Mass spectrum-based functional distinguishing method for various active oxygen species

Abstract

The invention belongs to the field of mass spectrometry analysis method research, and particularly relates to a mass spectrometry-based multi-active oxygen species functional distinguishing method, which comprises the following steps of: adding active oxygen species or an active oxygen species quencher into a reaction system at any time in an electrochemical luminescence reaction process; based on changes of electrochemical signals, electrochemical luminescence signals and mass spectrum signals before and after addition, functions of different active oxygen species in electrochemical luminescence reaction are distinguished; the invention also constructs a multi-active oxygen species functional distinguishing platform based on mass spectrum and a reaction decoupling monitoring device. According to the invention, various short-life reaction intermediates, reaction products and reaction byproducts generated after introduction of different ROS and ROS quenchers can be captured, and different roles played by various ROS in ECL reaction are disclosed from the molecular level; the method has remarkable advantages in the aspect of research on a complex chemical reaction system in which ROS participates, and shows a wide application prospect in the research on an ECL reaction mechanism.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry analysis methods, and specifically relates to a method for distinguishing the functions of multiple reactive oxygen species based on mass spectrometry. Background Technology

[0002] Reactive oxygen species (ROS) are a class of oxygen-containing substances activated by oxygen (O2) and possessing high chemical reactivity. Their unstable nature allows ROS to rapidly react with other molecules. In recent years, ROS-triggered electrochemiluminescence (ECL) reactions have gained widespread application in biosensing, cell imaging, and disease diagnosis and treatment due to their advantages of high sensitivity, low background interference, and electrochemical controllability. In ECL reactions using ROS as a co-reactant, ROS can be generated through the decomposition of hydrogen peroxide (H2O2), the oxygen reduction reaction (ORR), or the water oxidation reaction (WOR), ultimately reacting with a luminescent agent to produce ECL emission. Therefore, elucidating the specific role of ROS in the ECL reaction process is of great significance for studying the ECL reaction mechanism, improving ECL luminescence efficiency, and constructing ECL reaction devices.

[0003] In traditional ECL mechanism studies based on electrochemical and spectroscopic analysis methods, researchers indirectly explore the specific roles of each ROS component by introducing specific ROS scavengers into the ECL system to quench ROS and detecting the final ECL signal. Further spectroscopic analysis methods, such as electron spin resonance (EPR) and in-situ Raman spectroscopy, are used to characterize the ROS in the reaction system. Recently, Zhu Chengzhou's research group, by precisely controlling the oxygen reduction reaction pathway of MNC single-atom catalysts (SACs) and using Cu-NC materials with moderate oxygen affinity as the core reaction accelerator, achieved effective control of reactive oxygen species generation in a single luminol-dissolved oxygen system. Through in-situ characterization techniques (including electron paramagnetic resonance spectroscopy, Fourier transform infrared spectroscopy, and scanning electrochemical microscopy) combined with theoretical calculations, they revealed that •OH dominates cathodic electrochemiluminescence, and O2… •− The intrinsic mechanism by which synergistic electro-oxidation processes jointly trigger anodic luminescence (J. Am. Chem. Soc. 2024, 146, 12197-12205). However, the above-mentioned electrochemical and spectroscopic analysis methods still have the following problems: First, unlike ECL reaction systems that use organic molecules as co-reactants, ECL reactions using ROS as co-reactants often involve multiple ROS, including superoxide anion (O2). •− Hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen ( 1The presence of substances such as O2 makes it difficult to accurately elucidate the interaction mechanisms between each component and the luminescent material or intermediates; furthermore, electrochemical and spectroscopic analysis methods cannot directly reveal the role of ROS in the ECL reaction process at the molecular level, thus limiting a deeper understanding of its reaction mechanism.

