A method for detecting trimethylamine oxide based on electrochemiluminescence and its electrochemiluminescence sensor

Abstract

This invention discloses a method for detecting trimethylamine oxide based on electrochemiluminescence and its electrochemiluminescence sensor, belonging to the field of analytical detection technology. Based on a terpyridine-ruthenium electrochemiluminescence system, this invention uses trimethylamine, generated from the reduction of trimethylamine oxide, as a co-reactant. Utilizing the electrochemiluminescence reaction between trimethylamine and terpyridine-ruthenium, the relationship between trimethylamine oxide and the electrochemiluminescence signal intensity was successfully established, indirectly detecting trimethylamine oxide. This represents the first application of electrochemiluminescence methods in the analysis and detection of trimethylamine oxide. The method and sensor of this invention are safe, accurate, effective, and simple. The linear detection range for trimethylamine oxide is 30–1000 μM, with a detection limit of 26.79 μM (S / N = 3), exhibiting a wide linear range and high sensitivity. The actual sample spiked recovery rate is 96.90%–105.10%, demonstrating high reliability.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for detecting trimethylamine oxide based on electrochemiluminescence and its electrochemiluminescence sensor. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Trimethylamine oxide (TMAO) is an endogenous natural umami component found in aquatic products such as fish and shrimp, and it is widely distributed in nature. Under anaerobic conditions, TMAO can decompose into trimethylamine (TMA). Since TMA is the source of the distinctive odor of spoiled aquatic products, TMAO can be used as one of the indicators for evaluating the freshness of aquatic products. In many marine species, TMAO has important physiological functions, such as stabilizing proteins, regulating osmotic pressure, and regulating buoyancy. Currently, the classic method for determining TMAO in aquatic products is spectrophotometry, which involves reducing TMAO to trimethylamine using titanium trichloride, and then converting the trimethylamine content using the picric acid colorimetric method. However, the hazards and toxicity of TiCl3, as well as its storage, handling, and subsequent processing, are very challenging. Furthermore, this method is cumbersome, requiring repeated shaking extraction, resulting in poor reproducibility and low efficiency. Other methods for TMAO detection include chromatography, capillary electrophoresis, and nuclear magnetic resonance (NMR). However, these methods typically require expensive large-scale testing instruments, complex sample processing, and professional operation, which complicates analytical work. Therefore, it is of great significance to develop a simple, fast, accurate and widely applicable method for the detection of trimethylamine oxide. Summary of the Invention

[0004] To address the shortcomings of existing trimethylamine oxide detection technologies, the present invention aims to provide a trimethylamine oxide detection method and its electrochemiluminescence sensor based on electrochemiluminescence (ECL). The detection method and electrochemiluminescence sensor provided by the present invention are based on the ruthenium terpyridine electrochemiluminescence system. By using trimethylamine generated from the reduction of trimethylamine oxide as a co-reactant, the ECL reaction between trimethylamine and ruthenium terpyridine is utilized to indirectly detect trimethylamine oxide, enabling rapid and convenient detection of trimethylamine oxide.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a method for detecting trimethylamine oxide based on electrochemiluminescence, comprising the following steps:

[0007] (1) Trimethylamine oxide is reduced to trimethylamine using a reducing agent, and the reaction solution containing the reducing agent and trimethylamine oxide is added to the electrolyte solution;

[0008] (2) The solid electrochemiluminescence probe of terpyridine ruthenium is used as the working electrode. The working electrode, reference electrode and auxiliary electrode are installed in the device containing the solution obtained in step (1). The electrochemiluminescence signal detection system is connected. The electroluminescence signal is generated by the excitation of the electrical signal. The electrochemiluminescence signal is recorded. The linear relationship between the luminescence intensity and the concentration of trimethylamine oxide is established, and the corresponding linear regression equation is obtained.

[0009] A second aspect of the present invention provides an electrochemiluminescence sensor for detecting trimethylamine oxide, the sensor comprising a signal detection system, an electrode system, and a signal generation system;

[0010] The signal generation system includes a reducing agent, trimethylamine oxide, an electrolyte solution, and ruthenium tripyridine.

[0011] The beneficial effects of this invention are as follows:

[0012] This invention employs a three-electrode system, designing and fabricating a solid-state electrochemiluminescence probe of ruthenium terpyridine as the working electrode, an Ag / AgCl electrode as the reference electrode, and platinum as the auxiliary electrode. Electrochemiluminescence signals are generated by electrical signal excitation. TMAO is pre-reduced to TMA using the relatively safe and mild Fe(II)-EDTA as a reducing agent. The reduction product TMA serves as a co-reactant in the ruthenium terpyridine system to generate an electrochemiluminescence signal, enabling the detection of TMAO through electrochemiluminescence technology. Higher TMAO concentrations result in higher TMA concentrations and stronger electrochemiluminescence signal intensity, thus establishing a correlation between TMAO and the intensity of the electrochemiluminescence signal. The detection method of this invention is a rapid method for detecting TMAO in seafood such as shrimp meat. It is safe, accurate, effective and simple. The linear detection range of TMAO is 30-1000 μM, the detection limit is 26.79 μM (S / N=3), the detection linear range is wide and the sensitivity is high. The actual sample spiked recovery rate is 96.90%-105.10%, which shows good reliability. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 This is a schematic diagram illustrating the detection principle of using the ECL reaction to detect trimethylamine oxide in the detection method of the present invention;

[0015] Figure 2This is an electrochemiluminescence graph showing the change in electrochemiluminescence signal intensity with TMAO concentration in an embodiment of the present invention; wherein the buffer solution is a phosphate buffer with pH 10, and the TMAO concentrations are 30, 50, 160, 320, 640, and 1000 μM; the cyclic voltammetry scan range is 0-1.4 V, and the scan rate is 0.1 V / s;

[0016] Figure 3 This is a standard curve showing the change in electrochemiluminescence signal intensity as a function of TMAO concentration. Detailed Implementation

[0017] In view of the shortcomings of existing trimethylamine oxide detection technologies, this invention proposes a trimethylamine oxide detection method based on electrochemiluminescence and its electrochemiluminescence sensor.

