A method for electrochemically determining hydroxyl radicals and dmsos in real time

Abstract

The present application belongs to the field of electrochemical analysis, and provides a method for electrochemical real-time determination of hydroxyl radicals (·OH) and DMSO. The method is characterized in that the detection of the two target substances is realized by the oxidation of the intermediate product methane sulfinic acid produced by the reaction of ·OH and DMSO on the electrode surface and the generation of an electrochemical signal. The scheme is summarized as follows: DMSO is used as an electrochemical probe, and when the target substance (·OH) is added to the electrolyte, a voltammetry scan is immediately performed to record the signal, and the signal is positively correlated with the concentration of ·OH. Conversely, when a fixed concentration of ·OH exists in the electrolyte, after the target substance (DMSO) is added, the signal is recorded by voltammetry scan, and the determination of DMSO can also be realized. The present application adopts electrochemical method, and can realize in-situ and real-time determination of ·OH and DMSO without separation step; the working electrode adopts glassy carbon electrode, and does not need electrode modification process, and has good stability. The determination method is suitable for a wide pH range (2.2-6.5), and has the advantages of simple operation, high sensitivity and low cost.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis. Its feature is that it utilizes the intermediate product of the reaction between hydroxyl radicals and DMSO, methanesulfinic acid, to be oxidized on the electrode surface and generate an electrochemical signal to achieve the detection of two target substances. Background Technology

[0002] Dimethyl sulfoxide (DMSO), known as a universal solvent, is an organic solvent widely used in chemistry, pharmacology, and biology. In cell biology, DMSO is often used as a medium for cell cryopreservation because it can limit ice crystal damage and improve cell survival after thawing. After cell resuscitation, the DMSO concentration should be immediately reduced to below 0.1% to prevent its toxic side effects. Therefore, rapid and sensitive determination of DMSO content is of great importance in cell culture. DMSO is also a highly efficient hydroxyl radical scavenger. Hydroxyl radicals (·OH) are the most potent oxidizing oxygen species (ROS) and are found in human cells, plant cells, atmospheric particulate matter, cigarette smoke, and tobacco extracts. Excessive ·OH can cause cellular oxidative stress, leading to damage to DNA, amino acids, carbohydrates, and lipids. However, ·OH has an extremely short lifetime (approximately 10^6 days). -10 Making its quantitative detection a huge challenge.

[0003] Currently, a series of detection methods exist for the determination of ·OH. For example, electron spin resonance (ESR) is considered the most reliable technique for ·OH determination, and high-performance liquid chromatography (HPLC) is also commonly used. However, the expensive equipment, complex operation, and long processing time of these methods limit their application. Furthermore, these methods require the addition of spin trapping agents or other reagents to initiate a series of complex reactions, making in-situ, real-time determination of ·OH difficult. Electrochemical technology, with its fast response, ease of operation, and high sensitivity, has broad application prospects in the analytical field. To date, the development of electrochemical methods for ROS determination is relatively rare. One of the biggest challenges in electrochemical ROS determination is distinguishing ROS with similar properties based on changes in electrochemical potential, current, and impedance. Introducing specific recognition elements can improve the selectivity of electrochemical detection. For example, electrochemical sensors based on cerium oxide nanomaterials with enzyme-mimicking properties are used to determine ·OH, but these sensors cannot exclude interference from other ROS. Biorecognition elements (antibodies, enzymes, DNA, or aptamers) are also used for electrochemical determination of ·OH. However, electrode fabrication requires a time-consuming modification process, and recognition elements are expensive and prone to deactivation during storage.

[0004] Therefore, there is an urgent need for a simple, selective, and highly sensitive electrochemical method to achieve rapid determination of ·OH. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a real-time, in-situ method for the determination of ·OH and DMSO, which combines the advantages of simple operation, high sensitivity, and good selectivity. The constructed electrochemical method eliminates the need for complex material preparation, electrode modification, and separation processes, enabling sensitive and rapid quantitative determination.

[0006] The proposed method can be summarized as follows: using DMSO as an electrochemical probe, voltammetry is used to scan and record the signal immediately after the target substance (·OH) is added to the electrolyte. This signal is related to the concentration of ·OH. Conversely, if a certain amount of ·OH is present in the electrolyte, the target substance (DMSO) can also be added, and the signal can be recorded by voltammetry to determine DMSO. The principle of both target substance determinations is the same: the electrochemical signal originates from the oxidation of methanesulfinic acid (MSIA) produced by the reaction of DMSO with ·OH on the electrode surface.