[0004] Mass spectrometry (MS) enables simultaneous detection and structural characterization of multiple components in complex systems, offering advantages such as high sensitivity, high specificity, and multi-component detection. Electrochemical mass spectrometry (EC-MS) coupled with other techniques allows for in-situ capture of intermediates generated in electrochemical reactions, making it a powerful tool for studying electrochemical reaction mechanisms. Hu et al. constructed a non-contact EC-MS device based on a bipolar electrode (BUME) to capture the cationic radical of the amine co-reactant tripropylamine (TPrA) in an ECL reaction system (Angew. Chem. Int. Ed., 2021, 60, 18494). While devices integrating electrochemical reactions into the electrospray process can achieve rapid capture of transient electrochemical intermediates, the use of several kilovolts in the electrospray process cannot replicate the actual ECL reaction conditions. To this end, Shao Yuanhua's research group proposed an ultra-micro hybrid electrode EC-MS device using a quartz dual-channel θ capillary as both a micro-electrolysis cell and a spray emitter. The carbon layer deposited in one channel of the θ capillary serves as the working electrode, while the other channel is inserted with an Ag / AgCl electrode and injected with a reaction solution. When a voltage is applied, an electrochemical reaction occurs in the thin liquid layer at the capillary tip. Electrospraying is triggered by a piezoelectric gun, allowing newly formed species on the electrode surface to rapidly enter the mass spectrometer. This device was used to study Ru(bpy)3. 2+ The / TPrA system successfully captured [Pr2N=CHCH2CH3]. + [NHPr2] + and Ru(bpy)3 + Key intermediates (Chemical Science, 2016, 7, 6684-6688). Min et al. recently developed an electrochemical real-time mass spectrometry monitoring platform that can simultaneously acquire electrical, optical, and mass spectrometric signals during the ECL reaction. Using this platform, they successfully detected multiple reaction intermediates in the luminol ECL reaction and validated two luminol ECL reaction pathways at the molecular level (Chemical Science, 2022, 13, 6244-6253).

[0005] Existing analytical methods using mass spectrometry to explore the ECL reaction mechanism mostly focus on capturing reaction intermediates generated at the electrode interface by luminescent agents or amine co-reactants. They fail to acquire the electrochemical and optical signals during the ECL reaction, making it challenging to confirm how these intermediates participate in the reaction and generate the ECL signal. Although existing methods can achieve simultaneous acquisition of optical, electrochemical, and mass spectrometric signals in the ECL reaction, current mass spectrometry-based analytical techniques still have the following drawbacks when dealing with ROS-involved ECL reaction systems:

[0006] 1. The ECL reaction system using ROS as a co-reactant contains superoxide anion (O2). •− Hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen ( 1 Multiple ROS such as O2 are detected, but existing mass spectrometry methods are difficult to flexibly introduce a single type of ROS or ROS-selective quenchers, thus making it impossible to explore the role of each ROS in the reaction.

[0007] 2. Existing mass spectrometry methods are difficult to separate and decouple the electrochemical reaction of the luminescent material in the ECL reaction from the homogeneous chemical reaction of the subsequent reaction intermediates and various ROS, thus making it impossible to investigate the interaction between ROS and reaction intermediates in detail.

[0008] 3. In order to achieve rapid capture of reaction intermediates, existing mass spectrometry analysis platforms highly integrate electrochemical reaction cells into the capillary tip. However, this highly integrated design makes it impossible to modulate multiple parameters (such as solution pH, reaction atmosphere, and reaction solvent) during the reaction process. Summary of the Invention

[0009] This invention addresses the aforementioned technical problems in existing technologies by providing a mass spectrometry method that can reveal the roles of multiple ROS in the ECL reaction system at the molecular level. This method constructs a real-time ECL monitoring mass spectrometry platform by coupling a capillary electrochemical microreactor, a photomultiplier tube (PMT), and a Venturi acoustic spray mass spectrometer (V-EASI-MS). It allows for the flexible introduction of different ROS and ROS quenchers into the ECL reaction system, and simultaneous, real-time monitoring of electrochemical signals, ECL signals, and mass spectrometry signals before and after reagent introduction. Furthermore, it can capture various short-lived reaction intermediates, reaction products, and reaction byproducts generated after the introduction of different ROS and ROS quenchers, thereby elucidating the key roles of each ROS component in the ECL reaction at the molecular level. In addition, this invention can also modulate various conditional parameters during the ECL process. Therefore, this invention is of great significance for the study of ECL reaction mechanisms, the improvement of ECL luminescence efficiency, and the construction of ECL reaction devices.

[0010] The technical solution of the present invention is as follows:

[0011] A method for functional differentiation of multiple reactive oxygen species based on mass spectrometry includes the following steps:

[0012] At any point during the electrochemiluminescence reaction, reactive oxygen species or reactive oxygen species quenchers are added to the reaction system.

[0013] Real-time acquisition of electrochemical signals, electrochemiluminescence signals, and mass spectrometry signals of the reaction system before and after the addition of reactive oxygen species or reactive oxygen species quenchers;

[0014] Based on the changes in the electrochemical signal, electrochemiluminescence signal, and mass spectrometry signal, the functions of different reactive oxygen species in the electrochemiluminescence reaction can be distinguished.

[0015] Using the ROS-involved luciferin (Luc) ECL reaction as a model reaction, multiple short-lived intermediates, such as Luc... •+ (m / z 386.1770), protonated Luc(O) (m / z 403.1797), protonated Luc(OO) (m / z 419.1743), etc.

