Method for detecting oxidation-reduction state of FDX1 in cells

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for detecting the oxidation-reduction state of FDX1 in cells. The method comprises the following steps: detecting a characteristic signal of reduction-state FDX1 in a to-be-detected cell sample by adopting an electron paramagnetic resonance method, and determining the condition of the reduction-state FDX1 in the to-be-detected cell sample according to a detection result, the g value of the characteristic signal is 1.94 and / or 2.02. According to the method, the EPR technology is introduced into FDX1 oxidation-reduction state detection, the oxidation-reduction state of FDX1 in whole cells can be directly detected without marking the cells, real-time monitoring is facilitated, and operation is simple.

Description

Technical Field

[0001] The present application belongs to the field of biological detection technology, and specifically relates to a method for detecting the redox state of FDX1 in cells. Background Art

[0002] Ferredoxins (Fd) are an ancient, ubiquitous class of iron-sulfur (Fe-S) proteins that participate in numerous important physiological and biochemical processes, such as photosynthesis, nitrogen metabolism, steroid synthesis, drug metabolism, and reactive oxygen species detoxification. Fd proteins can be divided into several types based on their origin and function. FDX1 (Ferredoxin 1) is a key protein in mammalian cells, involved in redox reactions and electron transport.

[0003] Studies have shown that FDX1 is widely expressed in a variety of mammalian tissues and cells, and is involved in key metabolic pathways such as steroid hormone synthesis, bile acid synthesis, and drug metabolism. As an iron-sulfur protein in mitochondria, FDX1 plays a core role in maintaining mitochondrial electron transfer, regulating protein acylation, and mediating copper death. The redox state of its [2Fe-2S] cluster has a direct regulatory effect on the energy metabolism and oxidative stress response of the cell. FDX1 contains a [2Fe-2S] cluster, and its redox reaction is mediated by Fe 2+ / Fe 3+ The conversion between the two achieves electron transfer. In the reduced state, FDX1 accepts electrons from iron-sulfur protein reductase (FDXR) and transfers electrons to mitochondrial cytochrome P450, participating in redox processes such as steroid hormone synthesis. The redox state of FDX1 plays a key role in disease-related pathways such as copper death. Its function is regulated by multiple factors such as the intracellular redox environment, substrate concentration, and coenzyme levels, directly affecting related metabolic pathways. Therefore, detecting the redox state of FDX1 in cells is of great significance for studying the function and regulatory mechanism of FDX1 protein and the occurrence and development of related diseases.

[0004] Currently, there are several methods for detecting the redox state of FDX1:

[0005] Biochemical methods involve extracting intracellular FDX1, treating it with redox reagents, and then detecting its redox state via spectrophotometry or electrophoresis. This method is cumbersome and requires cell lysis to extract protein, disrupting redox homeostasis and preventing real-time detection of intracellular FDX1 redox state.

[0006] Fluorescent probe method: FDX1 is labeled with a fluorescent probe (such as roGFP) and the redox state of FDX1 is reflected by detecting changes in fluorescence intensity. Although this method can achieve real-time detection of the redox state of FDX1 in cells, it requires genetic engineering labeling. In addition, the additional label may interfere with the native conformation of FDX1. In addition, the fluorescent probe is easily interfered with by other substances in the cell and only reflects the GSH / GSSG ratio, which cannot specifically detect FDX1 (the quantitative relationship between changes in fluorescence intensity and the redox state of FDX1 is not clear).

[0007] In general, there is currently a lack of a simple, cell-disruption-free, and specific method for real-time detection of the redox state of intracellular FDX1. Summary of the Invention

[0008] Based on this, one or more embodiments of the present application provide a method for detecting the redox state of FDX1 in cells, including the following technical solutions:

[0009] One or more embodiments of the present application provide a method for detecting the redox state of FDX1 in a cell, the method comprising the following steps:

[0010] Using electron paramagnetic resonance to detect the characteristic signal of reduced FDX1 in the cell sample to be tested, and determining the status of reduced FDX1 in the cell sample to be tested based on the detection result;

[0011] The g value of the characteristic signal is 1.94 and / or 2.02.

[0012] In some embodiments of the present application, the electron paramagnetic resonance method satisfies one or more of the following conditions:

[0013] (1) The temperature is 77K-200K; optionally, the temperature is 100K-120K;

[0014] (2) microwave power of 1 mW-75 mW; optionally, microwave power of 5 mW-20 mW; and,

[0015] (3) The modulation amplitude is 1G-10G; optionally, the modulation amplitude is 1G-5G.

[0016] In some embodiments of the present application, the electron paramagnetic resonance method further satisfies one or more of the following conditions:

[0017] (4) Center field strength: 3300G-3400G;

[0018] (5) Magnetic field scanning range: 300mT-370mT;

[0019] (6) Microwave frequency: 9.4 GHz-9.6 GHz;

[0020] (7) Detector gain: 1.00×10 3 -1.00×10 5 ;

[0021] (8) Modulation frequency: 100kHz;

[0022] (9) Time constant: 100ms-300ms;

[0023] (10) Conversion time: 10ms-50ms; and,

[0024] (11) Signal accumulation times: 1 to 10 times.

[0025] In some embodiments of the present application, the method includes quantitatively detecting the reduced FDX1 in the cell sample to be tested.

[0026] In some embodiments of the present application, quantitative detection comprises the following steps:

[0027] grouping the cell samples to be tested;

[0028] One group was subjected to reduction treatment to reduce the oxidized FDX1 in the cells to the reduced FDX1, and was recorded as the completely reduced group;

[0029] The other group was not subjected to reduction treatment and was recorded as the test group;

[0030] The electron paramagnetic resonance technique is used to detect the test group and the complete reduction group to obtain the test group I 3494±1G , Test group I 3469±1G , Completely restored group I 3494±1G and fully restored group I 3469±1G , the proportion of reduced FDX1 is calculated by the following formula:

[0031] The proportion of reduced FDX1 = (test group I 3469±1G -Test Group I 3494±1G ) / (Completely restored group I 3469±1G -Fully restore group I 3494±1G )×100%,

[0032] Among them, the test group I 3469±1G and fully restored group I 3469±1G is the peak signal intensity value of the characteristic signal with a g value of 1.94, and the test group I 3494±1G and fully restored group I 3494±1G It is the peak-to-valley signal intensity value of the signal peak of the characteristic signal with a g value of 1.94.

