CN111323464B - System for quantitative detection of NQO1 enzyme activity and drug screening, and electroluminescence sensing method and application thereof - Google Patents

System for quantitative detection of NQO1 enzyme activity and drug screening, and electroluminescence sensing method and application thereof Download PDF

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CN111323464B
CN111323464B CN202010338008.4A CN202010338008A CN111323464B CN 111323464 B CN111323464 B CN 111323464B CN 202010338008 A CN202010338008 A CN 202010338008A CN 111323464 B CN111323464 B CN 111323464B
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nqo1
hydrogen peroxide
enzyme
electrode
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CN111323464A (en
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沈艳飞
郑永军
张袁健
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Southeast University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a system for quantitatively detecting the activity of NQO1 enzyme and screening drugs, an electroluminescence sensing method and application thereof, wherein the system for detecting the activity of the NQO1 enzyme and screening drugs comprises a three-electrode system, signal molecules and reaction liquid, wherein the reaction liquid is generated by reacting the NQO1 enzyme with a substrate capable of producing hydrogen peroxide with the NQO1 enzyme. In the invention, a simple and easily obtained substrate is utilized to simulate the in-vivo metabolic process of NQO1, the in-vivo metabolic process of NQO1 is introduced into an electroluminescence detection platform, and the activity change of NQO1 is indirectly reflected by utilizing hydrogen peroxide generated in the electroluminescence detection of the NQO1 and the substrate metabolic process, so that the detection of the NQO1 is realized. In addition, hydrogen peroxide generated based on NQO1 substrate metabolism is an important antitumor active ingredient, and when the NQO1 amount is fixed, different substrates and NQO1 react to generate hydrogen peroxide with different concentrations to evaluate the antitumor activity of different substrates, so that the antitumor activity screening of related medicines is realized.

Description

System for quantitative detection of NQO1 enzyme activity and drug screening, and electroluminescence sensing method and application thereof
Technical Field
The invention belongs to the field of electrochemical electroluminescence sensing, relates to construction and application of an electroluminescence sensing system for detecting NQO1 enzyme and screening related medicines, and in particular relates to a system for quantitatively detecting NQO1 enzyme activity and screening medicines and application thereof.
Background
Tumors have become a large killer that threatens human survival. The tumor is difficult to cure, and has a great relationship with the early detection of the tumor and the timely use of effective medicines. The effective early detection of tumor markers can help doctors predict the occurrence and development of tumors as early as possible, thereby providing effective treatment means. The rapid screening in the early stage of the medicament can also greatly promote the effective medicament to enter the clinic rapidly, thereby leading the patient to obtain the treatment opportunity earlier. Despite the great efforts invested by many researchers in this regard, the current survival needs of oncology patients are still not met. Therefore, the development of early diagnosis tools and preclinical drug screening tools has great significance for the diagnosis and treatment of tumors. NQO1 is an enzyme that is closely related to tumors and is an effective early diagnostic marker for tumors and drug targets.
NQO1 is a two-electron reductase mainly involved in detoxification and drug activation of quinone compounds in vivo. NQO1 is expressed in tumor tissues at 5-200 fold higher than normal tissues, and thus it is considered as an effective tumor marker. In addition, because of the many links between the compound and the occurrence and development of tumors, the compound is also developed into an effective drug action target, and various related drugs are being developed.
Currently, the detection mechanism for NQO1 is mainly based on its reduction of quinones, releasing optical signaling molecules. The method has certain defects in stability of the probe, accuracy and sensitivity of detection due to the existence of ester bonds or amide bonds in the probe and an optical mechanism. In addition, designing and synthesizing probes like this requires skilled synthetic skills and associated theoretical knowledge. These greatly limit the popularization and detection performance of this type of method.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a detection system for the activity of NQO1 enzyme, and provides a brand-new detection thought and detection method, which effectively solve the problems that the technical method for detecting NQO1 is difficult to design and synthesize, difficult to popularize and use, high in background signal, relatively unstable in probe, poor in detection performance and the like. In the invention, a simple and easily obtained substrate is utilized to simulate the in-vivo metabolic process of NQO1, the in-vivo metabolic process of NQO1 is introduced into an electroluminescence detection platform, and hydrogen peroxide generated in the metabolic process of NQO1 and the substrate is detected by utilizing an electroluminescence technology to indirectly reflect the activity change of NQO1, so that the detection of NQO1 is realized. In addition, hydrogen peroxide generated based on NQO1 substrate metabolism is an important antitumor active ingredient, and when the adding amount of NQO1 is controlled and fixed, different substrates and NQO1 act to generate hydrogen peroxide with different concentrations to evaluate the antitumor activity of different substrates, so that the antitumor activity screening of related medicines is realized.