[0007] Therefore, the technical solution of the present invention is as follows:

[0008] The first aspect of this invention is to provide a method for real-time electrochemical determination of hydroxyl radicals and DMSO, comprising a method for determining hydroxyl radicals and a method for determining DMSO; wherein in both the method for determining hydroxyl radicals and the method for determining DMSO, the electrochemical signal originates from the oxidation of methanesulfinic acid produced by the reaction of DMSO with ·OH on the electrode surface; the method for determining hydroxyl radicals comprises the following steps:

[0009] Step S11: A certain amount of DMSO is dissolved in the electrolyte;

[0010] Step S12: Add the OH source substance to be tested;

[0011] Step S13: Perform electrochemical voltammetry scanning, record the signal, and calculate the ·OH concentration by substituting it into the standard curve;

[0012] The method for determining DMSO includes the following steps:

[0013] Step S21: A certain amount of ·OH source substance is dispersed in the electrolyte;

[0014] Step S22: Add the target substance to the DMSO to be tested;

[0015] Step S23: Perform an electrochemical voltammetry scan, record the signal, and substitute it into the standard curve to calculate the DMSO concentration.

[0016] Further, in step S13, the establishment of the standard curve includes using the square wave voltammetry intensity (0.8V) caused by different concentrations of ·OH, after subtracting the background signal, as the ordinate, and the ·OH concentration as the abscissa to establish the standard curve.

[0017] In one implementation, in step S13, the established standard curve is ΔI1 = 0.57 + 11.28c ·OH .

[0018] Further, in step S13, an electrochemical voltammetry scan is performed to record the signal intensity of the oxidation peak (0.8V); based on the measured signal intensity, the concentration of ·OH in the unknown sample is obtained according to the standard curve.

[0019] Further, in step S23, the establishment of the standard curve includes using the square wave voltammetry intensity (0.8V) caused by different concentrations of DMSO, after subtracting the background signal, as the ordinate, and the DMSO concentration as the abscissa to establish the standard curve.

[0020] In one implementation, in step S23, the established standard curve is ΔI2 = -0.02 + 5.06c DMSO .

[0021] Further, in step S23, an electrochemical voltammetry scan is performed, and the signal intensity of the oxidation peak (0.8V) of the square wave voltammetry is recorded. Based on the measured signal intensity, the concentration of DMSO is obtained according to the standard curve.

[0022] Furthermore, in step S11 or step S21, the electrolyte in the electrolyte solution is an electrolyte that is inert in the voltammetric test.

[0023] In some preferred embodiments, the electrolyte in the electrolyte solution is selected from one or a combination of KCl, Na2SO4, NaCl, and K2SO4.

[0024] Furthermore, in step S11 or step S21, the solvent in the electrolyte is an electrochemically inert solvent; either organic or inorganic solvents are acceptable.

[0025] Furthermore, in step S11 or step S21, the pH of the electrolyte solution is 2.2-6.5.

[0026] Furthermore, the concentration range of DMSO in step S11 is 0.0001-1 mol / L.

[0027] Further, in step S12 or step S21, the ·OH source includes Fenton system, Fenton-like system, enzyme catalysis system, nanomaterial catalysis system, atmospheric particulate matter, cigarette smoke or filter extract, and intracellular source.

[0028] Furthermore, in step S13 or step S23, the electrochemical voltammetry includes cyclic voltammetry, linear scan voltammetry, square wave voltammetry, and pulse voltammetry.

[0029] A second aspect of the present invention is to provide an electrochemical system for real-time determination of hydroxyl radicals and DMSO, using the electrochemical real-time determination method for hydroxyl radicals and DMSO described above; the electrochemical system includes a reference electrode, a counter electrode, and a working electrode.

[0030] Furthermore, the reference electrode is selected from saturated calomel electrode, silver / silver chloride electrode, and mercury oxide electrode;

[0031] Furthermore, the counter electrode is selected from platinum wire electrodes and graphite electrodes;

[0032] Furthermore, the working electrode is selected from glassy carbon electrode, gold electrode, carbon rod electrode, indium tin oxide electrode, and fluorine-doped SnO2 transparent conductive glass;

[0033] In a preferred embodiment, the reference electrode is a saturated calomel electrode;

[0034] In a preferred embodiment, the counter electrode is a platinum wire electrode;

[0035] In a preferred embodiment, the working electrode is a glassy carbon electrode.

[0036] Furthermore, the detection range for ·OH is 0.4–5120 μM.

[0037] Furthermore, the detection range of DMSO is 40-320 μM.