[0033] In some embodiments of the present application, the cell samples of the test group are added with a cryoprotectant and frozen;

[0034] Optionally, the cryoprotectant comprises one or more of glycerol, sucrose and dimethyl sulfoxide;

[0035] Optionally, the amount of the cryoprotectant is 10%-20% (v / v) of the cell sample;

[0036] Optionally, the freezing method includes quick freezing using liquid nitrogen.

[0037] In some embodiments of the present application, the processing steps of the complete reduction group include: destroying the cell membrane under anaerobic conditions, adding a reducing agent to perform a reduction reaction, and freezing;

[0038] Optionally, the cell membrane is destroyed in an oxygen-free environment; Optionally, the cell membrane is destroyed in an atmosphere of inert gas; Optionally, the cell membrane is destroyed in an atmosphere of argon and / or nitrogen;

[0039] Optionally, cell membrane disruption may include repeated freezing and thawing;

[0040] Optionally, the reducing agent includes one or more of sodium dithionite and sodium ascorbate;

[0041] Optionally, the reduction reaction conditions include: temperature of 20°C-30°C, time of 5 min-15 min;

[0042] Optionally, the freezing method includes quick freezing using liquid nitrogen.

[0043] In some embodiments of the present application, the method satisfies one or more of the following conditions:

[0044] 1) The cell sample to be tested includes cells to be tested and PBS buffer; optionally, the amount of PBS buffer corresponding to each gram of cells to be tested is 0.5 mL-5 mL; optionally, the amount of PBS buffer corresponding to each gram of cells to be tested is 1 mL-2 mL;

[0045] 2) The cell sample to be tested includes recombinant cells that highly express FDX1;

[0046] 3) The FDX1 includes human FDX1; and FDX1 proteins and homologous proteins from different species or expression systems (such as bacteria, fungi or other non-human sources); and,

[0047] 4) The cell sample to be tested includes bacteria;

[0048] Optionally, IPTG is used to induce the recombinant cell to express FDX1; optionally, one or more of the following conditions are met during the induction process:

[0049] (A) The concentration of IPTG is 0.1 mM-1 mM; alternatively, the concentration of IPTG is 0.4 mM-0.8 mM;

[0050] (B) the induction time is 3 hours to 24 hours; alternatively, the induction time is 10 hours to 16 hours; and,

[0051] (C) L-cysteine ​​and one or more of ammonium ferric citrate and ammonium ferrous sulfate are added to the induction system.

[0052] In some embodiments of the present application, the cell sample to be tested is treated with a drug;

[0053] Optionally, the drug used in the drug treatment includes a copper death inducer;

[0054] Optionally, the drug treatment conditions include: shaking and a temperature of 35°C-39°C.

[0055] In some embodiments of the present application, the test also includes a negative control group;

[0056] Optionally, the negative control group treats the cell sample to be tested with solvent DMSO instead of the drug.

[0057] Compared with traditional technologies, the advantages of this application include:

[0058] This application introduces electron paramagnetic resonance (EPR) technology into the detection of the redox state of FDX1. It does not require labeling of the whole cell and can directly detect the redox state of FDX1 in the whole cell, which is conducive to real-time monitoring and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0060] Figure 1 Schematic diagram of the EPR signal differences in the redox states of the [2Fe-2S] cluster of FDX1.

[0061] Figure 2 Schematic diagram of the EPR process for detecting reduced FDX1 in Escherichia coli cells.

[0062] Figure 3 Comparison of the signals of overexpressed reduced FDX1 in E. coli whole cell samples and purified reduced FDX1.

[0063] Figure 4 To use Elesclomol-Cu 2+ Comparison of EPR spectra of treated cells at different time points.

[0064] Figure 5 The linear relationship between the concentration of FDX1 and the EPR signal intensity.

[0065] Figure 6 Comparison of EPR spectra of FDX1 whole cell samples with and without IPTG induction during overexpression.

[0066] Figure 7 The effect of different induction time treatments on the EPR signal intensity of FDX1 whole cell samples during overexpression.

[0067] Figure 8 The figure shows the effect of different concentrations of IPTG on the EPR signal intensity of FDX1 whole cell samples during overexpression.

[0068] Figure 9 The effect of supplementation of iron-sulfur precursors (L-cysteine ​​and ferrous ammonium sulfate) on the EPR signal intensity of FDX1 whole-cell samples during overexpression.

[0069] Figure 10 This is the effect of different concentrations of whole cell samples on the EPR signal intensity of FDX1 whole cell samples during sample preparation.

[0070] Figure 11 This is the effect of different microwave powers on the EPR signal intensity of FDX1 whole-cell samples during EPR testing.

[0071] Figure 12 This is the effect of different temperatures on the EPR signal intensity of FDX1 whole-cell samples during EPR testing.

[0072] Figure 13 This is the effect of different modulation amplitudes on the EPR signal intensity of FDX1 whole-cell samples during EPR testing. DETAILED DESCRIPTION

[0073] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0075] the term

[0076] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0077] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0078] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0079] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0080] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0081] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0082] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0083] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0084] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0085] In this application, 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.

[0086] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0087] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0088] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0089] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0090] Traditional EPR technology is primarily used for in vitro detection of purified proteins, but specific detection in whole-cell samples is difficult due to high background noise and insufficient sensitivity. Currently, there is no ideal method for sensitive, specific, real-time, and non-invasive detection of the redox state of FDX1 in cells.