[0016] Preferably, during the test, mass spectrometry data acquisition is started first, followed by electrochemical methods such as CA and CV. The time point at which the electrochemical method is started is recorded. In subsequent data processing, the recorded time point is used to synchronize the optical, mass spectrometry, and electrical signals.

[0017] A mass spectrometry-based platform for distinguishing the functions of multiple reactive oxygen species includes a capillary electrochemical flow microreactor, an optical detection unit, and a mass spectrometer;

[0018] The capillary electrochemical flow microreactor is used to perform electrochemiluminescence reactions;

[0019] The optical detection unit is located below the capillary electrochemical flow microreactor and is used to detect the electrochemiluminescence signal in real time.

[0020] The mass spectrometer is connected to the capillary electrochemical flow microreactor via an interface for real-time detection of mass spectrometry signals of reaction intermediates and products.

[0021] Preferably, the capillary electrochemical flow microreactor includes a reservoir and has a built-in counter electrode, reference electrode, and working electrode.

[0022] The working electrode is an embedded working electrode, which is embedded in the molten capillary. The surface of the section of the molten capillary located in the liquid storage tank is treated as an optically transparent window so that the optical detection unit can directly detect the light emitted from the working electrode.

[0023] Preferably, the molten capillary is coaxially sleeved with a sheath gas capillary through a three-way connector. The sheath gas capillary is connected to the port of the three-way connector near the mass spectrometer. The end of the molten capillary is adjusted to extend 0.5-2 mm beyond the sheath gas capillary and to be 1-5 mm away from the mass spectrometer inlet to ensure stable spray formation and mass spectrometry signal acquisition.

[0024] Preferably, the remaining port of the three-way connector is used as an air inlet, allowing for free adjustment of the reaction atmosphere to detect the impact of different atmospheric conditions on the ECL reaction system.

[0025] More preferably, the reservoir is a self-made polydimethylsiloxane (PDMS) reservoir (600 μL), containing a platinum counter electrode (500 μm in diameter) and a silver reference electrode (500 μm in diameter); a fused capillary (200 μm inner diameter, 360 μm outer diameter, 10 cm length) with an embedded platinum working electrode (100 μm in diameter); the polyimide coating on the inner part of the fused capillary in the PDMS reservoir is removed so that the optical detection unit below can directly detect the ECL emission light from the Pt working electrode. The capillary inserted into the platinum working electrode is passed through a stainless steel tee connector and a sheath gas capillary (530 μm inner diameter, 700 μm outer diameter, 1 cm length). The end of the inner capillary is adjusted to extend 1 mm beyond the sheath gas capillary and to be 2 mm away from the mass spectrometer inlet to ensure stable spray formation and mass spectrometry signal acquisition.

[0026] Preferably, the optical detection unit is an MPI-E type electrochemiluminescence analyzer, and the operating voltage of the photomultiplier tube (PMT) is set to 600 V and 800 V respectively for ECL signal acquisition; the mass spectrometer is a Venturi atmospheric pressure acoustic spray mass spectrometer (V-EASI-MS). More preferably, the Venturi atmospheric pressure acoustic spray mass spectrometer uses nitrogen-driven V-EASI to minimize the influence of high voltage on the electrochemical reaction.

[0027] Preferably, the platform further includes a sample introduction module connected to the capillary electrochemical flow microreactor, used to introduce target reagents into the reaction system during the reaction process. The target reagents can be different types of ROS or ROS quencher solutions, which are introduced into the ECL reaction system as needed to study the interaction mechanisms between different ROS and luminescent materials.

[0028] The ECL reaction is initiated in the capillary electrochemical microreactor. During the reaction, specific reactive oxygen species or quencher solutions are injected into the reaction system through the injection module. The chemiluminescence and mass spectrometry signals of the system before and after the injection operation are collected synchronously and in real time using the optical detection unit and mass spectrometer. Based on the synergistic or antagonistic relationship between the changing trends of one or more specific ion chromatograms in the mass spectrometry signal and the changing trends of the electrochemiluminescence signal, the functions of different reactive oxygen species in the ECL reaction are distinguished.

[0029] Preferably, the injection module is a capillary tube (inner diameter 200 μm, outer diameter 360 μm, length 6 cm) connected to the syringe. The capillary tube is inserted into the reservoir. This design allows ROS or ROS quencher to be precisely and flexibly injected into the ECL reaction system at any time during the ECL reaction process via the capillary tube.