The invention also provides a system and a method for screening the medicines.
The technical scheme is as follows: in order to achieve the above purpose, the quantitative detection system for the activity of the NQO1 enzyme comprises a three-electrode system, signal molecules and a reaction solution, wherein the reaction solution is generated by reacting the NQO1 enzyme with a substrate capable of producing hydrogen peroxide with the NQO1 enzyme.
The three-electrode system comprises a glassy carbon electrode, a gold electrode, an ITO electrode, a foam nickel electrode and a foam nickel modified electrode which are used as working electrodes, a platinum wire is used as a counter electrode, and Ag/AgCl is used as a reference electrode. Preferably, a foam nickel electrode is used as a working electrode, a platinum wire is used as a counter electrode, and Ag/AgCl is used as a counter electrode.
Wherein the signal molecule is luminol or isoluminol.
Preferably, the signal molecule is 0.1M luminol.
Wherein the substrate is beta-lappactone, tan shinone IIA, mendione or (+ -) -Dunnione.
The detection method for quantifying the activity of the NQO1 enzyme comprises the following steps:
and (3) reacting NQO1 with a substrate capable of producing hydrogen peroxide with NQO1 enzyme to generate a reaction solution containing hydrogen peroxide, and testing the luminous intensity of the reaction solution by utilizing three electrodes and signal molecules, thereby realizing quantitative detection of the NQO1 enzyme.
The quantitative detection system for the activity of the NQO1 enzyme is applied to quantitative and rapid detection of the NQO 1.
The system for screening the medicines comprises a three-electrode system, signal molecules and reaction liquid, wherein the reaction liquid is generated by reacting NQO1 enzyme with a target medicine to be screened. Wherein, the NQO1 enzyme reacts with the target drug to be screened to generate hydrogen peroxide or other active oxygen which is the NQO1 related drug.
The three-electrode system comprises a glassy carbon electrode, a gold electrode, an ITO electrode, a foam nickel electrode and a foam nickel modified electrode which are used as working electrodes, a platinum wire is used as a counter electrode, and Ag/AgCl is used as a reference electrode.
Wherein the signal molecule is luminol or isoluminol.
The method for screening the medicine comprises the following steps:
and reacting NQO1 with the target drug to be screened to generate a reaction solution containing hydrogen peroxide, and testing the luminous intensity of the reaction solution by utilizing three electrodes and signal molecules, thereby realizing analysis and evaluation of the antitumor activity of the drug.
The drug screening system disclosed by the invention is applied to screening and evaluating the anti-tumor activity of NQO1 related drugs.
In the quantitative detection method for detecting the activity of the NQO1 enzyme, the first part is a part in which NQO1 reacts with a substrate to generate hydrogen peroxide, and the principle is shown in figure 1.
In the preferred detection process of the invention, beta-lapacone is used as a model substrate, and under the action of enzyme and coenzyme (NADPH), quinone is changed into phenol, and the phenol is unstable, and then returns to the raw material beta-lapacone under the participation of oxygen, and superoxide anion free radicals can be generated. The unstable superoxide anion radical becomes stable hydrogen peroxide over time.
Wherein, the concentration of beta-lappactone is 0-30 mu M (wherein 0 is blank), the preferred concentration is 20 mu M, the preferred concentration is 50-250 mu M, the preferred concentration is 100 mu M, the concentration of NQO1 is 0-50 mu g/mL, the reaction time is 5-30mins, and the preferred time is 20min. The reaction temperature is 10-50℃and preferably 37 ℃.
The second part is the part of the signal molecule which acts on the object to be detected, and the principle of the second part is shown in figure 2.
In the invention, a glassy carbon electrode, a gold electrode, an ITO electrode or a foam nickel electrode is used as a working electrode, preferably a foam nickel electrode, a platinum wire is used as a counter electrode, and an Ag/AgCl electrode is used as a counter electrode. The electrochemical scanning rate is 0.01-0.5V/s, preferably 0.2V/s. The scanning potential is-0.5 to 1V, preferably 0 to 0.6V. The photoelectric multiple boost is 300-900, preferably 700. The luminol or the isoluminol thereof is taken as a signal molecule, and the concentration of the signal molecule is 10-500mM, preferably 100mM luminol. The hydrogen peroxide and luminol generated by the first part generate optical signals under the action of the electrode.