[0091] This application aims to establish a novel method for detecting the redox state of intracellular FDX1 based on electron paramagnetic resonance (EPR) technology. This method utilizes the principle that EPR technology responds to the reduced state of FDX1 but not the oxidized state, allowing direct detection of the redox state of intracellular FDX1 without labeling or processing the cells. This method has the advantages of high sensitivity, strong specificity, good real-time performance, and non-invasiveness. It provides a new technical means for studying the redox state of intracellular FDX1 and is suitable for fields such as cell metabolism research, oxidative stress assessment, and drug screening.

[0092] One or more embodiments of the present application provide a method for detecting the redox state of FDX1 in a cell, the method comprising the following steps:

[0093] Using electron paramagnetic resonance to detect the characteristic signal of reduced FDX1 in the cell sample to be tested, and determining the status of reduced FDX1 in the cell sample to be tested based on the detection result;

[0094] The g value of the characteristic signal is 1.94 and / or 2.02.

[0095] In some examples of the present application, the electron paramagnetic resonance method satisfies one or more of the following conditions:

[0096] (1) The temperature is 77K-200K (e.g., 77, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200K); optionally, the temperature is 100K-120K;

[0097] (2) microwave power of 1 mW to 75 mW (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 mW); optionally, microwave power of 5 mW to 20 mW; and,

[0098] (3) The modulation amplitude is 1G-10G (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10G); optionally, the modulation amplitude is 1G-5G.

[0099] In some examples of the present application, the electron paramagnetic resonance method further satisfies one or more of the following conditions:

[0100] (4) Center field strength: 3300G-3400G (for example, 3300, 3310, 3320, 3330, 3340, 3350, 3360, 3370, 3380, 3390, 3400G);

[0101] (5) Magnetic field scanning range: 300mT-370mT (for example, 300, 310, 320, 330, 340, 350, 360, 370mT);

[0102] (6) Microwave frequency: 9.4 GHz to 9.6 GHz (e.g., 9.4, 9.5, 9.6 GHz);

[0103] (7) Detector gain: 1.00×10 3 -1.00×10 5 ; (for example, 1.00×10 3 , 2.5×10 3 , 5×10 3 , 7.5×10 3 , 1.00×10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 1.00×10 5 )

[0104] (8) Modulation frequency: 100kHz;

[0105] (9) Time constant: 100ms-300ms (e.g., 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300ms);

[0106] (10) Conversion time: 10 ms-50 ms (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 ms); and,

[0107] (11) Number of signal accumulation times: 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times).

[0108] In some examples of the present application, the method includes quantitatively detecting the reduced FDX1 in the cell sample to be tested.

[0109] In some examples of this application, quantitative detection includes the following steps:

[0110] grouping the cell samples to be tested;

[0111] One group was subjected to reduction treatment to reduce the oxidized FDX1 in the cells to the reduced FDX1, and was recorded as the completely reduced group;

[0112] The other group was not subjected to reduction treatment and was recorded as the test group;

[0113] The electron paramagnetic resonance technique is used to detect the test group and the complete reduction group to obtain the test group I 3494±1G , Test group I 3469±1G , Completely restored group I 3494±1G and fully restored group I 3469±1G , the proportion of reduced FDX1 is calculated by the following formula:

[0114] The proportion of reduced FDX1 = (test group I 3469±1G -Test Group I 3494±1G ) / (Completely restored group I 3469±1G -Fully restore group I 3494±1G )×100%,

[0115] Among them, the test group I 3469±1G and fully restored group I 3469±1G is the peak signal intensity value of the characteristic signal with a g value of 1.94, and the test group I 3494±1G and fully restored group I 3494±1G It is the peak-to-valley signal intensity value of the signal peak of the characteristic signal with a g value of 1.94.

[0116] In some examples of the present application, a cryoprotectant is added to the cell sample of the test group and frozen. The present application does not specifically limit the type of cryoprotectant, which may include but is not limited to glycerol, sucrose and dimethyl sulfoxide. One or more types may be selected. The present application does not specifically limit the amount of cryoprotectant used. Just select a suitable amount, which may include but is not limited to: the amount of the cryoprotectant is 10%-20% (v / v) of the cell sample, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. The present application does not specifically limit the freezing method, which may include but is not limited to quick freezing with liquid nitrogen.

[0117] In some examples of the present application, the treatment steps of the complete reduction group include: disrupting the cell membrane under anaerobic conditions, adding a reducing agent to carry out a reduction reaction, and freezing. Optionally, the cell membrane is disrupted in an anaerobic environment, including but not limited to: disrupting the cell membrane in an inert gas atmosphere. Further, optionally, the cell membrane is disrupted in an argon and / or nitrogen atmosphere. The present application does not specifically limit the method of disrupting the cell membrane, which may include but is not limited to repeated freezing and thawing. The present application does not specifically limit the type of reducing agent, and a suitable strong reducing agent can be selected. Strong reducing agents may include but are not limited to sodium disulfite and sodium ascorbate. One or more can be selected. Based on the selection of a suitable reducing agent, appropriate reduction conditions can be selected accordingly. For example, the reduction reaction conditions include: a temperature of 20°C-30°C (for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C) and a time of 5 min-15 min (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min). The present application does not particularly limit the freezing method, which may include but is not limited to quick freezing with liquid nitrogen.

[0118] In some examples of the present application, the method satisfies one or more of the following conditions 1) to 4):

[0119] 1) The cell sample to be tested includes cells to be tested and PBS buffer; optionally, the amount of PBS buffer corresponding to each 1g of cells to be tested is 0.5mL-5mL (for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mL); optionally, the amount of PBS buffer corresponding to each 1g of cells to be tested is 1mL-2mL;

[0120] 2) The cell sample to be tested includes recombinant cells that highly express FDX1;

[0121] 3) This application does not specifically limit the species origin of FDX1, and it may be derived from animals (such as mammals), bacteria, fungi, etc. In one example, the FDX1 includes animal-derived FDX1; further, the FDX1 includes human-derived FDX1; and,

[0122] 4) The cell sample to be tested includes bacteria.

