Electrochemical luminescence biosensor for evaluating activity of chemical substance estrogen

Abstract

The invention discloses an electrochemical luminescence biosensor for evaluating the activity of a chemical substance estrogen. According to the electrochemical luminescence biosensor, an activated estrogen receptor is used as a biomarker of a chemical substance estrogen effect, and a polypeptide molecule containing an estrogen receptor co-activator conserved sequence is used as a recognition element of the activated estrogen receptor; and evaluating the estrogen activity of the chemical substance through a detection signal of the electrochemical luminescence labeled molecule. In the operation process of the biosensor, polypeptide molecules are fixed on an electrode, a chemical substance to be detected and an estrogen receptor are incubated and then added on the electrode to react with the polypeptide molecules, then an antibody marked by electrochemical luminescence molecules is added to react with the estrogen receptor, and finally signal detection is carried out in a detection pool containing an electrochemical luminescence co-reactant. The electrochemical luminescence biosensor disclosed by the invention has the advantages of simplicity in operation, short detection time, high sensitivity and the like, and can be used for rapidly screening and evaluating the activity of a chemical substance estrogen.

Description

Technical Field

[0001] The invention relates to an electrochemiluminescence biosensor for evaluating the estrogen activity of chemical substances, belonging to the technical field of biological detection. Background Art

[0002] Environmental estrogens (EEs) are natural or synthetic compounds found in the environment that can affect the physiological functions of estrogen in the body and are a typical class of environmental endocrine disruptors (EDCs). By mimicking the effects of endogenous estrogens, environmental estrogens interact with estrogen receptors in the body, inducing endocrine abnormalities and other adverse effects. Studies have shown that environmental estrogens are closely associated with the development of malignant tumors and diseases of the reproductive, nervous, and immune systems.

[0003] Environmental estrogens are widely distributed and structurally diverse, including polychlorinated biphenyls (PCBs), bisphenols, organophosphorus pesticides, and herbicides. These substances are widely used in daily life. In addition to these chemicals and industrial products, some drugs used to regulate endocrine processes and some personal care products have also been found in recent years to have estrogen-disrupting effects. Considering the serious harm caused by environmental estrogens, many countries and organizations have implemented measures to restrict the production of typical environmental estrogens, such as bisphenol A, polychlorinated biphenyls (PCBs), alkylphenols, and organophosphorus flame retardants. However, some environmental estrogens already present in the environment have long degradation cycles and pose persistent environmental and biological hazards. At the same time, an increasing number of alternative chemicals are being widely used, and the toxic effects of these alternatives are still unknown. Therefore, identifying, detecting, and quantifying environmental estrogens is extremely important for assessing and monitoring their impact on public health and the environment.

[0004] Existing methods for detecting substances, such as large-scale instrumentation or biological and chemical sensors used for chemical analysis, can only detect target molecules, but cannot detect unknown compounds or screen for emerging nuclear receptor ligands. Furthermore, methods for screening unknown ligands using cell-based and animal experiments suffer from long detection cycles and poor stability. As a result, biological methods for identifying and evaluating EEs cannot meet the growing demand for evaluating estrogen activity. However, applying biosensors to EE evaluation could meet this demand. By mimicking the activity of estrogen receptors within cells, sensors can be constructed in cell-free systems to detect estrogen activity.

