Polypeptide probe fluorescent biosensor for detecting ERRgamma active substance

Abstract

The invention discloses a polypeptide probe fluorescent biosensor for detecting an ERRgamma active substance, which adopts a polypeptide probe containing an ERRgamma co-activator conserved sequence as an element for identifying an activated state ERRgamma, and realizes high-sensitivity detection of the ERRgamma active substance by virtue of a signal of a fluorescence labeled molecule. In the operation process, the activated state ERRgamma recruits and is combined with the polypeptide probe marked with the fluorescent molecules, and after a chemical substance to be detected is added, the inhibited state ERRgamma is separated from the polypeptide probe marked with the fluorescent molecules. By detecting and comparing the fluorescence polarization values of the system before and after the chemical substance to be detected is added, the detection of the ERRgamma active substance is realized. The fluorescent biosensor disclosed by the patent can simulate a biological action process, and can quickly detect and evaluate known and unknown ERRgamma active substances. The sensor has the remarkable advantages of low cost, high sensitivity, good selectivity, simplicity and convenience in operation, high response speed, convenience in on-site instant detection and the like, is expected to promote technical development in the field of pollutant sensing detection, and has important significance in commercialization and practical application.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a method for detecting ERRγ active substances. Background Art

[0002] my country currently faces multiple challenges to its water environment, with the long-term retention of endocrine disrupting chemicals (EDCs) in water bodies being a particular concern. Estrogen-related receptor γ (ERRγ) active substances, a typical class of EDCs, include organophosphates, benzophenones, polybrominated diphenyl ethers and their metabolites, halogenated bisphenol A derivatives, polychlorinated biphenyl metabolites, and phytoestrogens. These substances pose multiple threats to environmental safety and human health by disrupting the endocrine system, disrupting ecological balance, and bioaccumulating and transmitting through the food chain. These pollutants exhibit typical "triple effects" (carcinogenicity, teratogenicity, and mutagenicity) and multi-system toxicity. They can accumulate and amplify through the food chain, causing irreversible damage to aquatic organisms and posing a potential threat to human health. Therefore, establishing an efficient pollutant identification and assessment technology system, especially for the accurate identification and risk warning of emerging organic pollutants in water, has become a core scientific issue for safeguarding public health and sustainable development strategies. Detection of ERRγ active substances not only helps identify potential environmental problems but also provides an important basis for scientific decision-making and ecological governance. By establishing a systematic detection and evaluation system, the potential safety risks of ERRγ active substances can be effectively controlled, and the establishment of an early risk warning mechanism can reduce the probability of adverse effects of ERRγ active substances.

[0003] To reduce the environmental hazards of ERRγ-active substances, sensitive detection methods are needed to monitor ERRγ-active substances in the environment and assess their potential risks to ecosystems and human health. Currently, the assessment of the toxic effects of ERRγ-active substances relies primarily on traditional toxicological methods, such as whole-body biological testing based on experimental animal models. These methods are characterized by lengthy cycles and significant resource consumption, making them inadequate for rapid screening of ERRγ-active substances. While cytotoxicity assays offer high-throughput detection, they are limited by significant deviations from living organisms in their biological response characteristics, poor stability, and long cycle times. The relevance of these results to human exposure risk remains controversial. Toxic effect biosensor technology offers an innovative approach to addressing this challenge. By integrating a biomolecular recognition element with a signal transducer, this technology can monitor endocrine disrupting effects in environmental samples in real time. Compared to traditional toxicological methods, this technology demonstrates significant advantages in detection sensitivity, response speed, and simultaneous multi-parameter analysis, and is expected to become a core technology for early warning of safety risks associated with ERRγ-active substances.

[0004] Traditional toxicological risk assessment methods (such as animal experiments and cytotoxicity tests) face technical bottlenecks such as long detection cycles, high costs, insufficient dynamic response capabilities, and difficulty in analyzing molecular-level mechanisms when faced with complex chemical exposure scenarios. Fluorescent biosensors, with their advantages of minimal sample preparation, short measurement time, high specificity and sensitivity, and low detection limits, have become a new analytical technology that breaks through the above limitations. Its core detection mechanism is to utilize the excitation-emission spectral characteristics of fluorescent groups to monitor the conformational changes caused by the binding of chemicals to receptors in real time, and convert the intermolecular interactions into quantifiable fluorescent signal outputs. This sensing system that integrates molecular recognition elements and fluorescent detection modules not only overcomes the limitations of traditional methods in real-time monitoring and high-throughput screening, but also provides technical support with both sensitivity and specificity for early warning of the toxic effects of chemicals, analysis of the mechanism of action, and risk grading, promoting the development of toxicological assessments towards precision and dynamism.