[0123] Optionally, IPTG is used to induce the recombinant cell to express FDX1; optionally, one or more of the following conditions are met during the induction process:

[0124] (A) The concentration of IPTG is 0.1 mM-1 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mM); optionally, the concentration of IPTG is 0.4 mM-0.8 mM;

[0125] (B) the induction time is 3 hours to 24 hours (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours); optionally, the induction time is 10 hours to 16 hours; and,

[0126] (C) L-cysteine ​​and one or more of ammonium ferric citrate and ammonium ferrous sulfate are added to the induction system. For example, L-cysteine ​​and ammonium ferric citrate are added to the induction system. Another example is L-cysteine ​​and ammonium ferrous sulfate are added to the induction system. Another example is L-cysteine, ammonium ferric citrate, and ammonium ferrous sulfate are added to the induction system.

[0127] In some examples of the present application, the cell sample to be tested is treated with a drug;

[0128] Optionally, the drug used in the drug treatment includes a copper death inducer;

[0129] Optionally, the drug treatment conditions include: shaking, and a temperature of 35° C.-39° C. (eg, 35, 36, 37, 38, 39° C.).

[0130] In some examples of this application, the test also includes a negative control group;

[0131] Optionally, the negative control group treats the cell sample to be tested with solvent DMSO instead of the drug.

[0132] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0133] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0134] The present invention provides a label-free detection method for the redox state of FDX1 based on EPR technology. By optimizing the whole-cell sample processing process and EPR detection parameters, the characteristic signals of the reduced state of FDX1 (g = 1.94 and 2.02) are specifically captured, overcoming the defects of traditional methods that require labeling or damage to cell integrity. The examples show that this method has high sensitivity (detection limit 10μM) and good reproducibility (RSD <5%) in the E. coli model, making it suitable for dynamic monitoring of cellular oxidative stress and metabolic regulation research.

[0135] Technical principle: The active center of FDX1 (ferredoxin 1) is a [2Fe-2S] cluster, which consists of two iron atoms and two sulfur atoms. This iron-sulfur cluster can reversibly transform between the oxidized and reduced states through a single electron transfer, showing redox activity. 3+ state, the [2Fe-2S] cluster is in an oxidized state (i.e., [2Fe-2S] 2+ ), all electrons are paired, so no EPR signal is generated; when an iron atom is in Fe 2+ state, the other iron atom is in Fe 3+ state, the [2Fe-2S] cluster is in the reduced state (i.e., [2Fe-2S] + ), there is an unpaired electron, so it can produce a characteristic paramagnetic signal, the g value of which is usually around 1.94 and 2.02, see Figure 1 , which can be used to distinguish the redox state of FDX1.

[0136] Electron paramagnetic resonance (EPR) is a technique specifically used to detect substances containing unpaired electrons. When a sample is placed in a strong magnetic field, the spin magnetic moment of the unpaired electrons will undergo energy level splitting. Under specific microwave frequency radiation, electrons can jump from low energy levels to high energy levels, generating a resonant absorption signal. By analyzing this signal, information such as the structure and electronic environment of paramagnetic species (such as free radicals or transition metal centers) in the sample can be obtained. This application utilizes the principle that EPR technology has a signal response to reduced FDX1 but no signal response to oxidized FDX1. The redox state of FDX1 in living cells can be directly detected without labeling and processing the cells. Specifically, the cell sample to be tested is placed in an EPR spectrometer, and by collecting its EPR signal, the redox level of FDX1 can be evaluated. The intensity of the EPR signal is positively correlated with the content of reduced FDX1. By comparing the differences in EPR signal intensities between samples treated under different conditions, the changes in the redox state of FDX1 can be understood.

[0137] To obtain high-quality EPR signals, whole-cell samples need to be optimized and appropriate EPR parameters need to be selected. For example, the concentration of FDX1 in overexpressed cells can be increased as much as possible to make it easier to detect; low-temperature EPR technology can be used to increase signal intensity and sensitivity; and different microwave powers and modulation amplitudes can be used to optimize signal quality. See the process for details. Figure 2 .

[0138] Example 1: EPR detection of FDX1 redox state in cells

[0139] 1. Strain construction: The human FDX1 gene (UniProt number: P10109) was cloned into the pET-28a(+) vector and transformed into E. coli BL21(DE3). Positive clones were screened with kanamycin (50 μg / mL).

[0140] 2. Protein expression:

[0141] (1) Seed solution preparation: Pick a single colony and inoculate it into 5 mL of LB medium (containing kanamycin) and culture it at 37°C and 220 rpm for 12 hours.

[0142] (2) Expansion culture: Transfer the seed solution to 1 L sterile LB medium (supplemented with kanamycin) at a 1% (v / v) inoculum volume and culture at 37°C until the OD 600 =1.0-1.5.

[0143] (3) Induction of expression: Add IPTG to a final concentration of 0.4 mM, 1 mM L-cysteine ​​(L-Cys) and 0.1 mg / mL ferrous ammonium sulfate (FAS, Fe(NH4)2·(SO4)2·6H2O), cool to 18°C ​​and induce for 14 h (220 rpm).

[0144] 3. Sample processing:

[0145] (1) Cell collection and resuspension: Cells were collected by centrifugation at 6000 × g for 10 min at 4°C, washed twice with PBS buffer (pH 7.4), and then fully dispersed and suspended in PBS buffer at a ratio of 1 mL PBS buffer / 1 g cells.

[0146] (2) Sample grouping:

[0147] Test group: Take 120 μL of bacterial solution, add 20 μL of glycerol as a cryoprotectant, mix thoroughly, and then directly load into a quartz EPR tube and quick-freeze in liquid nitrogen until testing.