[0038] Furthermore, in step S13 or step S23, the electrochemical signal peak appears at 0.7-0.9 V; the electrochemical signal peak typically appears in the range of 0.7-0.9 V. It should be noted that the exact location of the electrochemical signal is affected by solution conditions.

[0039] In a preferred embodiment, in step S13 or step S23, the electrochemical signal peak appears at 0.8V.

[0040] Beneficial effects:

[0041] This invention utilizes the same mechanism, namely, the electrochemical oxidation of methanesulfinic acid (MSIA) produced by the reaction of DMSO with ·OH to generate a corresponding electrical signal, which indicates the concentration of ·OH or DMSO, thereby achieving the detection of two target analytes. Its features and advantages are described below:

[0042] (1) The present invention uses an electrochemical method, which does not require any separation steps and can realize in-situ, real-time determination of ·OH and DMSO.

[0043] (2) The working electrode used in this invention is a glassy carbon electrode, which does not require any time-consuming modification process and has extremely high stability.

[0044] (3) The present invention uses commercially available reagents as electrochemical probes, which avoids the cumbersome material preparation process and ensures good selectivity.

[0045] (4) The determination method proposed in this invention is applicable to a wide pH range (2.2-6.5).

[0046] (5) The determination method proposed in this invention has the advantages of simple operation and low cost.

[0047] (6) The electrochemical method proposed in this invention achieves sensitive detection of ·OH and DMSO, with detection ranges of 0.4-5120 μM and 40-320 μM, respectively. Attached Figure Description

[0048] Figure 1 The effect of different electrolyte compositions: (a) KCl, (b) Na2SO4, (c) NaCl, (d) K2SO4;

[0049] Figure 2 The effect of different solvent compositions: (A) 0.1 mol / L isopropanol aqueous solution, (B) 0.1 mol / L tert-butanol aqueous solution;

[0050] Figure 3 Cyclic voltammogram of DMSO versus ·OH (produced in Fenton);

[0051] Figure 4 Cyclic voltammogram of DMSO versus ·OH (produced by horseradish peroxidase);

[0052] Figure 5 Cyclic voltammogram of DMSO versus ·OH (produced in tobacco oil extract);

[0053] Figure 6 Effect of DMSO concentration, (a)-(g) 0, 0.0001, 0.001, 0.01, 0.04, 0.1, 1 mol / L;

[0054] Figure 7 Square wave voltammograms of DMSO on ·OH (produced in Fenton) at different pH values, (a)-(h): 2.2, 2.8, 3.1, 3.6, 4.2, 4.6, 5.5, 6.5;

[0055] Figure 8 Square wave voltammetry of OH (produced in Fenton) versus DMSO in cell cryopreservation solution;

[0056] Figure 9Square wave voltammetry and corresponding linear correction plots of DMSO against different concentrations of ·OH (produced in Fenton), (a)-(l): 0, 0.4, 0.8, 1.6, 16, 80, 160, 320, 640, 1280, 2560, 5120 μM;

[0057] Figure 10 Square wave voltammograms and corresponding linear correction plots of OH (produced in Fenton) against different DMSO concentrations, (a)-(h): 0, 20, 40, 80, 120, 160, 240, 320 μM. Detailed Implementation

[0058] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0059] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0060] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0061] Example 1: Determination of ·OH using DMSO as a probe in different electrolyte compositions

[0062] (1) Dissolve KCl, Na2SO4, NaCl, and K2SO4 in water to prepare a 0.1 mol / L electrolyte. Add DMSO (0.04 mol / L) to complete the detection system. The reference electrode, counter electrode, and working electrode in the electrochemical test are a saturated calomel electrode, a platinum wire electrode, and a glassy carbon electrode, respectively. Adding a substance that generates ·OH to the electrolyte and recording the electrochemical signal can achieve real-time, in-situ determination of ·OH. None of the above electrolytes affect the generation of characteristic signals; such as Figure 1 As shown.

[0063] (2) Na₂SO₄ was dissolved in water, water / isopropanol, and water / tert-butanol respectively to prepare a 0.1 mol / L electrolyte. DMSO (0.04 mol / L) was then added, completing the detection system. The reference electrode, counter electrode, and working electrode in the electrochemical test were a saturated calomel electrode, a platinum wire electrode, and a glassy carbon electrode, respectively. A substance that generates ·OH was added to the electrolyte, and the oxidation peak (0.8 V) signal intensity of the square wave voltammetry was recorded.

[0064] Based on the established standard curve (ΔI1=0.57+11.28c) ·OH This allows for real-time, in-situ determination of ·OH.