[0005] Electrochemiluminescence is a combination of chemiluminescence and electrochemistry. Compared with chemiluminescence, electrochemiluminescence has stronger controllability and selectivity; compared with photoluminescence, electrochemiluminescence does not require a light source; compared with electrochemical methods, electrochemiluminescence has stronger selectivity and less electrode contamination. Therefore, electrochemiluminescence not only solves problems such as scattered light and luminescent impurities, but also has many advantages such as good applicability, simple instrumentation, low background signal, wide linear working range, and high sensitivity, making electrochemiluminescence widely used in analytical chemistry and biosensing fields (Zhang JJ, et al. Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiolbased on a competitive assay with cDNA amplification [J]. Analytical Methods, 2014, 6 (17): 6796-6801.). Electrochemiluminescence technology has become a relatively mature analytical technology and is widely used in clinical testing, immunoassays, food and water quality testing, biological agent testing, and drug analysis (Jiang Q et al. An antibody-free and signal-on type electrochemiluminescence sensor for diethylstilbestrol detection based on magnetic molecularly imprinted polymers-quantum dots labeled aptamer conjugated probes [J]. Journal of Electroanalytical Chemistry, 2017, 789: 1-8.). However, the inventors found that there is currently no biosensor in China that uses polypeptide probes as recognition molecules and uses electrochemiluminescence detection technology to evaluate the estrogen activity of chemical substances. Therefore, there is an urgent need for a sensor that uses polypeptides to recognize estrogen receptors in an activated conformation to detect the binding of nuclear receptors to ligand small molecules, thereby indirectly screening and evaluating the activity of compounds. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to evaluate the estrogenic activity of test compounds at the molecular level, based on low cost, simple operation procedures, and a wide range of applications. The present invention provides a sensor based on electrochemiluminescence detection mode, using a polypeptide molecule as a recognition element for activated estrogen receptors, labeling the estrogen receptors with electrochemiluminescent signal molecules, and evaluating the estrogenic activity of chemical substances by detecting the electrochemiluminescent signal. The method provided by the present invention comprises the following steps:

[0007] (1) Apply the peptide solution dropwise to the surface of a freshly cleaned working electrode to immobilize the peptide. The electrode is then washed with a large amount of wash buffer. The electrode is then treated with a blocking agent to remove weakly adsorbed peptides and block vacant sites, and then washed.

[0008] (2) The test compound and the estrogen receptor are mixed and incubated. After the incubation is completed, the compound is dropped onto the working electrode to be incubated with the polypeptide molecule. After the incubation is completed, the compound is washed again.

[0009] (3) Add the antibody labeled with electrochemiluminescent molecules onto the working electrode and incubate it with the estrogen receptor on the electrode surface. After incubation, wash it again.

[0010] (4) The working electrode is placed in a detection cell containing an electrochemiluminescent co-reactant, and an electrochemiluminescence detection instrument is used to detect the signal.

[0011] (5) By comparing the electrochemiluminescence signal of the test compound system with the electrochemiluminescence signal of the blank system to determine whether it is increased, the activation effect of the test compound on the estrogen receptor can be determined. Preferably, the polypeptide comprises a core sequence that recognizes a nuclear receptor and contains a functional group that can bind to a working electrode. In some embodiments, the probe is characterized by a polypeptide comprising a core sequence LXXLL and a length of 18 amino acids.

[0012] Preferably, the functional group to which the polypeptide is linked may be a sulfhydryl group or biotin.

[0013] Preferably, the estrogen receptor is directly labeled with an electrochemiluminescent molecule through a covalent or non-covalent binding reaction or indirectly labeled through a biospecific reaction.

[0014] Preferably, the estrogen receptor carries a His tag, and the electrochemiluminescent signal molecule indirectly labels the estrogen receptor via an anti-His tag antibody.

[0015] Preferably, the electrochemiluminescent molecule may be ruthenium terpyridine, luminol, or quantum dots. Preferably, the electrochemiluminescent liquid may be tripropylamine, diethylaminoethanol, or 2-(dibutylamino)ethanol.

[0016] Preferably, PBS buffer is used to prepare the solutions of the polypeptide and estrogen receptor and the solution of the estrogen active compound.

[0017] Preferably, an electrochemical workstation is used to detect the incubated working electrode, and after applying voltage, the electrochemiluminescence signal value of the compound is read and quantitative analysis is performed.

[0018] The beneficial effects of the present invention are:

[0019] Compared with the traditional technology, the present invention has the following beneficial effects:

[0020] 1. The method of the present invention performs signal detection based on the electrochemiluminescence detection mode. Ordinary electrochemical workstations can be equipped with this function, making the method easy to promote and use.

[0021] 2. The method of the present invention can detect the activation effect of compounds on estrogen receptors at the molecular level, and has the advantages of good specificity and simple operation.

[0022] 3. The method provided by the present invention for detecting the binding of nuclear receptors and small molecule compounds is suitable for high-throughput detection.

[0024] 4. The method of the present invention can evaluate the activation effect of a compound on estrogen receptors and has important application value in evaluating the human health risks of the compound.

[0025] 5. The present invention is a heterogeneous system, and cleaning is performed at each step to avoid interference from matrix effects.

