified on the surface of the HEMT device. When a test drug (such as a ligand) is added and interacts, the two-dimensional electron gas density (2DEG) in the HEMT channel changes, enabling detection of the target drug through changes in the electrochemical signal. This invention, by modifying ERα on a HEMT device, constructs an ERα-functionalized HEMT biosensor for detecting potential ERα antagonists, providing technical support for high-throughput drug screening and new drug development.

[0011] Furthermore, in step S01:

[0012] The quartz glass tube has an inner diameter of 5 mm and a height of 15 mm;

[0013] The gate region includes a gate, and a surface of the gate is coated with a gold film.

[0014] Furthermore, in step S02:

[0015] The concentration of the 3-mercaptopropionic acid aqueous solution is 15-25 mM;

[0016] The reaction time is 10-14 h, and the reaction temperature is 10-30 ℃.

[0017] Furthermore, in step S03:

[0018] The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 15-25 mM, the concentration of the N-hydroxysuccinimide is 40-60 mM, and the volume ratio is 1:1;

[0019] The activation time is 15-30 min, and the activation temperature is 10-30 ℃.

[0020] Furthermore, in step S04:

[0021] The concentration of the ERα protein aqueous solution is 8-12 μg / mL;

[0022] The reaction time is 10-14 h, and the reaction temperature is 2-8 ℃.

[0023] In a second aspect, the present invention provides an ERα-functionalized HEMT biosensor constructed according to the above method.

[0024] In a third aspect, the present invention provides a use of a HEMT biosensor constructed according to the above method in detecting potential ERα antagonists, the use comprising:

[0025] The test compound solution was loaded onto the constructed ERα-functionalized HEMT biosensor;

[0026] The current intensity between the source and drain electrodes of the ERα-functionalized HEMT biosensor loaded with the test compound solution was scanned and recorded using an electrochemical workstation. The measurement was repeated three times to obtain three current intensity values, I1, I2, and I3.

[0027] 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 ;

[0028] The three current intensity values ​​I1, I2, and I3 are the first set of data, and the three current intensity values ​​I 01 , I 02 , I 03 For the second set of data, the t-test method was used to determine whether there was a significant difference between the first and second sets of data;

[0029] If there is a significant difference, the test compound is a potential ERα antagonist.

[0030] Furthermore, the detection method is linear sweep voltammetry;

[0031] The linear sweep voltammetry was set with a constant voltage range of 0–2 V, a current limit of 0.1 A, and a step size of 2 mV.

[0032] In a fourth aspect, the present invention provides a use of the above-mentioned ERα-functionalized HEMT biosensor in detecting the strength of the interaction between ERα protein and a potential ERα antagonist, wherein the use comprises:

[0033] Weigh a certain amount of potential ERα antagonist and prepare a stock solution with a concentration of 10 mM;

[0034] The stock solution was diluted 10-fold to obtain a set of ERα potential antagonist solutions with gradient concentrations;

[0035] On the constructed ERα-functionalized HEMT biosensor, different concentrations of ERα potential antagonist solutions were loaded in order from low to high concentration. The current intensity I between the source and drain electrodes was scanned and recorded using an electrochemical workstation. The measurement was repeated three times, and the average value was calculated to obtain I.

[0036] The solvent used to prepare the ERα potential antagonist solution was used as a blank control. The current intensity between the source and drain of the ERα functionalized HEMT biosensor loaded with the blank control was scanned and recorded using an electrochemical workstation. The measurement was repeated three times, and the average value was calculated to obtain I0.

[0037] The logarithm of the concentration of the ERα potential antagonist solution is Lg[A g ] as the horizontal axis, the relative value of current change (I-I0) / I0 as the vertical axis, select at least five consecutive data points for a linear fit, obtain at least one linear fitting function, and select R from the at least one linear fitting function. 2 >0.9 target linear fitting function;

[0038] Obtain two data points belonging to the endpoints in the target linear fitting function, and generate a target linear range with the concentrations of the ERα potential antagonist solution corresponding to the two data points belonging to the endpoints. Within the target linear range, the concentration of the ERα potential antagonist solution [A g ] is the horizontal axis, with concentration [A g The ratio of the current change value I-I0 is taken as the ordinate, and a quadratic linear fitting is performed to obtain a quadratic linear fitting function, and the dissociation equilibrium constant K of the interaction between ERα protein and ERα potential antagonist is calculated based on the quadratic linear fitting function. D value;

[0039] According to the dissociation equilibrium constant K D The size of the value determines the strength of the interaction between ERα protein and potential ERα antagonists. The smaller the value, the stronger the interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1The electrochemical signals and X-ray photoelectron spectroscopy (XPS) results of the HEMT biosensor before and after modification with 3-mercaptopropionic acid and ERα protein, where (a) is the electrochemical signal characterization result, (b) is the XPS total scan spectrum, (c) is the XPS carbon fine scan spectrum, and (d) is the XPS nitrogen fine scan spectrum.

