Dual-mode probe for detecting hydrogen peroxide as well as preparation method and application of dual-mode probe

Abstract

The invention discloses a dual-mode probe for detecting hydrogen peroxide (H2O2) as well as a preparation method and application of the dual-mode probe, and belongs to the technical field of application of organic probes in analysis and detection. The molecular structural formula of the probe is as follows: # imgabs0 #, in the presence of H2O2, the boric acid ester group of the probe reacts with H2O2, and the generated product is firmly adsorbed on the surface of a carbon nanotube enhanced screen-printed electrode through a pi-pi stacking effect. Then, when differential pulse voltammetry is used for detection, it is observed that the oxidation peak intensity of an original probe is remarkably reduced, and a new oxidation peak appears along with the original probe. The ratio type electrochemical detection of H2O2 is realized by measuring the change of the peak area ratio of the product to the probe oxidation peak current, and the linear range is 10-150 [mu] M; meanwhile, fluorescence spectrum analysis is displayed at the wavelength of 585 nm, the intensity of a fluorescence emission peak is gradually increased along with the increase of the H2O2 concentration, and the linear response range is expanded to 10-300 mu M. The electrochemical ratio-type signal and fluorescence opening detection mode not only can effectively correct errors caused by system or background noise, but also greatly improves the accuracy, reproducibility and sensitivity of detection. Therefore, the method has the potential of being practically applied to quantitative analysis of H2O2 in complex samples.

Description

Technical Field

[0001] The present invention relates to a ratiometric electrochemical and fluorescence dual-mode detection probe, in particular to a dual-mode probe for detecting hydrogen peroxide (H2O2) and a preparation method and application thereof, belonging to the technical field of application of organic probes in analysis and detection. Background Art

[0002] As an important biological active oxygen molecule, H2O2 is widely involved in the free radical scavenging and tissue maintenance process in cells. Its concentration imbalance is closely related to serious health problems such as diabetes, Parkinson's disease and Alzheimer's disease. Although the importance of H2O2 in the biomedical field is self-evident, its comprehensive impact mechanism on human health remains to be explored in depth. In view of this, it is particularly important to develop a simple, economical and highly selective H2O2 analysis technology.

[0003] At present, traditional detection methods such as high performance liquid chromatography, electrochemical method, colorimetry and fluorescence method are used in H2O2 analysis, but they are generally faced with challenges such as cumbersome operation, expensive equipment and long detection cycle. Electrochemical method has shown great potential in H2O2 detection due to its advantages of high sensitivity, good selectivity, low cost and easy miniaturization; while fluorescence method is favored by its convenience and high sensitivity of real-time monitoring. The combination of dual-mode detection strategies is expected to further improve the detection accuracy and reduce system or background interference through mutual correction. Studies have used electrochemical sensors combined with organic metal frameworks, precious metals or carbon-based materials to detect H2O2, but these materials are often limited in their wide application due to high preparation costs, low selectivity and accuracy. Therefore, the development of an economical and efficient H2O2 analysis method with excellent selectivity and accuracy has become an urgent need in the current research field. This will not only promote a deeper understanding of the biological functions of H2O2, but will also provide strong technical support for the early diagnosis and treatment of related diseases. Summary of the invention

[0004] In order to break through the bottleneck of the existing technology, the present invention provides a dual-mode ratiometric electrochemical and fluorescence detection method integrating single-probe technology, aiming at detecting H2O2 with high accuracy, high selectivity and high sensitivity; at the same time, the hydrogen peroxide dual-mode probe and its construction method are provided.

[0005] In order to achieve the above object, the first aspect of the present invention provides a method for preparing probe BB: dissolving resorufin and 4-bromomethylphenylboronic acid pinacol ester in an organic solvent, adding an alkaline substance and a dehydrating agent, and heating and stirring in an oil bath to react. Subsequently, thin layer chromatography is used to detect the reaction process. After the reaction is complete, it is purified by vacuum filtration and column chromatography, and dichloromethane: methanol is used as an eluent to obtain a BB probe, and its molecular structure is as follows:

[0006]

[0007] The organic solvent is acetonitrile.

[0008] The alkaline substance is cesium carbonate, and the dehydrating agent is anhydrous sodium sulfate.

[0009] The molar ratio of resorufin to 4-bromomethylphenylboronic acid pinacol ester is 1:1 to 1:3, preferably 1:1.5.

[0010] The volume ratio of the eluent dichloromethane to methanol is 10:1 to 500:1, preferably 100:1.