[0030] A reaction decoupling monitoring device integrates a three-way hybrid connector into the mass spectrometry-based multi-reactive oxygen species functional differentiation platform, the three-way hybrid connector including channel 1, channel 2 and channel 3;

[0031] The channel 1 is connected to the outlet of the capillary electrochemical flow microreactor and is used to transport the active intermediates generated by the electrochemical reaction.

[0032] Channel 2 is connected to the sample injection module;

[0033] Channel 3 is connected to the ion source of the mass spectrometer;

[0034] The active intermediates generated by the electrochemical reaction in the capillary electrochemical flow microreactor enter through channel 1, where they mix with reactive oxygen species entering from channel 2 at the three-way mixing connector and undergo a homogeneous chemical reaction. The reaction products are then transported to a mass spectrometer for detection via channel 3. The heterogeneous electrode reactions of the reactants and various ROS or ROS quenchers can be independently introduced using a decoupling device to achieve spatial separation.

[0035] Preferably, the tee-type hybrid connector is a PEEK tee-type connector.

[0036] The aforementioned device can also be used to screen reactive oxygen species quenchers or inhibitors.

[0037] Compared with the prior art, the advantages of the present invention are as follows:

[0038] (1) The method of the present invention can flexibly introduce a single type of ROS or a ROS selective quencher for various ECL reaction systems with ROS as a co-reactant, thereby exploring the role of each ROS in the reaction.

[0039] (2) The method of the present invention can separate and decouple the electrochemical reaction of the luminescent body in the ECL reaction from the homogeneous chemical reaction of the subsequent reaction intermediates and various ROS, thereby enabling a detailed investigation of the interaction between ROS and reaction intermediates.

[0040] (3) The method of the present invention separates the electrochemical reaction cell from the electrospray capillary, which can achieve rapid capture of reaction intermediates and modulate multiple parameters (such as solution pH, reaction atmosphere and reaction solvent) during the reaction process. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the Luc cathode ECL reaction path proposed in this invention.

[0042] Figure 2 A schematic diagram of the ECL real-time monitoring mass spectrometry platform constructed in Example 1.

[0043] Figure 3 The mass spectra of nitrogen saturation and oxygen saturation under the operation of Example 2 are obtained at potentials of 0 V and −2 V, respectively.

[0044] Figure 4 The graphs show the changes in step potential, ECL signal, and Luc under nitrogen-saturated and oxygen-saturated conditions applied to the reaction system in Example 2. •+ Selected ion chromatograms of protonated Luc(O) (m / z 386.1770), protonated Luc(OO) (m / z 403.1797), protonated Luc(OO) (m / z 419.1743), and protonated NMA (m / z 210.0909).

[0045] Figure 5 The following are the potential scans, corresponding current and ECL signal variation curves over time under nitrogen saturation and oxygen saturation conditions in Example 3, and the corresponding Luc values ​​under the same conditions. •+ Selected ion chromatograms of protonated Luc(O) (m / z 386.1770), protonated Luc(OO) (m / z 403.1797), protonated Luc(OO) (m / z 419.1743), and protonated NMA (m / z 210.0909) are shown. The right-hand figure is a magnified view of the selected ion chromatograms in the time range of 5-15 s.

[0046] Figure 6 This is a schematic diagram of the improved device for the ECL real-time monitoring mass spectrometry platform constructed in Example 4.

[0047] Figure 7 This is a positive ion mode mass spectrum recorded at a potential of -2 V after injecting a mixed solution of different free radical scavengers and Luc in Example 5.

[0048] Figure 8 The curves showing the changes in applied potential and ECL signal over time under the corresponding conditions in Example 6, and the curve Luc under the corresponding conditions. •+ Selected ion chromatograms of protonated Luc(OO) (m / z 386.1770), protonated NMA (m / z 419.1743), and protonated NMA (m / z 210.0909).

[0049] Figure 9 A schematic diagram of the reaction decoupling monitoring device constructed in Example 7.

[0050] Figure 10 This is a positive ion mode mass spectrum recorded at a potential of -2 V after different ROS solutions were introduced into the Luc system in Example 8.

[0051] Figure 11 Example 9: After introducing different ROS solutions into the Luc system, the potential-time curves and Luc were obtained under conditions of 0 V and -2 V step potentials. •+ Selected ion chromatograms of protonated Luc(OO) (m / z 386.1770), protonated NMA (m / z 419.1743), and protonated NMA (m / z 210.0909).