[0041] Figure 2 The response of the ERα-functionalized HEMT sensor to a concentrated solution of the test compound, wherein (a) to (e) are the responses of the ERα-functionalized HEMT sensor of the present invention to 1 pM concentrations of tamoxifen, toremifene, raloxifene, sucrose, and sodium chloride, respectively;

[0042] Figure 3 The results of tamoxifen detection using ERα-functionalized HEMT biosensor, where (a) is I DS -V DS The signal changes with the concentration of tamoxifen. (b) The relative current change (I-I0) / I0 and the logarithmic concentration of tamoxifen Lg[A g ], (c) is the ratio of tamoxifen concentration and current change [A g ] / ΔI and tamoxifen concentration [A g ];

[0043] Figure 4 The results of detecting toremifene using ERα-functionalized HEMT biosensor, where (a) is I DS -V DS Signal changes with the concentration of toremifene, (b) is the relative current change (I-I0) / I0 and the logarithmic concentration of toremifene Lg[A g ], (c) is the ratio of toremifene concentration and current change [A g ] / ΔI and toremifene concentration ([A g ]) linear relationship between them;

[0044] Figure 5 The results of raloxifene detection using ERα-functionalized HEMT biosensor, where (a) is I DS -V DS Signal changes with raloxifene concentration, (b) is the relative current change (I-I0) / I0 and raloxifene logarithmic concentration Lg[A g ], (c) is the ratio of raloxifene concentration and current change [A g ] / ΔI and toremifene concentration ([A g ]) is a linear relationship between them.

[0045] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0046] In order to make the purpose, 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the invention belongs. The words "including" and similar words used in this article mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0047] Example 1

[0048] (1) Obtain a quartz glass tube (inner diameter 5 mm × height 15 mm) with both ends open, and use the HEMT device as a carrier. Glue one of the open ends of the quartz glass tube to its gate region to form a sample pool. The gate region includes a gate, and its surface is coated with a gold film. Then, add 50 μL of a 20 mM 3-mercaptopropionic acid aqueous solution to the sample pool and soak it at room temperature for 12 h to form a self-assembled monolayer. After the Au-S bond is formed, wash the chip surface three times with water to remove the physically adsorbed 3-mercaptopropionic acid.

[0049] (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 groups. The reaction was allowed to proceed at room temperature for 30 min and then rinsed three times with water. After the activation reaction was completed, a stable amine activation product was generated on the chip surface.

[0050] (3) After diluting the ERα protein with water to a concentration of 10 μg / mL, 50 μL was added to the sample pool and reacted at 4 °C for 12 h. After the reaction was completed, the ERα-functionalized HEMT biosensor was obtained.

[0051] Example 2

[0052] An electrochemical workstation and X-ray photoelectron spectrometer (XPS) were used to characterize the electrochemical signals and element changes of the HEMT biosensor before and after modification with 3-mercaptopropionic acid and ERα protein in the construction process of Example 1. Figure 1 The electrochemical signals and XPS measurement results of HEMT biosensors before and after modification with 3-mercaptopropionic acid and ERα protein are shown. Figure 1 As shown in (a), compared with the bare chip, the current changes significantly after modification with 3-mercaptopropionic acid and ERα protein. Figure 1 (b) to Figure 1 As shown in (d), after modification with 3-mercaptopropionic acid, an Au-S (S 2p) peak was formed at 162.5 eV, and a new carbonyl peak (OC=O) appeared at 289.0 eV, indicating the formation of SAM. After modification with ERα protein, two new N1s peaks appeared, located at 401.4 eV and 403.5 eV, corresponding to the neutral amino group (NH2) and protonated amino group (NH3 + ), a C1s peptide bond peak (-CONH-) appeared at 286.5 eV. These results indicate that 3-mercaptopropionic acid and ERα protein were successfully modified on the HEMT device, and the ERα-functionalized HEMT biosensor was successfully constructed.