The design principle of the invention is as follows:
based on the current detection of NQO1, a response type probe method is mainly used, and a fluorescence method is mainly used, wherein the method has the defects of low detection background signal, unstable probe molecules and the like, and the final detection performance is relatively poor (poor sensitivity) directly. In addition, it is difficult to solve the above problems by the fluorescent probe method due to the detection and light emission mechanism of the fluorescent probe itself. The invention combines the fact that NQO1 generates hydrogen peroxide in the metabolic process in vivo, and the detection advantage of electroluminescence itself compared with a fluorescence method and the high-efficiency application in the aspect of hydrogen peroxide detection, and provides the method for detecting the metabolic generation of the hydrogen peroxide by using the electroluminescence platform so as to indirectly reflect the activity of NQO1 enzyme.
The detection mechanism of the present invention is shown in fig. 3.
Firstly, the substrate, NQO1 and NADPH are opened in a buffer solution, the reaction is carried out for 20 minutes at 37 ℃, a large amount of hydrogen peroxide is generated in the process, then the reaction solution is transferred into an ECL test pool, all electrodes and signal molecules are added, and the electroluminescent intensity is read and recorded under the set condition. According to the recorded values, the linear relation between the light intensity and different enzyme concentrations can be obtained, so that the quantitative detection of the NQO1 enzyme activity is realized. In addition, when the enzyme concentration is constant, different substrates are added, and optical signals with different intensities can be generated. The concentration of the active molecular hydrogen peroxide generated by the substrate can be obtained according to the optical signal intensity, the activity of the substrate is obtained, and the drug screening is realized.
The detection method and the detection system can be used for quantitative detection of the activity of NQO1 and also can be used for screening the anti-tumor activity of related drugs.
The invention mainly transfers the metabolic process of the NQO1 in vitro by referring to the metabolic pathway of the NQO1 in vivo, and utilizes a simple electroluminescent detection platform to construct an NQO1 detection system, so that the efficient detection of the NQO1 is realized, compared with the earlier stage of the detection by complex design of the synthesized substrate, the metabolic substrate is a natural and easily obtained substrate, and the detection system has lower detection background signal and better stability, sensitivity and selectivity. Provides a brand new strategy and tool system for in vitro rapid diagnosis and drug screening of NQO 1. Therefore, the detection system and the detection method provided by the invention are necessary and significant.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
firstly, the invention provides a system for quantitatively detecting the activity of NQO1 enzyme and screening drugs, which has the advantages of easily available metabolic substrates, low detection background signals, high stability, sensitivity, high selectivity, excellent detection performance and the like.
Secondly, the method for rapidly detecting the activity of the NQO1 enzyme based on the enzyme metabolism mechanism by utilizing the system of the invention widens the enzyme detection means. The detection method based on the system of the invention effectively improves the detection performance of the enzyme, the detection limit is 10ng/mL, the signal to noise ratio is 460, and the method has great advantages compared with the prior method (figure 8).
Based on the NQO1 detection system and the detection method, the invention constructs an anti-tumor activity screening system of NQO1 related drugs, and the invention realizes drug activity screening for the first time by using the electroluminescent sensing platform, widens the application direction of electroluminescent sensing and realizes drug screening of electroluminescent sensing for the first time. The invention provides a new idea for simultaneously realizing enzyme activity detection and drug screening for electroluminescent sensing.
Drawings
FIG. 1 is a schematic diagram of the principle of generating hydrogen peroxide by the action of NQO1 and a substrate;
FIG. 2 is a schematic diagram showing the principle of action of a signal molecule and an analyte;
FIG. 3 is a schematic diagram of a detection mechanism according to the present invention;
FIG. 4 is a graph showing the verification of hydrogen peroxide generation after reaction of NQO1 with a substrate;
FIG. 5 is a schematic diagram of the intensity of light generated after different concentrations of NQO1 react with a substrate;
FIG. 6 is a graph of the response of a detection system to other active substances;
FIG. 7 is a schematic diagram of the assay system for assessing drug activity and activity of different compounds;
FIG. 8 is a summary of reported methods for assessing NQO1 activity.
Detailed Description
The invention is further described below with reference to specific embodiments and figures.