[0005] Traditional fluorescent biosensor detection methods have significant technical bottlenecks: fluorescent biosensors based on fluorescence resonance energy transfer (FRET) technology (Gowda, Krishne, et al. "Development of a coactivator displacement assay for the orphan receptor estrogen-related receptor-γusing time-resolved fluorescence resonance energy transfer." Analytical biochemistry 357.1 (2006): 105-115.), which detect the complex of ERRγ-LBD and ligand, use the polypeptide probe PGC-1α as the recognition molecule, titanium labeled on the ERRγ antibody and fluorescein FI labeled on the polypeptide as signal molecules, and the signal conversion mechanism is that when ERRγ-LBD is not bound to the ligand, ERRγ-LBD itself is in an activated conformation and binds to the polypeptide probe PGC-1α, and the titanium labeled on the ERRγ antibody and the fluorescein FI labeled on the polypeptide are activated. The fluorescein FI on the peptide is close, generating a FRET signal. When 4-hydroxytamoxifen (4-OHT) is added, the conformation of ERRγ-LBD is inhibited by 4-OHT, and the inhibited ERRγ-LBD is separated from the polypeptide probe PGC-1α. The titanium labeled on the ERRγ antibody and the fluorescein FI labeled on the polypeptide are far apart, and the FRET signal decreases. Based on the change in the FRET signal, the detection of an ERRγ active substance 4-hydroxytamoxifen (4-OHT) is achieved (EC50 = 130nM). There is another fluorescent biosensor based on fluorescence resonance energy transfer (FRET) technology (Coward, Peter, Doris Lee, Mitchell V. Hull, and Jürgen M. Lehmann. "4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptorγ." Proceedings of the National Academy of Sciences 98, no. 15 (2001): 8880–8884.). Its detection object and signal conversion mechanism are the same as those of the above-mentioned sensor. It uses the polypeptide probe SRC-1.2 as the recognition molecule, europium labeled on the ERRγ antibody and allophycocyanin labeled on the polypeptide probe as signal molecules. The EC50 values ​​for detecting diethylstilbestrol (DES), tamoxifen (TAM) and 4-hydroxytamoxifen (4-OHT) are 700 nM, 400 nM and 50 nM, respectively.

[0006] The aforementioned fluorescent biosensor based on the FRET principle has shown promising application in the detection of ERRγ-active substances. However, this technology faces numerous challenges in practical application. First, the construction of a FRET system requires the careful design and selection of appropriate fluorescent donor and acceptor pairs, which not only increases the difficulty and cost of experimental operations, but also the complexity and cost of the experiment. Second, the fluorescence quantum efficiency, a key parameter measuring the luminescence capacity of a fluorescent substance, directly affects the intensity of the FRET signal. A low quantum efficiency can significantly reduce the detection sensitivity. Third, the FRET process is extremely sensitive to the spatial distance between the donor and acceptor. When the distance between the two exceeds the effective range of 1-10 nanometers, the energy transfer efficiency decreases exponentially, making the stability and reproducibility of the experimental system difficult to ensure. In sharp contrast, fluorescence polarization technology relies on the rotational motion of fluorescently labeled molecules in solution. Only a single site of target molecule needs to be labeled, and the changes in the binding state of the molecule can be reflected by measuring the fluorescence polarization value. This simplified labeling method greatly reduces the complexity of the experimental operation. Furthermore, fluorescence polarization detection directly outputs numerical values, making experimental results intuitive and easy to quantify and analyze. Unlike FRET technology, which relies on complex spectral fitting and energy transfer efficiency calculations, this method provides a more reliable technical approach for high-throughput, high-sensitivity analysis of ERRγ active substances. Currently, fluorescence polarization has not been used as a screening and detection technology for ERRγ active substances. Given the need for rapid detection of ERRγ active substances and the shortcomings of existing methods, a new biosensor based on fluorescence polarization is urgently needed to provide an innovative solution for real-time monitoring of ERRγ active substances. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to solve the problem of the sensing method based on fluorescence resonance energy transfer technology for detecting ERRγ active substances, which has the following defects: limited action range (only 1-10nm), spectral overlap and easy crosstalk, fluorophore labeling that may interfere with molecular function, significant influence of environmental factors, complex quantitative analysis and limited application in living organisms. The main purpose of the present invention is to provide a fluorescent biosensor method for evaluating the activation and inhibition effects of test compounds on ERRγ at the molecular level, and detecting a batch of ERRγ active substances in unknown samples in the environment. The method is simple to operate and has good reproducibility.