[0053] Example 3

[0054] (1) It is known that sucrose and sodium chloride are not antagonists (ligands) of ERα, and tamoxifen, toremifene and raloxifene are all ERα antagonists. Tamoxifen, toremifene and raloxifene were prepared into a test solution with a concentration of 1 pM using 5% DMSO, and sucrose and sodium chloride were prepared into a test solution with a concentration of 1 pM using purified water. 50 μL of each of the above test solutions was loaded onto the ERα-functionalized HEMT biosensor prepared in Example 1, and the reaction was carried out 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 solutions was scanned and recorded using an electrochemical workstation. The measurement was repeated three times for each sample to obtain I1, I2 and I3;

[0055] (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 blank control. Repeat the measurement three times to obtain I 01 , I 02 and I 03 ;

[0056] (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 02and I 03 Perform a t-test.

[0057] The results are as follows Figure 2 As shown in (a) to (e), tamoxifen, toremifene and raloxifene solutions produced strong responses on the ERα-functionalized HEMT biosensor, and there was a significant difference in current intensity compared with the blank control (p < 0.01); while sucrose and sodium chloride solutions produced weak or no responses on the ERα-functionalized HEMT biosensor, and there was no significant difference in current intensity compared with the blank control. This indicates that the ERα-functionalized HEMT biosensor in this 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.

[0058] Example 4

[0059] (1) Preparation of test solution: Weigh 3.87 mg of tamoxifen and prepare a stock solution with a concentration of 10 mM with 5% DMSO aqueous solution. Then take the stock solution and dilute it 10 times in sequence to obtain a set of tamoxifen solutions with a gradient concentration range of 1 fM-1 nM.

[0060] (2) Sample measurement: 50 μL of tamoxifen solution was added to the ERα-functionalized HEMT biosensor constructed in Example 1 in descending order of concentration. The reaction was carried out at room temperature for 1 h. The current intensity between the source and drain of the ERα-functionalized HEMT biosensor loaded with tamoxifen solution of different concentrations was scanned and recorded using an electrochemical workstation. The measurement was repeated three times, and the average of the three current intensities was calculated to obtain I.

[0061] (3) Using 50 μL of 5% DMSO aqueous solution 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 5% DMSO aqueous solution. The measurement was repeated three times, and the average value was calculated to obtain I0;

[0062] (4) The logarithm of tamoxifen concentration (Lg[A g ]) as the horizontal axis, the relative value of current change (I-I0) / I0 as the vertical axis, select at least five consecutive data points for a linear fit, obtain at least one linear fitting function, and select R from the at least one linear fitting function. 2 >0.9 target linear fitting function;

[0063] (5) Obtain two data points belonging to the endpoints of the target linear fitting function, and generate a target linear range with the concentration of the ERα potential antagonist solution corresponding to the two data points belonging to the endpoints. Within the target linear range, the concentration of tamoxifen ([A g ]) as the horizontal axis, with concentration ([A g The ratio of the current change value (ΔI=I-I0) is the vertical coordinate, and a quadratic linear fit is performed to obtain the linear equation: =a +b, calculate the dissociation equilibrium constant K of the interaction between tamoxifen and ERα protein D =b / a.

[0064] Results: The I of ERα in the presence of different concentrations of tamoxifen solution DS -V DS Signals such as Figure 3 As shown in (a). When the concentration range is 1 fM to 1 nM, I DS The signal showed a regular downward trend as the concentration of tamoxifen increased. Figure 3 As shown in (b), the logarithm of tamoxifen concentration (Lg[A g ]) as the horizontal axis and the relative value of current change (I-I0) / I0 as the vertical axis, a linear fit was performed. When the concentration range was 10 fM-100 pM, a good linear relationship was shown, and the linear equation was: =-0.0192 -0.1617 (R 2 =0.9477). Figure 3 As shown in (c), the concentration of tamoxifen ([A g ]) as the horizontal axis, with concentration ([A g ]) / current change (I-I0, ΔI) as the vertical coordinate, perform linear fitting, and the linear equation is: =1726.7577 -1.0824×10 -9 (R 2 =0.9996), and the linear relationship was good. The dissociation equilibrium constant K of the interaction between tamoxifen and ERα protein was calculated. D is 6.2684×10 -13 M.

[0065] Dissociation equilibrium constant K D The calculation process of derivation:

[0066] ;

[0067] 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 current change value, is the maximum current change.