[0065] Even though isopropanol and tert-butanol are also scavengers of ·OH, they have only a weak effect on the determination of ·OH using DMSO as a probe; for example Figure 2 As shown in the figure. This also indicates that both electrochemically inert electrolytes and electrolyte solutions can be used to determine ·OH using this method.

[0066] Example 2: Determination of ·OH generated in different systems using DMSO as a probe

[0067] (1) Electrolyte: 0.1 mol / L Na2SO4 aqueous solution, DMSO concentration: 0.004 mol / L, H2O2 (1.6 mmol / L) added, followed by Fe... 2+ (0.32 mmol / L), the characteristic signal appearing at 0.8 V can be recorded to determine the ·OH generated in the Fenton system. Figure 3 ). Figure 3 The strong oxidation peak appearing at 0.8V is attributed to the reaction of DMSO with Fenton to produce MSIA, which is then oxidized on the surface of the working electrode.

[0068] (2) The electrolyte is a 0.1 mol / L Na2SO4 aqueous solution with a DMSO concentration of 0.04 mol / L. H2O2 (1.6 mmol / L) is added, followed by the addition of horseradish peroxidase (0.05 g / L). The characteristic signal appearing at 0.8 V is recorded, which allows for the determination of the ·OH produced by the horseradish peroxidase system. Figure 4 ). Figure 4 The weak oxidation peak that appears at 0.8V is attributed to the reaction of DMSO with horseradish peroxidase to produce MSIA, which is then oxidized on the working electrode surface.

[0069] (3) An electrolyte of 0.1 mol / L Na2SO4 aqueous solution and DMSO concentration of 0.04 mol / L were added to the cigarette filter extract. The characteristic signal appearing at 0.8 V was recorded, which can realize the determination of ·OH generated in the tobacco oil extract. Figure 5 ). Figure 5 The weak oxidation peak appearing at 0.8V is attributed to the reaction of DMSO with the ·OH produced by the e-liquid extract, which generates MSIA, and the MSIA is oxidized on the surface of the working electrode.

[0070] The determination of ·OH generated in different systems using DMSO as an electrochemical probe demonstrates the good reliability of this method.

[0071] Example 3: Determination of ·OH generated in Fenton system using DMSO of different concentrations as probes

[0072] (1) Electrolyte: 0.1 mol / L Na₂SO₄ aqueous solution; DMSO concentrations: 0, 0.0001, 0.001, 0.01, 0.04, 0.1, 1 mol / L; H₂O₂ (1.6 mmol / L); Fe₂O₃ added. 2+ (0.2 mmol / L) and recording the electrochemical signal, the ·OH generated in the Fenton system can be measured; such as Figure 6 As shown, the determination of ·OH can be achieved in the presence of different concentrations of DMSO, but a concentration of DMSO above 0.001 mol / L can react more fully with ·OH, thereby inducing a characteristic signal.

[0073] Example 4: Determination of ·OH generated in the Fenton system at different pH levels using DMSO as a probe

[0074] (1) Electrolyte: 0.1 mol / L Na2SO4 aqueous solution, pH values: 2.2, 2.8, 3.1, 3.6, 4.2, 4.6, 5.5, 6.5; DMSO concentration: 0.04 mol / L; H2O2 (16 mmol / L) added; Fe2O3 added. 2+ (0.32 mmol / L) and record the square wave voltammetry signal to determine the ·OH ( ) generated in the Fenton system. Figure 7 ). Figure 7 This indicates that changes in the pH of the electrolyte have no significant effect on the determination of ·OH by this method, and the relative standard deviation of the obtained signal intensity is 3.1%.

[0075] Example 5: Determination of DMSO using ·OH as a probe

[0076] (1) The electrolyte is a 0.1 mol / L Na2SO4 aqueous solution with a H2O2 concentration of 1.6 mmol / L, and Fe is added simultaneously. 2+ (0.32 mmol / L) and cell washing buffer containing DMSO were used, and the square wave voltammetry signal was recorded. Based on the linear equation (ΔI² = -0.02 + 5.06c),... DMSO The DMSO content can be determined by counting the DMSO molecules; for example... Figure 8 As shown. Figure 8 The oxidation peak appearing at 0.8 V is attributed to the reaction of ·OH generated by Fenton in the electrolyte with the target DMSO to produce MSIA, which is then oxidized on the surface of the working electrode.