[0008] This method, using fluorescence polarization (FP), offers advantages such as high sensitivity, rapid measurement, easy data analysis, simple operation, and excellent reproducibility. This technique utilizes a fluorescently labeled peptide probe and exploits the molecular weight difference between the peptide probe and the target molecule, ERRγ, which affects its rotational speed. This conformation-dependent binding of ERRγ allows real-time monitoring of ERRγ conformational changes, enabling high-throughput screening of ERRγ-active substances. Under baseline conditions, ERRγ is in an activated conformation, forming a stable complex with the fluorescently labeled peptide probe. This complex exhibits high molecular weight, slow rotational speed, and high FP values. When ERRγ inhibitors (such as 4-OHT) are present in environmental samples, 4-OHT binding induces a conformational shift of ERRγ to an inhibited state, leading to dissociation of ERRγ from the fluorescently labeled peptide probe and a subsequent decrease in FP values. Compared to traditional FRET techniques (which require dual fluorescent labeling), this method offers advantages such as the lack of complex calibration, rapid detection, and strong background immunity, providing a reliable technical support for the rapid risk assessment of ERRγ-active substances in water.

[0009] The present invention provides a fluorescent biosensor for sensitive detection of ERRγ active substances. The provided fluorescent biosensor has the following characteristics:

[0010] The fluorescent biosensor consists of a peptide probe labeled with a fluorescent molecule and the entire ERRγ protein.

[0011] In the embodiment, the ERRγ whole protein is characterized as an orphan nuclear receptor, whose domains include the transcriptional activation region of the N-terminal A / B region, the DNA binding domain (DNA-Binding Domain), the ligand binding domain (Ligand-Binding Domain) and the transcriptional regulatory region of the C-terminal E / F region.

[0012] In the Examples, the fluorescently labeled polypeptide probe is characterized by containing an LXXLL motif (also known as the LXXLL motif, NRbox, or Nuclear Receptor Box), a conserved short sequence consisting of five amino acids, where "L" represents leucine and "X" represents any amino acid. The polypeptide probe utilizes its LXXLL motif to bind to the activation function 2 (AF-2) of the ligand binding domain (LBD) of ERRγ.

[0013] The method provided by the present invention comprises the following steps:

[0014] 1. Detection of substances that inhibit ERRγ

[0015] ERRγ and a polypeptide probe labeled with a fluorescent molecule are system 1. ERRγ and a polypeptide probe labeled with a fluorescent molecule are mixed and incubated in a centrifuge tube, and then the fluorescence polarization value is detected.

[0016] The test compound, ERRγ and the polypeptide probe labeled with a fluorescent molecule are system 2. The test compound, ERRγ and the polypeptide probe labeled with a fluorescent molecule are mixed and incubated in a centrifuge tube, and then the fluorescence polarization value is detected.

[0017] All experiments were performed in triplicate, and results are expressed as mean ± SD (standard deviation). By comparing the fluorescence polarization values ​​of System 1 and System 2, if the fluorescence polarization value of System 2 is lower than that of System 1, it indicates that ERRγ in System 2 is dissociated from the fluorescent-labeled peptide probe, indicating that the test compound has an inhibitory effect on activated ERRγ, indicating that the test compound contains an ERRγ-active compound.

[0018] 2. Detection of substances that activate ERRγ

[0019] ERRγ, a polypeptide probe labeled with a fluorescent molecule, and an ERRγ inhibitor are system 1. ERRγ, a polypeptide probe labeled with a fluorescent molecule, and an ERRγ inhibitor are mixed and incubated in a centrifuge tube, and then the fluorescence polarization value is detected.

[0020] The test compound, ERRγ, a peptide probe labeled with a fluorescent molecule, and an ERRγ inhibitor are system 2. The test compound, ERRγ, a peptide probe labeled with a fluorescent molecule, and an ERRγ inhibitor are mixed and incubated in a centrifuge tube, and then the fluorescence polarization value is detected.

[0021] All experiments were performed in triplicate, and results are expressed as mean ± SD (standard deviation). By comparing the fluorescence polarization values ​​of System 1 and System 2, if the fluorescence polarization value of System 2 is higher than that of System 1, it indicates that ERRγ in System 2 is bound to the fluorescent-labeled peptide probe, and the test compound has an activating effect on the inhibited ERRγ, indicating that the test compound contains an ERRγ-active compound.