[0148] Complete reduction group: In an anaerobic glove box (95% N2 / 5% H2), the bacterial solution was repeatedly frozen and thawed 10 times to destroy the cell membrane. Subsequently, 50 μL of 200 mM sodium dithionite (Na2S2O4) was added and incubated at room temperature for 10 minutes to fully reduce the FDX1 in the system. After sealing, the system was transferred from the glove box to liquid nitrogen for quick freezing.

[0149] 4.EPR detection:

[0150] Instrument: Bruker A300 EPR spectrometer (equipped with liquid nitrogen cryogenic system).

[0151] The parameters were set as follows: central field strength: 3350 G; magnetic field scanning range: 300-370 mT; microwave frequency: 9.44 GHz; microwave power: 10.00 mW; detector gain: 1.00 × 10 4 Modulation frequency: 100kHz; Modulation amplitude: 3G; Time constant: 163.84ms; Conversion time: 16.00ms; Number of signal accumulations: 10 times; Temperature: 100K.

[0152] 5. Result analysis:

[0153] (1) Characteristic signal: Both the test group and the completely reduced group showed typical [2Fe-2S] cluster signal peaks at g = 1.94 and g = 2.02, which were consistent with the purified FDX1 standard ( Figure 3 ), confirming that FDX1 can be directly detected without disrupting cells, demonstrating the feasibility of directly detecting reduced FDX1 in whole cells.

[0154] Considering that there is a certain background signal in whole cell samples (from Mn 2+ , other iron-sulfur cluster signals in the cellular respiratory chain and other paramagnetic substances), if the secondary integral area of ​​the EPR spectrum is used to quantify the amount of FDX1, it may bring greater errors. Therefore, this application uses the signal intensity value of the peak tip of the signal peak near g = 1.94 (magnetic field strength: 3469 ± 1G) (i.e., the sample to be tested I 3469±1G ) and the peak-to-valley (magnetic field strength: 3494±1G) signal intensity (i.e., the sample to be tested I 3494±1G ) to quantify the reduced FDX1 in the cell (another advantage of this approach is that the quantification of the peak-to-peak-valley intensity difference can avoid the error caused by baseline drift compared to direct quantification using the peak-to-peak or peak-to-valley intensity). Accordingly, the peak-to-peak-valley signal intensity difference under the same magnetic field strength in the fully reduced sample is used to quantify the total amount of FDX1 in the cell (i.e., reduced FDX1 + oxidized FDX1), and the redox ratio is calculated using the following formula:

[0155]

[0156] (2) Quantitative calculation: The difference between the peak signal intensity and the valley signal intensity of the signal peak near g = 1.94 was used to quantify the reduced FDX1 in the cells. The proportion of natural reduced FDX1 in the whole cells reached 83 ± 3% (n = 5).

[0157] Example 2: EPR detection of the effect of copper death inducer treatment on the redox state of FDX1

[0158] Elesclomol (STA-4783) is a mitochondrial-targeted copper ionophore that binds to Cu 2+ The formed complex can specifically induce cuproptosis through the following mechanism and is directly related to the redox state of FDX1: Elesclomol-Cu 2+ After entering the cell, Cu is released inside the cell 2+ FDX1 is the core regulatory protein of copper death, and its reduced state can directly reduce Cu 2+ Cu + This process leads to the oxidation of the Fe-S cluster of FDX1, resulting in loss of electron transport capacity, ultimately triggering lipoylated protein aggregation and cell death. During copper death, the proportion of oxidized FDX1 increases significantly, resulting in a decrease in the EPR signal (g = 1.94 and g = 2.02). This change can serve as an early molecular marker of copper death. Compared to other methods, EPR technology can directly reflect the dynamic changes in FDX1 state.

[0159] The specific implementation details are described as follows:

[0160] (1) Drug treatment: Take the cell suspension prepared in step (1) of "3. Sample treatment" in Example 1 and add Elesclomol-Cu to a final concentration of 200 μM. 2+ The complex (prepared in DMSO) was shaken at 37°C.

[0161] (2) Negative control group: Add Elesclomol-Cu 2+ The complex solution was treated with an equal volume of DMSO (final concentration ≤ 0.1%).

[0162] (3) Sample preparation: Glycerol was added to the cells treated in steps (1) and (2) as in Example 1, and the reaction was terminated by quick freezing in liquid nitrogen at 0 h, 0.5 h, 1 h, 3 h, and 5 h, respectively. (4) EPR analysis: The same instrument and parameter settings as in Example 1 were used, focusing on analyzing the changes in the g = 1.94 signal intensity.

[0163] (5) Result analysis: The signal peak intensity of the treatment group g = 1.94 decreased compared with the control group ( Figure 4 ), indicating that the proportion of FDX1 reduced state was significantly reduced (decreased by 80% in 5 h).

[0164] Example 3: Methodology Validation

[0165] 1. Sensitivity Verification

[0166] a. Establishment of linear relationship: In order to evaluate the detection sensitivity and linear range of the EPR detection method for reduced FDX1 protein, a series of purified reduced FDX1 protein solutions with known concentrations were first used (the concentrations were 1000 μM, 800 μM, 500 μM, 400 μM, 200 μM, 100 μM, 50 μM, 20 μM and 10 μM, respectively), and 100 μL of each solution was taken and placed in a quartz EPR sample tube. Under fixed EPR parameter conditions (refer to the description in Example 1), the EPR signal was measured, and the peak-to-peak intensity of the main characteristic peak at each concentration was recorded. A standard curve was drawn with the FDX1 concentration as the horizontal axis and the EPR signal intensity as the vertical axis. The results showed that in the concentration range of 10-1000 μM, the EPR signal intensity was well linearly related to the FDX1 concentration, and the linear correlation coefficient (R 2 ) is 0.998 (see Figure 5 ), which can be used to estimate the concentration of FDX1 in the sample.