[0011] The second aspect of the present invention provides a dual-mode ratiometric electrochemical sensor for detecting H2O2 using a single probe, and the method is as follows: an ethanol solution of single-walled carbon nanotubes (SWCNT) is drop-coated on a screen-printed electrode (SPCE), and after natural drying, a H2O2 detection electrode SPCE / SWCNT is obtained for standby use; a control group solution and a test group solution are respectively prepared; the prepared SPCE / SWCNT electrode is immersed in the control group solution and the test group solution for incubation, the electrode is taken out, and after washing and drying, a modified electrode is obtained, defined as SPCE / SWCNT / BB (control group) and SPCE / SWCNT / BB+Re (test group), and the electrodes are used as working electrodes, Ag / AgCl is used as a reference electrode, and a Pt wire is used as a counter electrode. The DPV response in a 0.1M PBS (pH 7.4) solution is measured using a CHI1040C electrochemical workstation, and the potential window is: -0.6 to 0.2V.

[0012] The preparation of the control group solution and the test group solution: (1) adding the probe BB solution to the phosphate buffer solution, incubating, and obtaining the control group solution; (2) adding the probe BB solution and H2O2 solutions of different concentrations to the phosphate buffer solution, incubating, and obtaining the test group solution.

[0013] The third aspect of the present invention provides a method for detecting H2O2 by using a single probe for fluorescence detection, the method being as follows: adding the control group and the test group solutions into cuvettes respectively, performing fluorescence spectrum measurement using an Agilent fluorescence-spectrophotometer, and obtaining an emission spectrum in the range of 460 to 800 nm at an excitation wavelength of 405 nm; the slit width of the excitation monochromator is 5 nm, and the slit width of the emission monochromator is 10 nm.

[0014] Principle of the invention: The invention designs and synthesizes a dual-mode organic probe for detecting H2O2. The probe can be modified on the surface of a screen-printed electrode through JI-JI interaction. In the absence of H2O2, the electrode surface only has the oxidation peak of the probe BB measured by differential pulse voltammetry. When the probe reacts with H2O2, a new oxidation peak appears on the electrode surface, and the current gradually decreases as the current increases. The ratio electrochemical quantitative detection of H2O2 can be achieved according to the change in the peak area ratio between the oxidation peak of the new peak and the oxidation peak of BB. In addition, the probe generates fluorescence after reacting with H2O2, and the fluorescence quantitative detection of H2O2 can be achieved at the same time. The dual-mode H2O2 ratio electrochemical and fluorescence on-detection constructed by the invention can be used for the detection of H2O2 in serum. When used simultaneously and verified with each other, it can not only achieve high-selective detection of H2O2, but also achieve high-accuracy and high-sensitivity detection.

[0015] Compared with the prior art, the advantages or beneficial effects of the present invention are: Based on the organic probe molecule design strategy, the present invention successfully developed a dual-mode H2O2 detection technology integrating electrochemical and fluorescence detection, which provides strong technical support and solution strategies for biomedicine, environmental monitoring, etc. Compared with the previous H2O2 detection methods, this technology has the following significant advantages: (1) Boric acid-phenol "switch" is used as an electrochemical recognition mechanism, which has high analytical selectivity; (2) The probe or reaction product is modified on the electrode surface through π-π adsorption, and the electrode preparation process is simple and fast; (3) The probe itself has an electrochemical signal as a self-reference; after the reaction, the oxidation peak can be gradually shifted negatively, thereby realizing ratio detection; (4) The fluorescence of the probe and H2O2 reaction is turned on, and the background is small. At the same time, combined with dual-mode readout, the influence of system or background errors can be corrected, and higher accuracy can be obtained; (5) The preparation of screen-printed electrodes is simple, controllable, and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the organic probe BB prepared in Example 1 of the present invention.

[0017] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the organic probe BB prepared in Example 1 of the present invention.

[0018] Figure 3 This is a high-resolution mass spectrum of the organic probe BB prepared in Example 1 of the present invention.

[0019] Figure 4 The electrochemical response of the ratiometric electrochemical sensor constructed in Example 2 of the present invention to different concentrations of H2O2.

[0020] Figure 5 This is the high performance liquid chromatography before and after the reaction of the organic probe BB of the present invention with H2O2.

[0021] Figure 6 This is a fluorescence detection diagram of the organic probe BB of the present invention to different concentrations of H2O2.

[0022] Figure 7 This is an interference experiment of the ratiometric electrochemical sensor constructed by the present invention on different biological related substances.

[0023] Figure 8 This is a fluorescence interference experiment of the organic probe BB of the present invention on different biological related substances. DETAILED DESCRIPTION

[0024] The following examples are provided to provide further details for a better understanding of the present invention.