[0052] Figure 12 Example 10: O2 under conditions where no potential was applied •- Real-time changes in optical and mass spectrometry signals before and after injection. Detailed Implementation

[0053] In the following specific embodiments, the luciferin (Luc), potassium superoxide (KO2), 1,4-benzoquinone (BQ), isopropanol (IPA), DL-tryptophan (Trp), and lithium trifluoromethanesulfonate (LiOTf) involved were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Acetic acid and ferrous ammonium sulfate hexahydrate ((NH4)2Fe(SO4) 2• 6H2O), ammonium acetate (NH4AC), sodium hypochlorite (NaClO), and hydrogen peroxide (H2O2) were purchased from Shanghai Reagent Co., Ltd. (Shanghai, China). Polydimethylsiloxane (PDMS) was purchased from Dow Corning, Inc., Midland, Michigan, USA. Acetonitrile (ACN), methanol (MeOH), and ethanol (EtOH) were chromatographically pure reagents from Merck (Darmstadt, Germany). All laboratory water was prepared using the MilliQ ultrapure water system manufactured by Millipore (Massachusetts, USA). All other reagents were of analytical grade and were not subjected to additional purification steps before use.

[0054] Example 1:

[0055] The construction steps of the ECL real-time monitoring mass spectrometry platform developed in this invention are as follows:

[0056] The platform consists of a capillary electrochemical flow microreactor, a Venturi atmospheric pressure acoustic spray mass spectrometer (V-EASI-MS), and a photomultiplier tube (PMT). The capillary electrochemical microreactor comprises: a self-made PDMS reservoir (600 μL) housing a platinum counter electrode (500 μm diameter) and a silver reference electrode (500 μm diameter); a fused capillary (200 μm inner diameter, 360 μm outer diameter, 10 cm length) housing a platinum working electrode (100 μm diameter). Furthermore, the polyimide coating on the capillary section within the PDMS reservoir was removed to allow the PMT below to directly detect the ECL emission from the Pt working electrode. The device employs nitrogen-driven V-EASI to minimize the impact of high voltage on the electrochemical reaction. During assembly, the capillary inserted into the platinum working electrode is passed through a stainless steel tee connector and a sheath gas capillary (530 μm inner diameter, 700 μm outer diameter, 1 cm length). Adjust the inner capillary end to extend 1 mm beyond the sheath gas capillary and position it 2 mm from the mass spectrometer inlet to ensure stable spray formation and mass spectrometry signal acquisition. Based on the Venturi effect, newly generated active intermediates on the electrode surface can be rapidly atomized and transported to mass spectrometry detection.

[0057] Figure 2 This is a schematic diagram of the ECL real-time monitoring mass spectrometry platform constructed in Example 1. It can realize the simultaneous acquisition of optical, mass, and electrical signals.

[0058] Example 2:

[0059] The experimental steps for real-time monitoring of mass spectrometry signals under nitrogen and oxygen saturation environments are as follows:

[0060] Using the apparatus of Example 1, the mass spectrometry signals were observed by applying potentials of 0 V and -2 V to the reaction system under nitrogen-saturated and oxygen-saturated atmospheres, respectively. Similarly, the ECL signal and the selected ion chromatography signal of each intermediate were observed by applying step potentials from 0 V to -2 V to the reaction system under nitrogen-saturated and oxygen-saturated atmospheres, respectively. The reaction system was ACN / H₂O (volume ratio 1:1), with 0.01 mM Luc and 10 mM NH₄AC as supporting electrolytes.

[0061] Figure 3 The images show the mass spectra of nitrogen saturation and oxygen saturation under the conditions of 0 V and -2 V, respectively, as described in Example 2. From the images, it can be concluded that Luc(m / z) 193.0887 can be detected under both nitrogen and oxygen saturation conditions without an applied reduction potential. 2+Signal. When the potential is switched to -2 V, nitrogen gas shows a Luc signal at m / z 386.1776. •+ The mass spectrometry peaks were observed. Under oxygen saturation conditions, the key intermediate Luc(OO) (m / z 419.1755) and the product NMA (m / z 210.0915) were detected, and a new ion signal (Luc(O)) was detected at m / z 403.1755.

[0062] Figure 4 The graphs show the changes in step potential, ECL signal, and Luc under nitrogen-saturated and oxygen-saturated conditions applied to the reaction system in Example 2. •+ Selected ion current chromatograms of protonated Luc(O) (m / z 386.1770), protonated Luc(OO) (m / z 403.1797), protonated Luc(OO) (m / z 419.1743), and protonated NMA (m / z 210.0909) were obtained. The graphs show that when the potential is switched to -2 V, there is no obvious ECL signal under nitrogen saturation, but Luc(OO) appears at m / z 386.1776. •+ The mass spectrometry peaks were observed. Under oxygen-saturated conditions, not only were the key intermediate Luc(OO) (m / z 419.1755) and the product NMA (m / z 210.0915) reported in the literature detected, but a strong ECL signal was also observed. In addition, besides the above reaction intermediates, a new ion signal (Luc(O)) was also detected at m / z 403.1755 under oxygen-saturated conditions. The signal intensity of this new ion was significantly correlated with the applied potential and ECL emission under oxygen-saturated conditions. These experimental phenomena confirm that dissolved oxygen is a key co-reactant in the Luc cathode ECL reaction.