[0167] b. Detection Limit Assessment: Based on the establishment of a standard curve, Escherichia coli expressing FDX1 induced with IPTG was selected as the test subject. A two-fold serial dilution (i.e., 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32) was performed by volume using 50 mM PBS (pH 7.5). Samples were prepared as described in Example 1. EPR signal intensities were recorded after testing, and concentrations were converted using the standard curve. The results showed that under the optimized test conditions described in Example 1, the minimum detectable concentration of reduced FDX1 was 10 μM.

[0168] 2. Repeatability Verification

[0169] a. Intra-batch repeatability: The test sample was prepared according to Example 1, and the same sample was measured 5 times in a row. The relative standard deviation (RSD) of the g = 1.94 signal peak was 3.2%, as shown in the following table:

[0170] Table 1

[0171]

[0172]

[0173] b. Inter-batch reproducibility: Referring to Example 1, three batches of samples were prepared under the same conditions, and each batch was measured three times. The inter-batch RSD was 4.8%, as shown in the following table:

[0174] Table 2

[0175]

[0176] c. Inter-day repeatability: The test samples were prepared according to Example 1, and the same samples stored in liquid nitrogen were measured for 3 consecutive days. The inter-day RSD was 5.1%, as shown in the following table:

[0177] Table 3

[0178]

[0179] 3. Specificity Verification

[0180] When cells containing FDX1 expression plasmid but without induced expression were used, the EPR signal was significantly weakened, further confirming the specificity of the signal source. Figure 6 . Figure 6 In the experiment, the test sample corresponding to “+IPTG” was prepared with reference to Example 1, and the difference between “-IPTG” and “+IPTG” was that no IPTG was added to induce FDX1 expression.

[0181] Example 4: Impact of key technical points on FDX1 detection

[0182] The key technical points of this application method affect the intensity of the detected FDX1 EPR signal. This is mainly divided into two aspects: on the one hand, it is necessary to promote the overexpression of FDX1 as much as possible and increase the concentration of FDX1 in the cell; on the other hand, it is necessary to optimize the parameters to improve the detected signal intensity and sensitivity. The following points will be explained in detail:

[0183] 1. The criticality of intracellular FDX1 overexpression

[0184] a. Optimization of induction time: The E. coli BL21 (DE3) strain carrying pET28a-FDX1 was sampled 3 hours, 6 hours, 10 hours, 14 hours, 16 hours, and 24 hours after IPTG induction. The cells were collected and resuspended, cryoprotectant was added, and after thorough mixing, they were directly placed into a quartz EPR tube and quickly frozen in liquid nitrogen. The samples to be tested were prepared and the EPR signal intensity of FDX1 was detected. That is, in the optimization process, except for setting multiple levels of induction time, the rest of the operations were the same as in Example 1. The results are shown in FIG. Figure 7The results showed that the sample inducing for 14 hours had the strongest signal, with the signal intensity at g = 1.94 1.6 times that of the 6-hour group. However, increasing the induction time after 14 hours did not increase the EPR signal intensity, but instead decreased it. The signal intensity of the 24-hour group decreased by 52%. Therefore, the optimal induction time is 14 hours.

[0185] b. Gradient screening of IPTG concentration: The induction time was fixed at 14 hours, and the IPTG concentration was set at multiple levels (0.1mM, 0.2mM, 0.4mM, 0.6mM, 1.0mM) for induction culture. The cells were collected and resuspended, cryoprotectant was added, and after thorough mixing, they were directly loaded into a quartz EPR tube and quickly frozen with liquid nitrogen to prepare each test sample. The effect of IPTG concentration (0.1mM, 0.2mM, 0.4mM, 0.6mM, 1.0mM) on the FDX1 EPR signal intensity was tested. That is, in the optimization process, except for setting multiple levels of IPTG concentration, the rest of the operations were the same as in Example 1. The results are shown in FIG. Figure 8 The results showed that the EPR signal induced by 0.4 mM IPTG was the strongest. Therefore, 0.4 mM IPTG was selected as the optimal induction concentration.

[0186] c. Iron-sulfur precursor supplementation: Prepare the test sample with reference to Example 1. During the induction process, set up the following groups: add L-cysteine ​​and ferrous ammonium sulfate to the LB medium during induction; do not add L-cysteine ​​and ferrous ammonium sulfate to the LB medium during induction. Other than that, the rest of the operation is the same as in Example 1. Test the EPR spectrum of FDX1, the results are shown in Figure 9 Results: Adding L-cysteine ​​and ammonium ferrous sulfate significantly increased the signal intensity. The EPR signal intensity increased to 2.5 times that of the unoptimized group. Conclusion: Iron and sulfur source supplementation is essential for increasing the detection signal intensity and ensuring test accuracy.

[0187] 2. Criticality of sample preparation conditions

[0188] a. Necessity of a cryoprotectant: Samples were prepared as described in Example 1. During the preparation process, two groups were added with glycerol and one without. Results showed that the EPR tubes of samples without glycerol were prone to bursting during the quick freezing and removal from liquid nitrogen, often resulting in the inability to complete the test. Therefore, a cryoprotectant is crucial for testing.

[0189] b. Optimization of whole cell sample concentration: Prepare the test sample with reference to Example 1. In the step of "(1) Cell collection and resuspension", prepare whole cell samples according to the ratio of 5 mL buffer / 1 g cells, 4 mL buffer / 1 g cells, 3 mL buffer / 1 g cells, 2 mL buffer / 1 g cells, 1 mL buffer / 1 g cells, and 0.5 mL buffer / 1 g cells. Except for setting multiple levels of resuspension concentration, the rest are the same as Example 1. Test and compare the EPR signal intensity. The results are shown in Figure 10 The results showed that the EPR signal increased with increasing bacterial concentration, reaching its strongest signal at 1g of bacteria / 0.5mL. However, at this point, the bacterial solution was too viscous and easily adhered to the tube wall when loaded into the sample tube, resulting in a large error in quantification. Therefore, a ratio of 1mL of buffer / 1g of cells was selected for the preparation of whole-cell samples, which ensured both signal intensity and quantitative accuracy.