[0025] Example 1: Synthesis of dual-mode organic probe for detecting H2O2

[0026] First, dissolve resorufin (0.02132 g, 1 mmol) and excess 4-bromomethylphenylboronic acid pinacol ester (0.0446 g, 1.5 mmol) in 30 mL of acetonitrile. Subsequently, add an appropriate amount of cesium carbonate (81.5 mg, 2.5 mmol) and anhydrous sodium sulfate (355 mg, 2.5 mmol) to the reaction system, heat to 60 ° C in a constant temperature oil bath, and stir the reaction overnight. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the crude product was concentrated by vacuum distillation. Subsequently, a mixed solvent of dichloromethane and methanol (volume ratio of 100:1) was used as an eluent, and the crude product was purified by silica gel column chromatography to obtain the target probe BB, an orange-red powder. The product was further characterized by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrometry. Figure 1 , Figure 2 and Figure 3 Demonstrate the successful synthesis of H2O2 dual-mode organic probe BB.

[0027] Example 2: Construction of a ratiometric electrochemical sensor for H2O2 detection

[0028] First, the ethanol solution of carbon nanotubes (SWCNT) is drop-coated on the screen-printed electrode (SPCE), and after natural drying, a H2O2 detection electrode SPCE / SWCNT is obtained for use.

[0029] Solution preparation: (1) Add 100 μL of 0.1 M PBS buffer solution (pH = 7.4) and 10 μL of probe BB solution (5 mg mL -1), the above solution was incubated in a temperature incubator at 37°C for 30 min (control group); (2) 100 μL of 0.1 M PBS buffer solution (pH = 7.4), 10 μL of probe BB solution (5 mg mL -1 ) and H2O2 solutions of different concentrations, which were incubated in an incubator at 37°C for 30 min (test group).

[0030] Electrochemical detection: First, the prepared SPCE / SWCNT electrodes were immersed in the control group solution and the test group solution respectively and incubated at room temperature for 10 minutes. The electrodes were taken out, washed with distilled water and dried. The modified electrodes were defined as SPCE / SWCNT / BB (control group) or SPCE / SWCNT / BB+Re (test group). These electrodes were used as working electrodes, Ag / AgCl as reference electrodes, and Pt wire as counter electrodes. The DPV response in 0.1M PBS (pH 7.4) solution was measured using a CHI1040C electrochemical workstation. The potential window was -0.6 to 0.2V. Figure 4 In figure a, when there is no H2O2, there is only one oxidation peak at -0.273V, which is attributed to the electrochemical signal of BB itself. When H2O2 is added, the BB oxidation peak current gradually decreases, and a new oxidation peak appears at about 0.316V. This is mainly due to the electrochemical signal generated by the reaction of H2O2 and BB to form a resorufin structure. In addition, the ratiometric electrochemical quantitative detection of H2O2 can be achieved based on the change in the peak area ratio of the oxidation peak that generates the new peak and the BB oxidation peak. The detection shows two linear ranges, ranging from 10 to 150μM, and the detection limit is 0.5μM ( Figure 4 In addition, the product after the reaction was also verified by high performance liquid chromatography ( Figure 5 ). The above illustrates the successful construction of the H2O2 detection ratio electrochemical sensor of the present invention.

[0031] Example 3: Turn on fluorescence detection of H2O2

[0032] Solution preparation: Same as Example 2, (1) Add 100 μL of 0.1 M PBS buffer solution (pH = 7.4), 10 μL of probe BB solution (5 mg mL -1 ), the above solution was incubated in a temperature incubator at 37°C for 30 min (control group); (2) 100 μL of 0.1 M PBS buffer solution (pH = 7.4), 10 μL of probe BB solution (5 mg mL -1 ) and H2O2 solutions of different concentrations, which were incubated in an incubator at 37°C for 30 min (test group).

[0033] Fluorescence detection: The control group and test group solutions were added to cuvettes respectively, and the fluorescence spectrum was measured using an Agilent fluorescence-spectrophotometer to obtain an emission spectrum in the range of 460-800 nm at an excitation wavelength of 405 nm; the slit width of the excitation monochromator was 5 nm, and the slit width of the emission monochromator was 10 nm. Figure 6 In a, when there is no H2O2, there is a weak fluorescence signal at 585nm, which belongs to the fluorescence signal of BB itself. When H2O2 is added, the fluorescence signal at 585nm is enhanced, which is mainly due to the fluorescence signal generated by the reaction of H2O2 and BB to form a resorufin structure. According to the change of fluorescence intensity, the fluorescence quantitative detection of H2O2 can be realized. The detection shows two linear ranges, ranging from 10 to 300μM, and the detection limit is 0.2μM ( Figure 6 (b)

[0034] Example 4: Anti-interference experiment

[0035] The interference determination of the H2O2 detection ratio electrochemical sensor of the present invention was carried out using a CHI1040C electrochemical workstation. Referring to the solution electrochemical detection process of Example 2, the results are as follows Figure 7 As shown in the figure, other common biological active enzymes and small molecules (1-14 are glucose, adenosine triphosphate, glutathione, cysteine, ·OH, K + 、Na + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ , Mn 2+ , Fe 3+ 、Al 3+ ), the peak current ratio change caused by the method does not exceed 5.0%, indicating that the ratiometric electrochemical sensor of the present invention has high selectivity and anti-interference ability for H2O2 detection.