[0022] In the examples, a polypeptide probe containing a conserved sequence of ERRγ coactivators is used as a polypeptide probe to detect the binding and separation of ERRγ and the polypeptide probe to indirectly qualitatively or quantitatively describe the binding of ERRγ active substances to ERRγ.

[0023] In an embodiment, the mixed system is incubated for 1 hour.

[0024] In an embodiment, the concentration of the polypeptide probe used in the mixed system incubation is 3 nM.

[0025] In an embodiment, the ERRγ concentration used in the incubation of the mixed system is 1.5 nM.

[0026] Compared with the traditional technology, the present invention has the following beneficial effects:

[0027] (1) High sensitivity and specificity: Fluorescence resonance energy transfer (FRET) technology requires simultaneous labeling of the donor and the acceptor, and the signal is concentration-dependent. When the sample concentration is extremely low, the collision probability of the dual-labeled molecules decreases, and the signal intensity is insufficient. However, the fluorescence polarization method has a sensitive signal. Even at low concentrations, the change in rotation rate caused by molecular binding can still be detected. It can accurately identify the intermolecular interaction or small conformational changes between the peptide probe labeled with the fluorescent molecule and ERRγ. Traditional techniques, such as cell and animal experiments, usually rely on indirect indicators, have low sensitivity, and are difficult to detect trace molecules or rapid kinetic changes.

[0028] (2) Real-time dynamic detection: Fluorescent biosensors can monitor molecular dynamic changes in real time without the need for fixed samples. The molecular rotation speed is reflected by changes in fluorescence polarization. When the labeled molecule binds to the target molecule, the molecular rotation speed slows down, resulting in an increase in the fluorescence polarization value. There is no need for complex donor-acceptor distance effects like fluorescence resonance energy transfer (FRET) technology (which cannot detect long-range interactions). The transition of ERRγ between activation and inhibition conformations can be directly observed, and the results are more intuitive and easy to interpret.

[0029] (3) High-throughput screening capability: Fluorescence polarization technology only requires labeling one fluorophore on the peptide probe, avoiding the reliance on two fluorescent dyes in traditional fluorescence resonance energy transfer (FRET) technology, simplifying experimental design and operation procedures. Fluorescent biosensors are suitable for high-throughput screening and can simultaneously and rapidly evaluate the interaction between ERRγ and peptide probes labeled with fluorescent molecules, improving experimental efficiency and making them suitable for large-scale screening and optimization. Traditional experiments have significant limitations in operation and sample size, making it difficult to efficiently screen large quantities of compounds.

[0030] (4) Saving resources and costs: The experimental system of fluorescent biosensors is simple and requires less sample volume, thus avoiding the animal husbandry and ethical costs in animal experiments. Traditional animal experiments require high costs, including animal purchase, husbandry, experimental operation, and regulatory approval. Cell experiments require large volumes of culture medium, reagents, and consumables.

[0031] The technical principles of the detection method provided by the present invention include:

[0032] (1) ERRγ is an orphan nuclear receptor whose activity regulation does not rely on traditional ligands. In the absence of ligand, the ligand binding domain (LBD) of ERRγ is already in a stable activated conformation. The ERRγ activation function region 2 (AF-2) forms a specific hydrophobic surface that can directly recruit coactivators.

[0033] (2) Utilizing the principles of functional biomimetics and the ligand-induced conformational change characteristics of ERRγ, the activity state of ERRγ was monitored in real time using a fluorescently labeled coactivator peptide probe. Molecular mechanism studies have shown that ERRγ is in an activated conformation in the absence of ligand, and its ligand-binding domain (LBD) forms a stable complex with the coactivator.

[0034] (3) A fluorescently labeled peptide probe is used as a molecular recognition element, and the polarization value of the fluorophore is used to monitor the binding state of ERRγ and the peptide probe. Under baseline conditions (no chemical substances are present), ERRγ and the peptide probe form a stable complex, and its molecular rotation rate is slow, resulting in an increase in the fluorescence polarization value. When ERRγ active substances are present in the environmental sample, the binding of ERRγ active substances to ERRγ induces a conformational transition to an inhibitory state, causing the peptide probe to dissociate from the complex. The rotation rate of the free peptide probe molecule is significantly accelerated, and the fluorescence polarization value is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the principle of the peptide probe fluorescent biosensor used to detect ERRγ active substances.