[0077] Example 6: Establishment of linear relationship for ·OH determination using DMSO as a probe

[0078] (1) The electrolyte was a 0.1 mol / L Na2SO4 aqueous solution with a pH of 3.6 and a DMSO concentration of 0.04 mol / L. H2O2 (16 mmol / L) was added, and different concentrations of Fe were added. 2+ (0.32 mmol / L) and recorded the square wave voltammetry signal, with Fe 2+ A linear relationship was established between the concentration of ·OH and the oxidation peak intensity by square wave voltammetry (ΔI1=0.57+11.28c). ·OH ()( Figure 9 ΔI1=I1-I0, where I0 and I1 represent Fe, respectively. 2+ The voltage-voltage curve values ​​(0.8V) before and after addition.

[0079] Example 7: Establishment of linear relationship for DMSO determination using ·OH as a probe

[0080] (1) The electrolyte is a 0.1 mol / L Na2SO4 aqueous solution with a pH of 3.6 and a DMSO concentration of 0.04 mol / L. H2O2 (1.6 mmol / L) is added, along with Fe. 2+ (0.32 mmol / L) and different concentrations of DMSO were used to record square wave voltammetry signals. A linear relationship was established between DMSO concentration and the oxidation peak intensity of square wave voltammetry (ΔI2 = -0.02 + 5.06c). DMSO ()( Figure 10 ΔI2 = I2 - I0, where I0 and I2 represent the square wave voltage-ampere curve values ​​(0.8V) before and after the addition of DMSO, respectively.

[0081] In summary, this application provides a simple, sensitive, and selective real-time, in-situ method for the determination of ·OH and DMSO. This method eliminates the need for complex material preparation, electrode modification, and separation processes, enabling sensitive and rapid quantitative determination. In this scheme, the electrochemical signals all originate from the oxidation of methanesulfinic acid (MSIA) produced by the reaction of DMSO with ·OH on the electrode surface. When determining hydroxyl radicals, DMSO is used as the electrochemical probe. Upon adding the target substance (·OH) to the electrolyte, voltammetry is immediately performed to scan and record the signal. The ·OH concentration can be obtained by establishing a standard curve. Conversely, when a certain amount of ·OH is present in the electrolyte, the target substance (DMSO) can be added, and the signal can be scanned and recorded using voltammetry to determine DMSO.

[0082] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for real-time electrochemical determination of hydroxyl radicals and DMSO, characterized in that, This includes methods for determining hydroxyl radicals and methods for determining DMSO; in both methods for determining hydroxyl radicals and methods for determining DMSO, the electrochemical signal originates from the oxidation of methanesulfinic acid, produced by the reaction of DMSO with ·OH, on the electrode surface. The method for determining hydroxyl radicals includes the following steps: Step S11: A certain amount of DMSO is dissolved in an electrolyte, wherein the electrolyte in the electrolyte is an electrolyte that is inert in the voltammetric test, and the solvent in the electrolyte is an electrochemically inert solvent; Step S12: Add an OH source; Step S13: Use electrochemical voltammetry with a glassy carbon electrode as the working electrode to scan, record the signal, and substitute it into the standard curve to calculate the ·OH concentration; The method for determining DMSO includes the following steps: Step S21: A certain amount of ·OH source substance is dispersed in the electrolyte, wherein the electrolyte in the electrolyte is an electrolyte that is inert in the voltammetric test, and the solvent in the electrolyte is an electrochemically inert solvent; Step S22: Add the target substance containing DMSO; Step S23: Use electrochemical voltammetry with a glassy carbon electrode as the working electrode to scan, record the signal, and substitute it into the standard curve to calculate the DMSO concentration; The detection range of ·OH is 0.4-5120 μM, and the detection range of DMSO is 40-320 μM. The ·OH sources include Fenton systems, Fenton-like systems, enzyme catalysis systems, nanomaterial catalysis systems, atmospheric particulate matter, cigarette smoke or filter extracts, and endogenous substances; In step S13 or step S23, the electrochemical signal peak appears at 0.8V.

2. The method for real-time electrochemical determination of hydroxyl radicals and DMSO according to claim 1, characterized in that, In step S11 or step S21, the pH of the electrolyte solution is 2.2-6.

5.

3. The method for real-time electrochemical determination of hydroxyl radicals and DMSO according to claim 1, characterized in that, In step S11, the concentration range of DMSO is 0.0001-1 mol / L.

4. The method for real-time electrochemical determination of hydroxyl radicals and DMSO according to claim 1, characterized in that, In step S13 or step S23, the electrochemical voltammetry includes cyclic voltammetry, linear scan voltammetry, square wave voltammetry, and pulse voltammetry.

5. An electrochemical system for real-time determination of hydroxyl radicals and DMSO, characterized in that, The method for real-time electrochemical determination of hydroxyl radicals and DMSO according to any one of claims 1-4; the electrochemical system includes a reference electrode, a counter electrode, and a working electrode.

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