[0063] Example 3:

[0064] Under nitrogen-saturated and oxygen-saturated conditions, the reaction was performed from 0 V to -2 V at a scan rate of 100 mV / s. During this process, the applied potential, current response, ECL-time curves, and selected ion chromatograms of each intermediate under the corresponding conditions were recorded. The reaction system was ACN / H2O (volume ratio 1:1), with 0.01 mM Luc and 10 mM NH4AC as supporting electrolytes.

[0065] Figure 5 The following are the potential scans, corresponding current and ECL signal variation curves over time under nitrogen saturation and oxygen saturation conditions in Example 3, and the corresponding Luc values ​​under the same conditions. •+Selected ion current (CFC) chromatograms of protonated Luc(O) (m / z 386.1770), protonated Luc(OO) (m / z 403.1797), protonated Luc(OO) (m / z 419.1743), and protonated NMA (m / z 210.0909) are shown. The right-hand side is a magnified view of the CFC chromatogram within the time range of 5-15 s. From the figure, it can be concluded that under nitrogen saturation, the Luc molecule undergoes only a single-electron transfer reduction process. When the potential drops to -0.8 V, a Luc reduction peak appears in the CV curve. Simultaneously, the selected ion current chromatogram of Luc... •+ The signal at m / z 386.1776 changed with potential. Under oxygen saturation conditions, ECL emission occurred at -0.97 V, accompanied by significant enhancements in the Luc(OO) (m / z 419.1755) and NMA (m / z 210.0915) mass spectrometry signals. Furthermore, the Luc(O) ion signal at m / z 403.1755 began to rise at -0.85 V, earlier than the ECL signal, suggesting that Luc(O) may be the Luc ion responsible for the ECL reaction. •+ Byproducts generated from the reaction with ROS.

[0066] Example 4:

[0067] The construction steps of the improved ECL real-time monitoring mass spectrometry platform device are as follows:

[0068] To further investigate the interaction mechanisms between different ROS and luminescent materials, a sample introduction module was integrated into the original device. This module can deliver reactive oxygen species (ROS) scavengers or ROS solutions into the ECL reaction system as needed. Based on the existing device, a capillary tube (200 μm inner diameter, 360 μm outer diameter, and 6 cm length) connected to a syringe was inserted into the PDMS reservoir, with its end close to the inlet of the capillary electrochemical microreactor. This improved design allows for precise injection of the target solution into the reaction system via the capillary tube at any point during the ECL reaction process.

[0069] Figure 6 This is a schematic diagram of the improved ECL real-time monitoring mass spectrometry platform constructed in Example 4. This improved design allows for precise injection of the target solution into the reaction system via a capillary tube at any point during the ECL reaction process.

[0070] Example 5:

[0071] The experimental steps for ROS removal during the Luc cathode ECL process based on the improved device are as follows:

[0072] 50 μL of solution containing 0.1 mM BQ (O2) was injected into 500 μL of 0.01 mM Luc solution. •-Scavenger), 0.1 mM IPA (•OH scavenger), 0.1 mM TRP ( 1 After mixing the O2 scavenger with 0.01 mM Luc, a positive ion mode mass spectrum was recorded at a potential of -2 V.

[0073] Figure 7 This is a positive ion mode mass spectrum recorded at -2 V after injecting a mixed solution of different free radical scavengers and Luc in Example 5. From the figure, it can be concluded that the introduction of IPA (•OH scavenger) and Trp (… 1 After O2 scavenging, the mass spectrometry signal of the system showed no significant change, while BQ (O2 scavenger) showed no significant change. •- The addition of a scavenger significantly reduced the mass spectrometry signals of reaction intermediates Luc(OO) (m / z 419.1755) and NMA (m / z 210.0915).

[0074] Example 6:

[0075] Furthermore, an alternating step potential of 0 V and -2 V was used to trigger the ECL reaction of Luc in the capillary electrochemical microreactor, while an improved ECL real-time monitoring mass spectrometry platform system was used to simultaneously acquire current, mass spectrometry, and ECL signals. After two cycles, a mixed solution (50 μL) containing 0.01 mM Luc and 0.1 mM scavenger was rapidly injected into the PDMS reservoir using a syringe to achieve the removal of specific ROS.