[0026] The technical principles of the detection method provided by the present invention include: peptides containing conserved sequences of receptor coactivators can bind to nuclear receptors in a ligand-dependent manner. After ligand stimulation, the nuclear receptors will bind to the corresponding hormone response elements to start transcription. Using polypeptides that can recognize estrogen receptors in the activated conformation, the ligands of nuclear receptors are indirectly screened or detected and their effects are analyzed by quantitative methods. In some embodiments, the activity detection of nuclear receptor ligands is achieved by electrochemiluminescence signal values. When the concentration of the ligand compound is high, the number of estrogen receptors in the activated conformation will increase, and the number of signal marker molecules bound to the gold electrode will increase. The change in the electrochemiluminescence signal value is used to determine whether the polypeptide is bound to the nuclear receptor, thereby indirectly screening and evaluating estrogen-active compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of electrochemiluminescence sensor for identifying estrogenically active compounds.

[0028] Figure 2 These are the experimental results and fitting curves using endogenous estrogen E2 as a positive control.

[0029] Figure 3 The relative electrochemiluminescence signal values ​​of six perfluorinated compounds at different concentrations were detected by the peptide probe electrochemiluminescence sensor. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0033] Unless otherwise specified, the concentrations referred to in the present invention refer to the final concentration, which refers to the molar concentration of each component in the detection system.

[0034] 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.

[0035] The experiments in the following examples were all repeated three times in parallel, and the results were averaged.

[0036] Example 1: An electrochemiluminescent biosensor for environmental estrogen screening, comprising the following steps:

[0037] In this example, estrogen E2 is a product of Sigma and is the test compound in Example 1. DMSO was used to prepare the stock solution. E2 is a natural ligand for the estrogen receptor and is a positive compound for the feasibility of the assay. Estrogen receptor α protein is a product of abcam and is a complete protein containing the entire amino acid sequence. A coactivator peptide probe was used to detect the activation effect of estrogen E2 on the estrogen receptor.

[0038] The specific steps are as follows:

[0039] (1) A gold electrode (d = 2 mm) was polished with 0.05 μm Al2O3 powder on a suede polishing cloth, then rinsed thoroughly with ultrapure water, ultrasonicated in anhydrous ethanol and ultrapure water for 1 min respectively, and rinsed again with ultrapure water. Finally, an electrochemical method was used to remove the residual substances on the electrode surface to the maximum extent possible, and the gold electrode was activated by electrochemical methods. The specific steps for activating the gold electrode are as follows: using a CHI660E electrochemical workstation, oxidize at 2 V for 5 s, adjust to -0.35 V for 10 s, and then perform cyclic voltammetry in 3 mL of 0.5 M freshly prepared H2SO4 (potential -0.3~1.55 V, scan rate 4 V / s) for 5-10 times until it stabilizes; then, rinse the gold electrode with ultrapure water and perform cyclic voltammetry in freshly prepared 0.5 M H2SO4 (potential -0.3~1.55 V, scan rate 0.1 V / s) twice; finally, rinse the gold electrode with Milli-Q and blow dry with N2.

[0040] (2) Prepare a PBS solution containing 0.15M NaCl, 20mM NaH2PO4, pH 7.4 as a running buffer for subsequent experiments. Dilute the stored peptide stock solution to 10μM with running buffer. Apply the 10μM peptide solution to the surface of a freshly cleaned gold electrode and incubate at 25℃ for 2h to immobilize the peptide. Then wash the electrode with a large amount of washing buffer. Subsequently, treat the electrode with 10μM MCH for 2h to remove weakly adsorbed peptides and block vacant sites, and then wash again.

[0041] (3) The human recombinant estrogen nuclear receptor (ERα) was incubated with the experimental group containing gradient concentrations of E2 and the blank group without E2 in running buffer at 4°C for 90 minutes. 10 μL of the prepared mixture was added dropwise to the polypeptide self-assembled electrode and incubated at 37°C for 2 hours. After incubation, it was washed. Finally, 10 μL of the Ru labeled 2+ The antibody was added dropwise to the electrode and incubated for 1 hour, and then washed after incubation.