Materials, reagents and the like used in the examples are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Wherein: NQO1 (Sigma-Aldrich 100U/mg powder, 99% purity)
Luminol (Ala Ding Shiji, 99% purity)
Isoluminol (A Ding Shiji, 98% pure)
Beta-lapachone (Mecanline reagent, purity 99%), acenaphthenequinone (An Naiji reagent, purity 98%), 2, 6-diamantanthraquinone (An Naiji reagent, purity 98%), tanshinone IIA (carbofuran reagent, purity 98%), mendione (An Naiji reagent, purity 98%) or (. + -.) -Dunnione (source She Shiji, purity 98%)
Example 1
After confirming that NQO1 reacts with a substrate, hydrogen peroxide is generated
First, the total reaction solution was 3mL, and NQO1 (20. Mu.g/mL) was added to PBS buffer (0.01M, pH=7.4) containing NADPH (100. Mu.M) and β -lapachone (20. Mu.M) at a final concentration, and the mixture was allowed to react at 37℃for 20 minutes with an opening. The reaction solution was designated as "N". Then, TMB or TMB+HRP is added into the reaction liquid for color development detection of hydrogen peroxide, after 30 minutes, ultraviolet-visible light absorption and color images of each reaction liquid are recorded, and the reaction liquid is named as 'N+TMB' or 'N+TMB+HRP'. Blank control, test record as above.
As shown in FIG. 4, only after TMB and HRP are added simultaneously, the ultraviolet-visible light absorption of the reaction liquid is obviously changed, and the other reaction liquid is not changed, so that the enzyme and the substrate act to generate hydrogen peroxide.
Example 2
Detection of different concentrations of NQO1 by detection system to produce electroluminescent intensity measurements
The quantitative detection system for the activity of the NQO1 enzyme comprises: the three-electrode system foam nickel electrode is a working electrode, a platinum wire is a counter electrode, ag/AgCl is a reference electrode, and a signal molecule is 0.1M luminol, NQO1 and a substrate beta-lapachone. The detection process comprises the following steps: the total reaction solution was 3mL, and each of the substances was added to PBS buffer (0.01M, pH=7.4) containing NADPH (100. Mu.M) and β -lapachone (20. Mu.M) at a final concentration of 0. Mu.g/mL, 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, and 25. Mu.g/mL of NQO1, and reacted at 37℃for 20 minutes with an open reaction. And then, respectively transferring each reaction solution into an electroluminescent reaction tank, wherein a foam nickel electrode is used as a working electrode, a platinum wire is used as a counter electrode, ag/AgCl is used as a reference electrode, luminol with the final concentration of 0.1M is added as a signal molecule, and the intensity of optical signals (scanning potential is 0-0.6V, scanning speed is 0.2V/s and photoelectric multiple pressurization is 700) generated by each reaction solution is tested and recorded. Thereby fitting the linear relationship as shown in the results of fig. 5.
As shown in fig. 5, ECL intensity increased with increasing enzyme concentration and exhibited a better linear relationship. The detection limit of the system on NQO1 is 10ng/mL and the signal-to-noise ratio is 460. As can be seen from the comparison data of FIG. 8, the results are superior to the reported detection method for NQO1 by the fluorescent probe method. Therefore, the method provided by the invention can be used for quantitative detection of NQO1, and has better performance.
Example 3
Selectivity of detection system
In order to evaluate the selectivity of the detection system to NQO1, the invention selects some common active substances in organisms as interferents and tests the relevant response conditions. Specifically, each sample was added to a test solution of PBS buffer (0.01M, pH=7.4) at a predetermined concentration (wherein the final concentration of metal ions was 0.1mM, the final concentration of small molecules was 0.1mM, the concentration of comparative enzyme was 50. Mu.g/mL, and the concentration of NQO1 was 20. Mu.g/mL), and then the optical signal of the sample was measured by referring to the same detection method as in example 2.
As shown in FIG. 6, in the present invention, metal ions (Al 3+ ，Cu 2+ ，Fe 2+ ，Fe 3+ ) Small molecules (cysteine, glutathione, homocysteine, phenylalanine, serine, ascorbic acid) and some comparison enzymes (esterases, glucose oxidases) were used as control molecules to test the response of these molecules in the sensing system of the invention.
As shown in fig. 6, only NQO1 produces a distinct optical signal, and others have relatively little signal. Therefore, the detection platform has better selectivity to NQO 1.
Example 4
Drug screening application of detection system
The screening system includes: the three-electrode system foam nickel electrode is used as a working electrode, a platinum wire is used as a counter electrode, ag/AgCl is used as a reference electrode, and a signal molecule is 0.1M luminol, NQO1 and a drug to be screened (aceaphthane, 2, 6-diamantane, tan shinone IIA, mendione or (+ -) -Dunnione).