[0076] Figure 8 The curves showing the changes in applied potential and ECL signal over time under the corresponding conditions in Example 6, and the curve Luc under the corresponding conditions. •+ Selected ion current chromatograms of protonated Luc(OO) (m / z 386.1770), protonated NMA (m / z 419.1743), and protonated NMA (m / z 210.0909) were obtained. The figures show that the ECL and EIC signals of the system did not change significantly after the introduction of IPA and Trp, while the addition of BQ significantly quenched the ECL signal, proving that the O2 generated by ORR... •- It is the key ROS that triggers the Luc cathode ECL.

[0077] Example 7:

[0078] To elucidate the reaction pathways between the active intermediates generated during the electrochemical process and various ROS, a reaction decoupling monitoring device was constructed. Specifically, a PEEK T-connector was used to interconnect a capillary electrochemical microreactor (200 μm inner diameter, 360 μm outer diameter, 7 cm length), a capillary (200 μm inner diameter, 360 μm outer diameter, 8 cm length), and a V-EASI ion source. In this device, the active intermediates generated by the electrochemical reaction are transported to the PEEK T-connector and undergo homogeneous chemical reactions with various ROS. Through a spatial separation strategy, the heterogeneous electrode reaction and the homogeneous chemical reaction are decoupled.

[0079] Figure 9 This is a schematic diagram of the reaction decoupling monitoring device constructed in Example 7. This design achieves decoupling between heterogeneous electrode reactions and homogeneous chemical reactions through a spatial separation strategy.

[0080] Example 8:

[0081] The experimental steps for decoupling the electroreduction reaction and homogeneous chemical reaction in the Luc cathode ECL process are as follows:

[0082] First, different ROS were prepared using classical chemical methods: (1) O2 •- :2 mM potassium superoxide (KO2) was dissolved in ethanol to prepare it; (2)•OH: prepared by Fenton reaction, 1 mM H2O2 and 1 mM ferrous ammonium sulfate were added to an acetonitrile / water solution containing 10 mM NH4AC (1:1, v / v), and reacted for 30 min for later use; (3) 1 O2: Generated in an acetonitrile / water solution (1:1, v / v) containing 1 mM H2O2, 1 mM NaClO, and 10 mM NH4AC. After preparation, 50 μL of O2 was injected into 500 μL of a 0.01 mM Luc solution. •- Solution 1 O2 solution, •OH solution and H2O2 solution, positive ion mode mass spectra were recorded at a potential of -2 V.

[0083] Figure 10 This is a positive ion mode mass spectrum recorded at a potential of -2 V after introducing different ROS solutions into the Luc system in Example 8. From the figure, it can be concluded that, consistent with the results of the ROS quenching experiment, when O2 is introduced... •- Subsequently, Luc(OO) (m / z 419.1742) and NMA (m / z 210.0909) ion signals were detected. In contrast, upon introduction of •OH or 1At O2, almost no mass spectrometric signals of Luc(OO) and NMA were detected. Furthermore, the introduction of low concentrations of H2O2 (1 mM) also failed to induce the generation of Luc(OO) and NMA signals.

[0084] Example 9:

[0085] In the decoupling experiment, in order to separate the Luc radical cation (Luc... •+ An acetonitrile / water solution (1:1, v / v) containing 0.01 mM Luc and 10 mM NH4AC was injected into channel 1 (under nitrogen saturation), and a switching potential from 0 V to -2 V was applied to the platinum working electrode of channel 1. After two potential switching cycles, a specific ROS was introduced into channel 2, causing it to react with Luc in the mixing capillary. •+ The reaction proceeds. The reaction intermediates and products are then immediately transferred to the mass spectrometer inlet for detection.

[0086] Figure 11 Example 9: After introducing different ROS solutions into the Luc system, the potential-time curves and Luc were obtained under conditions of 0 V and -2 V step potentials. •+ Selected ion current chromatograms of protonated Luc(OO) (m / z 386.1770), protonated NMA (m / z 419.1743), and protonated NMA (m / z 210.0909). Notably, the figure shows that Luc(OO) without an applied reduction potential... •+ With O2 •- Luc can still be detected after the mixed capillary convergence. •+ The experimental results show that Luc(OO) and NMA signals are used to determine the Luc(OO) signal. •+ It may be generated through pathways other than electrochemical reduction. Because channel 1 contains Luc... •+ There is still unreacted Luc residue, suggesting that Luc may have directly reacted with O2. •- The reaction produces Luc •+ .