[0036] Figure 2 This is the standard curve for detecting 4-OHT using a peptide probe fluorescent biosensor for detecting ERRγ active substances. DETAILED DESCRIPTION

[0037] To facilitate understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the technical field of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments or embodiments and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, including any two related listed items, any more related listed items, or the combination of all related listed items.

[0039] The experimental equipment in the present invention includes but is not limited to a fluorescence spectrophotometer (FS5, Edinburgh Instruments).

[0040] In the detection methods provided herein, ERRγ-active substances primarily include chemical or biological molecules capable of modulating ERRγ activity. These substances are divided into two categories: agonists and inhibitors. Furthermore, some research tool molecules are also included, which can be synthetic small molecule compounds. These can be selected from, but are not limited to, DY131, GSK4716, and GSK5182.

[0041] The detection method provided by the present invention is mainly based on the binding and separation mechanism of ERRγ and polypeptide probes. Accordingly, the selected ERRγ needs to contain an activation function domain (AF-2) and a ligand binding domain (LBD).

[0042] In the embodiment of the present invention, the concentration of ERRγ used is 1.5 nM. The concentration of ERRγ in the present invention can be selected from, but not limited to, 1.5 nM, 3 nM, and 5 nM, preferably 1.5 nM.

[0043] In an embodiment of the present invention, the concentration of the fluorescent molecule-labeled polypeptide probe is 3 nM. The concentration of the fluorescent molecule-labeled polypeptide probe of the present invention can be selected from, but not limited to, 3 nM, 5 nM, and 10 nM, preferably 3 nM.

[0044] In an example of the present invention, the incubation time of the ERRγ and the fluorescent molecule-labeled polypeptide probe is 1 hour. The duration of the test of the present invention can be selected from, but not limited to: 1 hour, 1.5 hours, 2 hours, and 2.5 hours.

[0045] In the examples of the present invention, the fluorescent dye needs to have high fluorescence efficiency, optical stability, good water solubility, convenience of chemical modification, low toxicity and wide application applicability, and can be selected from, including but not limited to: FAM.

[0046] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0047] In one example, the polypeptide probe labeled with fluorescent molecules was synthesized by Wuhan Qiangyao Biotechnology Co., Ltd., the polypeptide sequence was EAEEPSLLKKLLLAPANTQ, and the N-terminus was labeled with fluorescein (FAM) dye.

[0048] The ERRγ is a product from abcam, with the product number ab152371, and is a human recombinant estrogen-related receptor γ complete protein with a GST tag at the N-terminus.

[0049] The 4-hydroxytamoxifen (4-OHT) is a product of MCE Company, with CAS No. 68047-06-3, and is the test compound in the examples. 4-Hydroxytamoxifen (4-OHT) is an inhibitor of ERRγ.

[0050] Example 1, Detection of 4-hydroxytamoxifen (4-OHT)

[0051] (1) ERRγ and peptide probes labeled with fluorescent molecules are used as system 1. ERRγ and peptide probes labeled with fluorescent molecules are mixed and incubated in a centrifuge tube: ERRγ and FAM-SRC-2 peptide probes of different concentrations are prepared in a buffer solution (150mM KCl, 0.1mM EDTA, 0.1mM DTT, 10mMTris-HCl). Select appropriate concentrations for subsequent incubation experiments, preferably 1.5nM for ERRγ and 3nM for FAM-SRC-2 peptide probe. During mixed incubation, 40μl of 4.5nM ERRγ, 40μl of 9nM FAM-SRC-2 peptide probe, and 40μl of running buffer are mixed in a 200μl brown centrifuge tube. Mix the solution with a vortex mixer to ensure that the solution is uniform. The shaking time is usually 5-10 seconds, which is adjusted according to the characteristics of the solution. Place the mixed centrifuge tube into a centrifuge for centrifugation, ensuring that the tubes are placed symmetrically to avoid sample displacement during centrifugation. After incubation at room temperature for 60 minutes, fluorescence polarization was measured on an FS5 fluorescence spectrophotometer. The FS5 fluorescence spectrophotometer was set to an excitation wavelength of 490 nm, an emission wavelength of 520 nm, and a slit width of 5 nm. Three replicate samples of System 1 were set up, and fluorescence polarization values ​​were measured for System 1.