[0068] Example 5

[0069] This example is essentially the same as Example 4, except that: (1) Preparation of the test solution: 4.06 mg of toremifene was weighed and prepared into a 10 mM stock solution with a 5% DMSO aqueous solution. The stock solution was then diluted 10-fold to obtain a series of toremifene solutions with a gradient concentration range of 10 fM to 100 nM.

[0070] Results: The I of ERα in the presence of different concentrations of toremifene solution DS -V DS Signals such as Figure 4 As shown in (a). When the concentration range is 10 fM to 100 nM, I DS The signal showed a regular downward trend as the concentration of toremifene increased. Figure 4 As shown in (b), the logarithm of the concentration of toremifene (Lg[A g ]) as the horizontal axis and the relative value of current change (I-I0) / I0 as the vertical axis, a linear fit was performed. When the concentration range was 1 pM-100 nM, a good linear relationship was shown, and the linear equation was: =-0.0058 -0.1449 (R 2 =0.9114). Figure 4 As shown in (c), the concentration of toremifene ([A g ]) as the horizontal axis, with concentration ([A g ]) / current change (I-I0, ΔI) as the vertical coordinate, perform linear fitting, and the linear equation is: =-430.7821 -7.1412×10 -8 (R 2 =0.9999), with a good linear relationship. Finally, the dissociation equilibrium constant K of the interaction between toremifene and ERα protein was calculated. D is 1.6577×10 -10 M.

[0071] Example 6

[0072] This example is essentially the same as Example 4, except that: (1) Preparation of the test solution: 4.06 mg of raloxifene was weighed and prepared into a 10 mM stock solution with a 5% DMSO aqueous solution. The stock solution was then diluted 10-fold to obtain a series of raloxifene solutions with a gradient concentration ranging from 1 fM to 10 nM.

[0073] Results: The I of ERα in the presence of different concentrations of raloxifene solution DS -V DS Signals such as Figure 5 As shown in (a). When the concentration range is 1 fM to 1 nM, I DS The signal showed a regular upward trend as the concentration of raloxifene increased. Figure 5 As shown in (b), the logarithm of raloxifene concentration (Lg[A g ]) as the horizontal axis and the relative value of current change (I-I0) / I0 as the vertical axis, a linear fit was performed. When the concentration range was 1 fM-100 pM, a good linear relationship was shown, and the linear equation was: =0.0152 +0.2341(R 2 =0.9688). Figure 5 As shown in (c), the concentration of raloxifene ([A g ]) as the horizontal axis, with concentration ([A g ]) / current change (I-I0, ΔI) as the vertical coordinate, perform linear fitting, and the linear equation is: =2719.8192 +2.6400×10 -10 (R 2 =0.9993), the linear relationship was good, and the dissociation equilibrium constant K of the interaction between raloxifene and ERα protein was finally calculated. D is 9.7065×10 -14 M.

[0074] It should also be noted that for Examples 2 to 6, the detection method for the ERα-functionalized HEMT biosensor was linear sweep voltammetry, with a constant voltage range of 0-2V, a current limit of 0.1A, and a step value of 2mV. During the test, the biosensor was placed on a probe station with the probe pressed against the source and drain electrodes, and placed in an electromagnetic shielding box. The current intensity (I DS ) and the corresponding voltage (V DS ).

[0075] From Examples 4 to 6, it can be seen that the ERα-functionalized HEMT biosensor prepared by the present invention can effectively detect the dissociation equilibrium constant K that reflects the interaction between ERα protein and ERα antagonist. D The smaller the dissociation equilibrium constant, the stronger the affinity between ERα protein and ERα antagonist, and vice versa.

[0076] In summary, from Examples 1 to 6, it can be seen that the ERα-functionalized HEMT biosensor constructed in the present invention has the advantages of high sensitivity (lowest detection limit as low as 1 fM), strong specificity, and a wide linear detection range (1 fM-100 nM), thereby enabling efficient and accurate detection of potential ERα antagonists, providing technical support for drug screening and research.

[0077] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. Application of an ERα-functionalized HEMT biosensor in detecting potential ERα antagonists, characterized in that: The applications include: The test compound solution was loaded onto the constructed ERα-functionalized HEMT biosensor; The current intensity between the source and drain electrodes of the ERα-functionalized HEMT biosensor loaded with the test compound solution was scanned and recorded using an electrochemical workstation. The measurement was repeated 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 ; The three current intensity values ​​I1, I2, and I3 are the first set of data, and the three current intensity values ​​I 01 , I 02 , I 03 For the second set of data, the t-test method was used to determine whether there was a significant difference between the first and second sets of data; If there is a significant difference, the test compound is a potential ERα antagonist.