[0087] Example 10:

[0088] To verify that Luc may be directly related to O2 •- The reaction produces Luc •+ Based on this assumption, we used the improved apparatus of Example 4 equipped with a sample injection module to inject 50 μL of O2 into the Luc system without applying an external potential. •- The solutions were analyzed, and their positive ion mode mass spectra before and after injection were measured, along with the corresponding potential-time curves, ECL-time curves, and Lucene-time curves. •+Selected ion chromatograms of protonated Luc(OO) (m / z 386.1770), protonated NMA (m / z 419.1743), and protonated NMA (m / z 210.0909).

[0089] Figure 12 Example 10: O2 under conditions where no potential was applied •- Real-time changes in optical and mass spectrometry signals before and after injection. From the graph, it can be concluded that: with O2... •- The injection resulted in significant ECL emission, while Luc... •+ The ion signals of Luc(OO) and NMA showed a significant increase, a phenomenon that confirms the interaction between Luc and O2. •- The direct homogeneous reaction contributes to the cathode ECL process.

[0090] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or alternatives made on the basis of the above shall all fall within the scope of protection of the present invention.

Claims

1. A method for functional differentiation of multiple reactive oxygen species based on mass spectrometry, characterized in that, Includes the following steps: Reactive oxygen species or reactive oxygen species quenchers are added to the reaction system at any time during the electrochemiluminescence reaction. Real-time acquisition of electrochemical signals, electrochemiluminescence signals, and mass spectrometry signals of the reaction system before and after the addition of reactive oxygen species or reactive oxygen species quenchers; Based on the changes in the electrochemical signal, electrochemiluminescence signal, and mass spectrometry signal, the functions of different reactive oxygen species in the electrochemiluminescence reaction can be distinguished.

2. A mass spectrometry-based platform for differentiating the functions of multiple reactive oxygen species, characterized in that, Includes capillary electrochemical flow microreactor, optical detection unit, and mass spectrometer; The capillary electrochemical flow microreactor is used to perform electrochemiluminescence reactions; The optical detection unit is located below the capillary electrochemical flow microreactor and is used to detect the electrochemiluminescence signal in real time. The mass spectrometer is connected to the capillary electrochemical flow microreactor via an interface for real-time detection of mass spectrometry signals of reaction intermediates and products.

3. The platform as described in claim 2, characterized in that, The capillary electrochemical flow microreactor includes a reservoir and has a built-in counter electrode, reference electrode, and working electrode. The working electrode is an embedded working electrode, which is embedded in the molten capillary. The surface of the section of the molten capillary located in the liquid storage tank is treated as an optically transparent window so that the optical detection unit can directly detect the light emitted from the working electrode.

4. The platform as described in claim 3, characterized in that, The molten capillary is passed through a tee connector and coaxially fitted with a sheath gas capillary. The sheath gas capillary is connected to the port of the tee connector near the mass spectrometer. The end of the molten capillary is adjusted to extend 0.5-2 mm beyond the sheath gas capillary and to be 1-5 mm away from the mass spectrometer inlet.

5. The platform as described in claim 2, characterized in that, The optical detection unit is an MPI-E type electrochemiluminescence analyzer; the mass spectrometer is a Venturi atmospheric pressure acoustic spray mass spectrometer.

6. The platform as described in claim 2, characterized in that, The platform also includes a sample introduction module, which is connected to the capillary electrochemical flow microreactor.

7. The platform as described in claim 6, characterized in that, The injection module is a capillary tube connected to the syringe, and the capillary tube is inserted into the liquid storage tank.

8. A reaction decoupling monitoring device, characterized in that, Integrate a tee hybrid connector into the platform as described in claim 6, the tee hybrid connector comprising channel 1, channel 2 and channel 3; The channel 1 is connected to the outlet of the capillary electrochemical flow microreactor and is used to transport the active intermediates generated by the electrochemical reaction. Channel 2 is connected to the sample injection module; Channel 3 is connected to the ion source of the mass spectrometer; The active intermediate generated by the electrochemical reaction in the capillary electrochemical flow microreactor enters through channel 1, mixes with the active oxygen species entering from channel 2 at the three-way mixing joint, and undergoes a homogeneous chemical reaction. The reaction product is then transported to the mass spectrometer for detection via channel 3.

9. The reaction decoupling monitoring device as described in claim 8, characterized in that, The tee-type hybrid connector is a PEEK tee-type connector.

10. The use of the device according to any one of claims 8-9 in screening reactive oxygen species quenchers or inhibitors.

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