Due to the antitumor activity of NQO 1-related drugs, most of them depend on the concentration of hydrogen peroxide produced. By evaluating the ECL intensities, i.e. hydrogen peroxide concentrations, generated by different substrates and NQO1 according to the principle shown in FIG. 7a, the antitumor activity of the different substrates can be evaluated. Thus, in the present invention, five compounds (acephthenequinone, 2, 6-diamantaquinone, tanshinone IIA, menadione or (±) -Dunnione) were selected as example drugs, which were tested for their reaction with NQO1, respectively, and the test methods and instrument parameters were the same as in example 2, and the electroluminescence intensity was tested and recorded, and the results are shown in fig. 7 b.
As shown in FIG. 7b, the first two general quinone compounds (2, 6-diamantaquinone) did not show significant ECL intensities, while the last three reported NQO 1-related drug candidates (tanshinone IIA, menadione or (+ -) -Dunnione) showed significant ECL signals. This result is consistent with the reported cell testing methods. The detection system is proved to be applicable to NQO1 related medicine antitumor activity screening.
Example 5
The quantitative detection system for NQO1 enzyme activity comprises; the three-electrode system foam nickel modified electrode is a working electrode, a platinum wire is a counter electrode, ag/AgCl is a reference electrode, and a signal molecule is isoluminol, NQO1 and a substrate tanshinone IIA.
Example 6
The drug screening system includes: the three-electrode system foam nickel modified electrode is a working electrode, a platinum wire is a counter electrode, ag/AgCl is a reference electrode, and the signal molecules are isoluminol, NQO1 and NQO1 which are related to the drugs to be screened.
Example 7
The quantitative detection system for NQO1 enzyme activity comprises; the three-electrode system gold electrode is a working electrode, the platinum wire is a counter electrode, the Ag/AgCl is a reference electrode, the signal molecule is luminol, NQO1 and a substrate (+ -) -Dunnione.
Example 8
The quantitative detection system for NQO1 enzyme activity comprises; the three-electrode system glassy carbon electrode is a working electrode, a platinum wire is a counter electrode, ag/AgCl is a reference electrode, luminol of a signal molecule, NQO1 and a substrate menozone.

Claims (5)

1. The quantitative detection system for the activity of the NQO1 enzyme is characterized by comprising a three-electrode system, a signal molecule and a reaction liquid, wherein the reaction liquid is generated by reacting NQO1 enzyme, NADPH and a substrate capable of producing hydrogen peroxide with the NQO1 enzyme; the signal molecule is luminol; the substrate is beta lapachone;
the three-electrode system foam nickel electrode is a working electrode, a platinum wire is a counter electrode, and Ag/AgCl is a reference electrode; the NADPH concentration is 100 mu M, the beta lapachone concentration is 20 mu M, and the luminol concentration is 0.1M;
the detection method of the system comprises the following steps: the NQO1 enzyme, NADPH and a substrate capable of producing hydrogen peroxide with the NQO1 enzyme are reacted to generate a reaction solution containing hydrogen peroxide, and the luminescence intensity of the reaction solution is tested by utilizing three electrodes and signal molecules, so that the quantitative detection of the NQO1 enzyme is realized.
2. A quantitative detection method of NQO1 enzyme activity using the quantitative detection system of NQO1 enzyme activity according to claim 1, characterized by comprising the steps of:
the NQO1 enzyme, NADPH and a substrate capable of producing hydrogen peroxide with the NQO1 enzyme are reacted to generate a reaction solution containing hydrogen peroxide, and the luminescence intensity of the reaction solution is tested by utilizing three electrodes and signal molecules, so that the quantitative detection of the NQO1 enzyme is realized.
3. Use of the quantitative detection system for NQO1 enzyme activity of claim 1 for quantitative rapid detection of NQO 1.
4. A system for drug screening based on the quantitative detection system for NQO1 enzyme activity according to claim 1, characterized by comprising a three-electrode system, a signal molecule and a reaction solution, wherein the reaction solution is generated by reacting NQO1 enzyme, NADPH and a target drug to be screened.
5. A method of drug screening using the system of drug screening of claim 4, comprising the steps of:
and reacting NQO1, NADPH and a target drug to be screened to generate a reaction solution containing hydrogen peroxide, and testing the luminous intensity of the reaction solution by utilizing three electrodes and signal molecules, thereby realizing analysis and evaluation of the antitumor activity of the drug.
CN202010338008.4A 2020-04-26 2020-04-26 System for quantitative detection of NQO1 enzyme activity and drug screening, and electroluminescence sensing method and application thereof Active CN111323464B (en)

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