[0190] 3. The criticality of EPR parameter setting

[0191] a. Microwave power optimization: Prepare the test sample with reference to Example 1, and collect signals at microwave powers of 1mW, 2mW, 5mW, 10mW, 20mW, 50mW, and 75mW. With respect to Example 1, except for setting multiple levels of microwave power, the other parameters are the same as Example 1. Figure 11 Although the results show that the higher the microwave power, the stronger the signal intensity when tested at 120K, increasing the power does not consistently improve the signal-to-noise ratio of the spectrum. In addition, considering that testing at lower temperatures may easily lead to signal saturation and reduce resolution, as well as the limitations of the instrument, a microwave power of 10mW was selected for safety reasons.

[0192] b. The criticality of temperature control: The samples to be tested were prepared with reference to Example 1 and measured at temperatures of 200K, 180K, 160K, 140K, 120K, and 100K. With respect to Example 1, except for the temperature being set at multiple levels, the other parameters were the same as in Example 1. The results are shown in Figure 12 The results show that the signal is optimal at 100K. Excessively high temperatures cause the iron-sulfur clusters to relax faster and the signal to weaken. Lowering the detection temperature within a certain temperature range can certainly increase the signal-to-noise ratio, but low-temperature testing below 100K requires the use of liquid helium as a refrigerant, which is expensive. In addition, when the temperature is too low, the signal is severely saturated, and the signal strength decreases, which is not conducive to testing. Therefore, considering the comprehensive consideration of cost and detection effect, the detection temperature is set at 100K.

[0193] c. Modulation amplitude optimization: Prepare the test samples with reference to Example 1 and compare the signal quality under 1G, 2G, 3G, 4G, 5G, 6G, 7G, 8G, 9G, and 10G modulation amplitudes. With respect to Example 1, except for setting multiple levels of modulation amplitude, the other parameters are the same as Example 1. The results are shown in Figure 13 Increasing the modulation amplitude can certainly increase signal strength, but the background signal will also increase. The 1G signal is too weak, and 10G causes the signal to be over-modulated and distorted, so the 3G modulation amplitude was chosen.

[0194] Example 5: Comparison with traditional EPR method

[0195] 1. Experimental Design

[0196] The same amount of FDX1-overexpressing Escherichia coli (prepared according to Example 1) was divided into two groups:

[0197] Traditional method group: cell disruption → ultracentrifugation → supernatant collection → protein extraction → centrifugal concentration → NaDT reduction → EPR detection. The specific process is as follows:

[0198] (1) The cells were resuspended in lysis buffer (pH 8.0, 20 mM Tris-HCl, 300 mM NaCl, 0.1% NP-40, 2 mM DTT, 1 mM PMSF, 10% glycerol, 5 mM imidazole), and then the cells were disrupted by ultrasound in an ice bath (power 30%, 3 seconds on / 3 seconds off, for a total of 10 minutes);

[0199] (2) Ultracentrifuge the lysate at 16,000 × g for 30 minutes at 4°C and collect the supernatant;

[0200] (3) Ni-NTA affinity chromatography filler (pre-equilibrated) was added to the supernatant for metal affinity purification. After binding for 60 minutes, non-specific binding proteins were washed with wash buffer (containing 30 mM imidazole) and then eluted with elution buffer (containing 250 mM imidazole) to collect the target protein;

[0201] (4) The eluate was concentrated to approximately 200 μL using a 10 kDa ultrafiltration centrifuge tube and replaced with an imidazole-free buffer;

[0202] (5) In an anaerobic glove box, add a final concentration of 50 mM NaDT (sodium dithionite) for protein reduction and react at room temperature for 10 minutes;

[0203] (6) The reduced protein solution was placed in an EPR quartz sample tube, sealed, taken out of the glove box, and quickly frozen in liquid nitrogen before low-temperature EPR detection (the detection conditions were described in Example 1).

[0204] The method set of the present application: preparation of whole cell samples → liquid nitrogen quick freezing → EPR detection, please refer to Example 1 for details.

[0205] 2. Comparison of results

[0206] a. Operation time: The traditional method takes 3-4 hours to complete sample preparation, while this method only takes 10-15 minutes.

[0207] b. Whether it reflects the redox state of intracellular FDX1: Direct testing of protein extracted using traditional methods did not detect a significant FDX1 signal, indicating that after cell disruption and subsequent extraction, FDX1 has become oxidized. Although it can be reduced using a reducing agent, the test results at this point do not reflect the original redox state of FDX1 in the cell. In contrast, under the same FDX1 concentration conditions, the proposed method can detect a significant EPR signal of reduced FDX1, indicating that the proposed method better preserves the reduced state of FDX1.

[0208] 3. Conclusion: Traditional methods require protein disruption and extraction, which is not only time-consuming but also unable to reflect the redox state within cells. Therefore, compared with traditional EPR detection methods, the method of this application shows significant advantages in ease of operation and can more accurately reflect the original redox state of FDX1 within cells.

[0209] The technical advantages of the embodiments of this application include:

[0210] (1) High sensitivity: EPR technology can detect the signal of unpaired electrons in the sample, so it can very sensitively detect changes in the redox state of FDX1.

[0211] (2) Strong specificity: The unique g-value characteristic of FDX1 is used for identification. At the same time, an overexpression system is adopted to greatly increase the content of FDX1 in the cell, making the signal significantly stronger than the background signal, thereby avoiding interference from other cellular signals. Therefore, the redox state of FDX1 can be specifically detected.

[0212] (3) Good real-time performance: EPR technology can realize real-time detection of the redox state of FDX1 in cells, so it can track the changes in the redox state of FDX1 under conditions such as drug treatment and hypoxia in real time.