[0042] (4) ECL measurements were performed on a CHI660E electrochemical analyzer. The electrodes were placed in 3 mL of prepared electrochemiluminescent solution (TPA buffer), with Pt as the counter electrode and Ag / AgCl (3 M KCl) as the reference electrode. The ECL signal was collected by a photomultiplier tube (PMT, H9307-03, Hamamatsu Photonics Co., Ltd., Japan) placed directly under the transparent electrochemical cell. Both the electrolytic cell and the PMT were enclosed in a light-tight box, and the voltage output of the PMT was sent to the CHI660E software for reading.

[0043] (5) Determine the activation effect of the test compound on the thyroid hormone receptor: By comparing whether the electrochemiluminescence signal of the test compound system is increased compared with the blank signal, the activation effect of the test compound on the estrogen receptor can be determined. All test concentrations are set up in triplicate and the results are averaged. Figure 2 As shown, the electrochemiluminescence signal for E2 increases with increasing concentration, reaching a maximum at around 100 nM, indicating that the test compound E2 has a significant activating effect on the estrogen receptor. To quantitatively describe the relationship between E2 concentration and electrochemiluminescence signal, a logistic model was used to fit the data, revealing an EC50 value of approximately 15.13 nM for E2. Based on the 90% signal difference calculation rule, the limit of detection (LOD) of the sensor constructed in this experiment for E2 is approximately 4.58 nM.

[0044] Example 2

[0045] In this example, the detection method is the same as that in Example 1, except that: in this example, the estrogen effect of six perfluoroalkyl compounds and the estrogen receptor inhibitor 4-OHT is evaluated, and the final concentration of all compounds is the concentration at which the effect plateau is reached. The experimental results are as follows: Figure 3 As shown, the signals of the six perfluoroalkyl compounds all increased, indicating that these six compounds all have estrogenic effects. The estrogen receptor inhibitor 4-OHT did not have a signal change, indicating that the sensor has good selectivity and only detects compounds with estrogenic effects.

Claims

1. An electrochemiluminescent biosensor for evaluating the estrogenic activity of a chemical substance, characterized in that: A polypeptide molecule containing a conserved sequence of an estrogen receptor coactivator is used as a recognition element for the activated estrogen receptor, and the estrogen receptor is labeled with an electrochemiluminescent signal molecule. The estrogen activity of the chemical substance is evaluated by detecting the electrochemiluminescent signal.

2. The electrochemiluminescent biosensor according to claim 1, characterized in that: The polypeptide molecule contains a LXXLL amino acid conservative sequence.

3. The electrochemiluminescent biosensor according to claim 1, characterized in that: The polypeptide molecules are fixed on the surface of the sensor electrode via the terminal modified functional groups.

4. The polypeptide molecule according to claim 3, characterized in that The terminal modified functional group is a thiol group.

5. The polypeptide molecule according to claim 3, characterized in that The terminal modified functional group is biotin.

6. The electrochemiluminescent biosensor according to claim 1, characterized in that: The electrochemiluminescent signal molecule directly labels the estrogen receptor through a covalent or non-covalent binding reaction.

7. The electrochemiluminescent biosensor according to claim 1, characterized in that: The electrochemiluminescent signal molecule indirectly labels the estrogen receptor through a biological specific reaction.

8. The electrochemiluminescent biosensor according to claim 7, characterized in that: The estrogen receptor has a His tag, and the electrochemiluminescent signal molecule indirectly labels the estrogen receptor through an anti-His tag antibody.

9. The electrochemiluminescent biosensor according to claim 7, characterized in that: The electrochemiluminescent signal molecule is terpyridine ruthenium, luminol or quantum dots.

10. The electrochemiluminescent biosensor according to claim 1, characterized in that The operation steps are: (a) Immobilizing the polypeptide molecules on the electrode surface and cleaning the electrode surface; (b) incubating the chemical substance to be tested with the estrogen receptor; (c) dropping the chemical substance / estrogen receptor mixed solution onto the electrode, incubating with the polypeptide molecules, and then cleaning the electrode surface; (d) dropping the electrochemiluminescent labeled antibody onto the electrode, incubating it with the estrogen receptor on the electrode surface, and then cleaning the electrode surface; (e) placing the electrode in a detection cell containing an electrochemiluminescent co-reactant, detecting the electrochemiluminescent signal, and evaluating the estrogenic activity of the chemical substance based on the detected signal value.

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