[0036] Similarly, the fluorescence detection interference determination of the BB probe of the present invention was carried out with reference to Example 3. The fluorescence spectrum was measured using an Agilent fluorescence-spectrophotometer, and the emission spectrum in the range of 460 to 800 nm was obtained at an excitation wavelength of 405 nm; the slit width of the excitation monochromator was 5 nm, and the slit width of the emission monochromator was 10 nm. The results are shown in FIG. Figure 8 As shown, other bioactive molecules and metal ions (1-14 are glucose, ATP, glutathione, cysteine, ·OH, K + 、Na + , Ca 2+ Mg 2+ , Cu 2+ 、Zn2+ , Mn 2+ , Fe 3+ 、Al 3+ ), the fluorescence change caused was no more than 4.5%, indicating that the BB probe of the present invention has high selectivity and anti-interference ability for H2O2 fluorescence detection.

Claims

1. A dual-mode organic probe for detecting H2O2, characterized in that: It has the following molecular structure: 。 2. The method for synthesizing the dual-mode organic probe for detecting H2O2 as claimed in claim 1, characterized in that: The following steps are involved: Resorufin and 4-bromomethylphenylboronic acid pinacol ester are dissolved in an organic solvent, and then an alkaline substance and a dehydrating agent are added, and the mixture is stirred and reacted in an oil bath, and real-time monitoring is performed by thin layer chromatography; after the reaction is completed, the mixture is purified by vacuum filtration and column chromatography, and eluted and purified by an eluent to obtain a H2O2 dual-mode organic probe, referred to as BB; The organic solvent is acetonitrile; the alkaline substance is cesium carbonate, and the dehydrating agent is anhydrous sodium sulfate.

3. The method for synthesizing a dual-mode organic probe for detecting H2O2 according to claim 2, characterized in that: The molar ratio of resorufin to 4-bromomethylphenylboronic acid pinacol ester is 1:1 to 1:

3.

4. The use of the dual-mode organic probe for detecting H2O2 as claimed in claim 1, characterized in that: It is used to construct a ratiometric electrochemical sensor for H2O2 detection, and quantitative detection of H2O2 is achieved through changes in electrochemical signals.

5. The use of the dual-mode organic probe for detecting H2O2 as claimed in claim 4, characterized in that: Here are the steps: (a) First, the ethanol solution of single-walled carbon nanotubes was evenly drop-coated on the surface of the screen-printed electrode and allowed to air-dry naturally to obtain an electrode for H2O2 detection, referred to as SPCE / SWCNT. (b) preparing a control group solution and a test group solution: adding the probe BB solution to the phosphate buffer solution, incubating, and obtaining a control group solution; adding the probe BB solution and H2O2 solutions of different concentrations to the phosphate buffer solution, incubating, and obtaining a test group solution; (c) The prepared SPCE / SWCNT electrodes were immersed in the control group solution and the test group solution respectively and incubated at room temperature. The electrodes were taken out, washed and dried to obtain the control group modified electrode SPCE / SWCNT / BB and the test group modified electrode SPCE / SWCNT / BB+Re; (d) using an electrochemical workstation, with the electrode obtained in step (c) as the working electrode, Ag / AgCl as the reference electrode, and Pt wire as the counter electrode, differential pulse voltammetry is used to record the corresponding differential pulse voltammetry curves before and after the addition of H2O2 standard solution, and the ratio of the peak area of ​​the generated oxidation peak to the peak area of ​​the probe oxidation peak and the standard curve of H2O2 concentration are plotted; the sample to be tested is tested according to the above method, and the quantitative detection of H2O2 is achieved according to the standard curve.

6. The use of the dual-mode organic probe for detecting H2O2 as claimed in claim 1, characterized in that: It is used for open-type fluorescence detection, and the quantitative detection of H2O2 is achieved through the change of fluorescence signal.

7. The use of the dual-mode organic probe for detecting H2O2 as claimed in claim 6, characterized in that: Here are the steps: (a) preparing a control group solution and a test group solution: adding a probe BB solution to a phosphate buffer solution, incubating, and obtaining a control group solution; adding a probe BB solution and H2O2 solutions of different concentrations to a phosphate buffer solution, incubating, and obtaining a test group solution; (b) Add the control group solution and the test group solution prepared in step (a) into a cuvette, use a fluorescence spectrophotometer, set the excitation wavelength to 405 nm, and perform fluorescence measurement; record the fluorescence curves under different concentrations of H2O2; draw a standard curve of fluorescence intensity and H2O2 concentration; detect the sample to be tested according to the above method, and achieve quantitative analysis of H2O2 based on the standard curve.

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