[0213] (4) Non-destructive: EPR technology does not damage cells and can be used for living cell detection.

[0214] (5) No labeling or processing required: This application utilizes the principle that EPR has a signal response to reduced FDX1 but no signal response to oxidized FDX1. The redox state of FDX1 in cells can be directly detected without labeling or processing the cells, and the operation is simple and quick.

[0215] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0216] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting the redox state of FDX1 in cells, characterized in that: The method comprises the following steps: Using electron paramagnetic resonance to detect the characteristic signal of reduced FDX1 in the cell sample to be tested, and determining the status of reduced FDX1 in the cell sample to be tested based on the detection result; The g value of the characteristic signal is 1.94 and / or 2.

02.

2. The method for detecting the redox state of FDX1 in cells according to claim 1, wherein The electron paramagnetic resonance method satisfies one or more of the following conditions: (1) The temperature is 77K-200K; optionally, the temperature is 100K-120K; (2) microwave power of 1 mW-75 mW; optionally, microwave power of 5 mW-20 mW; and, (3) The modulation amplitude is 1G-10G; optionally, the modulation amplitude is 1G-5G.

3. The method for detecting the redox state of FDX1 in cells according to claim 2, characterized in that: The electron paramagnetic resonance method further satisfies one or more of the following conditions: (4) Center field strength: 3300G-3400G; (5) Magnetic field scanning range: 300mT-370mT; (6) Microwave frequency: 9.4 GHz-9.6 GHz; (7) Detector gain: 1.00×10 3 -1.00×10 5 ; (8) Modulation frequency: 100kHz; (9) Time constant: 100ms-300ms; (10) Conversion time: 10ms-50ms; and, (11) Signal accumulation times: 1 to 10 times.

4. The method for detecting the redox state of FDX1 in cells according to any one of claims 1 to 3, characterized in that: The method comprises quantitatively detecting the reduced FDX1 in the cell sample to be tested.

5. The method for detecting the redox state of FDX1 in cells according to claim 4, characterized in that: Quantitative testing involves the following steps: grouping the cell samples to be tested; One group was subjected to reduction treatment to reduce the oxidized FDX1 in the cells to the reduced FDX1, and was recorded as the completely reduced group; The other group was not subjected to reduction treatment and was recorded as the test group; The electron paramagnetic resonance technique is used to detect the test group and the complete reduction group to obtain the test group I 3494±1G , Test group I 3469±1G , Completely restored group I 3494±1G and fully restored group I 3469±1G , the proportion of reduced FDX1 is calculated by the following formula: The proportion of reduced FDX1 = (test group I 3469±1G -Test Group I 3494±1G ) / (Completely restored group I 3469±1G -Fully restore group I 3494±1G )×100%, Among them, the test group I 3469±1G and fully restored group I 3469±1G is the peak signal intensity value of the characteristic signal with a g value of 1.94, and the test group I 3494±1G and fully restored group I 3494±1G It is the peak-to-valley signal intensity value of the signal peak of the characteristic signal with a g value of 1.

94.

6. The method for detecting the redox state of FDX1 in cells according to claim 5, characterized in that: The cell samples of the test group are added with a cryoprotectant and frozen; Optionally, the cryoprotectant comprises one or more of glycerol, sucrose and dimethyl sulfoxide; Optionally, the amount of the cryoprotectant is 10%-20% (v / v) of the cell sample; Optionally, the freezing method includes quick freezing using liquid nitrogen.

7. The method for detecting the redox state of FDX1 in cells according to claim 5, characterized in that: The treatment steps of the complete reduction group included: destroying the cell membrane under anaerobic conditions, adding reducing agents for reduction reaction, and freezing; Optionally, the cell membrane is destroyed in an oxygen-free environment; Optionally, the cell membrane is destroyed in an atmosphere of inert gas; Optionally, the cell membrane is destroyed in an atmosphere of argon and / or nitrogen; Optionally, cell membrane disruption may include repeated freezing and thawing; Optionally, the reducing agent includes one or more of sodium dithionite and sodium ascorbate; Optionally, the reduction reaction conditions include: temperature of 20°C-30°C, time of 5 min-15 min; Optionally, the freezing method includes quick freezing using liquid nitrogen.

8. The method for detecting the redox state of FDX1 in cells according to any one of claims 1 to 3 and 5 to 7, characterized in that: The method satisfies one or more of the following conditions: 1) The cell sample to be tested includes cells to be tested and PBS buffer; optionally, the amount of PBS buffer corresponding to each gram of cells to be tested is 0.5 mL-5 mL; optionally, the amount of PBS buffer corresponding to each gram of cells to be tested is 1 mL-2 mL; 2) The cell sample to be tested includes recombinant cells that highly express FDX1; 3) the FDX1 includes human FDX1; and 4) The cell sample to be tested includes bacteria; Optionally, IPTG is used to induce the recombinant cell to express FDX1; optionally, one or more of the following conditions are met during the induction process: (A) The concentration of IPTG is 0.1 mM-1 mM; alternatively, the concentration of IPTG is 0.4 mM-0.8 mM; (B) the induction time is 3 hours to 24 hours; alternatively, the induction time is 10 hours to 16 hours; and, (C) L-cysteine ​​and one or more of ammonium ferric citrate and ammonium ferrous sulfate are added to the induction system.

9. The method for detecting the redox state of FDX1 in cells according to any one of claims 1 to 3 and 5 to 7, characterized in that: The cell sample to be tested is treated with a drug; Optionally, the drug used in the drug treatment includes a copper death inducer; Optionally, the drug treatment conditions include: shaking and a temperature of 35°C-39°C.

10. The method for detecting the redox state of FDX1 in cells according to claim 9, characterized in that: The test also included a negative control group; Optionally, the negative control group treats the cell sample to be tested with solvent DMSO instead of the drug.

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