[0052] (2) 4-OHT, ERRγ and peptide probes labeled with fluorescent molecules are used as system 2. 4-OHT, ERRγ and peptide probes labeled with fluorescent molecules are mixed and incubated in a centrifuge tube: ERRγ, FAM-SRC-2 peptide probes and 4-OHT at different concentrations are prepared in a buffer solution (150mM KCl, 0.1mM EDTA, 0.1mM DTT, 10mMTris-HCl). Appropriate concentrations are selected for subsequent incubation experiments. ERRγ is preferably 1.5nM, FAM-SRC-2 peptide probe is preferably 3nM, and 4-OHT is preferably 1μM. During the mixed incubation, 40μl of 4.5nM ERRγ, 40μl of 9nM FAM-SRC-2 peptide probe, and 40μl of 3μM 4-OHT are mixed in a 200μl brown centrifuge tube. Use a vortex mixer to mix the solution to ensure that the solution is uniform. The shaking time is usually 5-10 seconds, which is adjusted according to the characteristics of the solution. Place the mixed tubes in a centrifuge, ensuring they are positioned symmetrically to prevent sample shifting during centrifugation. After incubation at room temperature for 60 minutes, measure fluorescence polarization on an FS5 fluorescence spectrophotometer. The FS5 fluorescence spectrophotometer was set to an excitation wavelength of 490 nm, an emission wavelength of 520 nm, and a slit width of 5 nm. Three replicate samples of System 2 were run, and fluorescence polarization values ​​were measured for System 2.

[0053] (3) By comparing the fluorescence polarization values ​​of system 1 and system 2, if the fluorescence polarization value decreases, it can be determined that 4-OHT is an ERRγ active compound and has an inhibitory effect on ERRγ. Figure 2 As shown in the figure, as the concentration of 4-OHT increases, the fluorescence anisotropy value decreases, indicating that 4-OHT is an ERRγ active compound and an ERRγ inhibitor. The measured EC50 value of 4-OHT is 21.2 nM.

Claims

1. A polypeptide probe fluorescent biosensor for detecting ERRγ active substances, characterized in that: A polypeptide probe labeled with a fluorescent signal molecule is used as the recognition element of the activated ERRγ, and the detection of ERRγ active substances is achieved through the change of fluorescence polarization when ERRγ binds to and separates from the polypeptide probe.

2. The polypeptide probe fluorescent biosensor according to claim 1, characterized in that: The ERRγ (estrogen-related receptor γ) belongs to the orphan nuclear receptor superfamily. Its ligand-binding domain (LBD) can maintain the transcriptional activation state through its inherent conformation in the absence of endogenous ligand binding, and can directly recruit coactivators (such as SRC family proteins) to the target gene promoter region to mediate the transcriptional regulation of downstream genes.

3. The polypeptide probe fluorescent biosensor according to claim 1, characterized in that: The polypeptide probe comprises a polypeptide sequence specifically binding to a fluorescent signal molecule and an LXXLL motif specifically binding to ERRγ.

4. The LXXLL motif (also known as LXXLL motif, NRbox, or Nuclear Receptor Box) according to claim 3 is a conserved short sequence consisting of five amino acids, where "L" represents leucine and "X" represents any amino acid. The polypeptide probe utilizes its LXXLL motif to bind to the activation function 2 (AF-2) of the ligand binding domain (LBD) of ERRγ.

5. According to claim 3, the fluorescent signal molecule includes but is not limited to FAM.

6. According to claim 5, the fluorescent molecule FAM is combined with the polypeptide probe mainly by forming an amide bond between the carboxyl group of FAM and the amino group of the polypeptide probe. 7 . The detection method according to claim 1 , wherein the detection method comprises drawing a fluorescence polarization standard curve and qualitatively or quantitatively describing the binding of the ERRγ to the chemical substance to be detected based on the fluorescence polarization standard curve.

8. According to claim 7, the steps of preparing a fluorescent biosensor for detecting ERRγ active substances are as follows: (a) providing a polypeptide probe labeled with a fluorescent molecule as a recognition molecule; (b) providing ERRγ as a detection molecule; (c) ERRγ is mixed with a fluorescently labeled polypeptide probe to form system 1, and the fluorescence polarization value of system 1 is detected; (d) ERRγ is mixed with a fluorescently labeled peptide probe and the chemical substance to be tested to form system 2, and the fluorescence polarization value of system 2 is detected. (e) By comparing the changes in fluorescence polarization values ​​of system 1 and system 2, it is determined whether the chemical substance to be tested is an ERRγ active substance. 9 . The method of claim 8 , wherein the detection of ERRγ active substances can be achieved by using a fluorescence spectrometer in a cuvette.

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