2. Use of the ERα-functionalized HEMT biosensor according to claim 1 in detecting potential ERα antagonists, characterized in that: The detection method was linear sweep voltammetry; The linear sweep voltammetry was set with a constant voltage range of 0–2 V, a current limit of 0.1 A, and a step size of 2 mV.

3. Application of an ERα-functionalized HEMT biosensor for detecting the strength of interaction between ERα protein and a potential ERα antagonist, characterized in that: The applications include: Weigh a certain amount of potential ERα antagonist and prepare a stock solution with a concentration of 10 mM; The stock solution was diluted 10-fold to obtain a set of ERα potential antagonist solutions with gradient concentrations; On the constructed ERα-functionalized HEMT biosensor, different concentrations of ERα potential antagonist solutions were loaded in order from low to high concentration. The current intensity I between the source and drain electrodes was scanned and recorded using an electrochemical workstation. The measurement was repeated three times, and the average value was calculated to obtain I. The solvent used to prepare the ERα potential antagonist solution was used as a blank control. The current intensity between the source and drain of the ERα functionalized HEMT biosensor loaded with the blank control was scanned and recorded using an electrochemical workstation. The measurement was repeated three times, and the average value was calculated to obtain I0. The logarithm of the concentration of the ERα potential antagonist solution is Lg[A g ] as the horizontal axis, the relative value of current change (I-I0) / I0 as the vertical axis, select at least five consecutive data points for a linear fit, obtain at least one linear fitting function, and select R from the at least one linear fitting function. 2 >0.9 target linear fitting function; Obtain two data points belonging to the endpoints in the target linear fitting function, and generate a target linear range with the concentrations of the ERα potential antagonist solution corresponding to the two data points belonging to the endpoints. Within the target linear range, the concentration of the ERα potential antagonist solution [A g ] is the horizontal axis, with concentration [A g The ratio of the current change value I-I0 is taken as the ordinate, and a quadratic linear fitting is performed to obtain a quadratic linear fitting function, and the dissociation equilibrium constant K of the interaction between ERα protein and ERα potential antagonist is calculated based on the quadratic linear fitting function. D value; According to the dissociation equilibrium constant K D The size of the value determines the strength of the interaction between ERα protein and potential ERα antagonists.

4. Use of the ERα-functionalized HEMT biosensor according to claim 1 in detecting potential ERα antagonists, characterized in that: The construction method of ERα-functionalized HEMT biosensor includes: Step S01: obtaining a quartz glass tube with two open ends, and using a HEMT device as a carrier, gluing one open end of the quartz glass tube to its gate region to form a sample cell; Step S02: adding a 3-mercaptopropionic acid aqueous solution into the sample pool to react with the gate region to generate Au-S bonds on the surface and form a self-assembled monolayer; Step S03: adding a mixed aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide for activation to generate an amine activated product; Step S04: After activation is completed, the pre-prepared ERα protein aqueous solution is added to the sample cell for reaction, thereby obtaining an ERα functionalized HEMT biosensor.

5. Use of the ERα-functionalized HEMT biosensor according to claim 4 in detecting potential ERα antagonists, characterized in that: In 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 a surface of the gate is coated with a gold film.

6. Use of the ERα-functionalized HEMT biosensor according to claim 4 in detecting potential ERα antagonists, characterized in that: In step S02: The concentration of the 3-mercaptopropionic acid aqueous solution is 15-25 mM; The reaction time is 10-14h, and the reaction temperature is 10-30°C.

7. Use of the ERα-functionalized HEMT biosensor according to claim 4 in detecting potential ERα antagonists, characterized in that: In step S03: The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 15-25 mM, the concentration of the N-hydroxysuccinimide is 40-60 mM, and the volume ratio is 1:1; The activation time is 15-30 minutes and the temperature is 10-30°C.

8. Use of the ERα-functionalized HEMT biosensor according to claim 7 in detecting potential ERα antagonists, characterized in that: In step S04: The concentration of the ERα protein aqueous solution is 8-12 μg / mL; The reaction time is 10-14h, and the reaction temperature is 2-8°C.

Images