[0018] A typical embodiment of the present invention provides a method for detecting trimethylamine oxide based on electrochemiluminescence, comprising the following steps:

[0019] (1) Trimethylamine oxide is reduced to trimethylamine using a reducing agent, and the reaction solution containing the reducing agent and trimethylamine oxide is added to the electrolyte solution;

[0020] (2) The solid electrochemiluminescence probe of terpyridine ruthenium is used as the working electrode. The working electrode, reference electrode and auxiliary electrode are installed in the device containing the solution obtained in step (1). The electrochemiluminescence signal detection system is connected. The electroluminescence signal is generated by the excitation of the electrical signal. The electrochemiluminescence signal is recorded. The linear relationship between the luminescence intensity and the concentration of trimethylamine oxide is established, and the corresponding linear regression equation is obtained.

[0021] This invention provides a method for detecting trimethylamine oxide based on a ruthenium terpyridine electrochemiluminescence (ECL) system. Trimethylamine, generated from the reduction of trimethylamine oxide, is used as a co-reactant. The ECL reaction between trimethylamine and ruthenium terpyridine indirectly detects trimethylamine oxide. This method enables rapid and convenient detection of trimethylamine oxide, and to the inventors' knowledge, this is the first time an electrochemiluminescence method has been applied to the analysis and detection of trimethylamine oxide.

[0022] In some embodiments of this implementation, the method for reducing trimethylamine oxide is as follows: 0.05-0.15M reducing agent aqueous solution is mixed with an equal volume of trimethylamine oxide aqueous solution (30-1000μM) of a certain concentration, and heated at 45-55°C for 5-20 minutes to reduce trimethylamine oxide to trimethylamine.

[0023] In some embodiments of this implementation, the reducing agent is complexed iron Fe(II)-EDTA.

[0024] Fe(II)-EDTA is relatively safe and mild. When used as a reducing agent, it can reduce trimethylamine oxide to trimethylamine without affecting the ECL reaction between trimethylamine and ruthenium terpyridine.

[0025] In some embodiments of this implementation, the electrolyte solution includes a buffer solution with a pH of 7-12. More preferably, the buffer solution includes, but is not limited to, phosphate buffer and carbonate buffer. pH affects the intensity of the ECL signal; for this system, the stronger the alkalinity, the stronger the ECL signal. Considering both signal intensity and the mildness of the detection conditions, the pH of the buffer solution is preferably 10.

[0026] In some embodiments of this implementation, the preparation method of the ruthenium terpyridine solid-state electrochemiluminescence probe is as follows:

[0027] Polish the surface of the glassy carbon electrode, then ultrasonically clean it with ultrapure water and ethanol in sequence, and blow it dry; drop a single-walled carbon nanotube-0.5% Nafion solution onto the electrode surface, dry it, and then immerse it in a terpyridine-ruthenium aqueous solution to obtain the final product.

[0028] In another embodiment of this method, the polishing is performed by sequentially polishing the surface of the glassy carbon electrode with 0.3μm and 0.05μm alumina powders. Polishing removes impurities from the surface of the glassy carbon electrode, giving it a mirror-like finish, which is beneficial for subsequent modification of the glassy carbon electrode with single-walled carbon nanotubes (Nafion).

[0029] In another embodiment of this method, the concentration of the single-walled carbon nanotube-0.5% Nafion solution is 0.5-2.0 mg / mL, and the volume is 5-15 μL. Modifying the glassy carbon electrode with single-walled carbon nanotubes and Nafion provides more open space structures on the electrode surface, which is beneficial for the immobilization of the terpyridine ruthenium electrochemiluminescent reagent and gives the probe better conductivity; ultimately making the electrochemiluminescent sensor more sensitive and stable.

[0030] In another embodiment of this method, the concentration of the ruthenium terpyridine aqueous solution is 0.5-10 mM, preferably 1 mM. A glassy carbon electrode modified with ruthenium terpyridine-Nafion single-walled carbon nanotubes is used, and the intensity of the luminescent signal is detected by utilizing the ECL reaction between ruthenium terpyridine and trimethylamine, thereby achieving rapid and accurate determination of the trimethylamine oxide concentration.

[0031] In another embodiment of this method, the immersion time in the ruthenium terpyridine aqueous solution is 5-24 hours, preferably 12 hours. Through immersion, ruthenium terpyridine is successfully deposited on the glassy carbon electrode modified with single-walled carbon nanotubes-Nafion.

[0032] In another embodiment of this invention, the method for exciting the electrochemiluminescence signal is not limited, as long as it can generate an electrochemiluminescence signal. In some embodiments, methods include cyclic voltammetry, linear scanning voltammetry, potentiostatic method, and potential step pulse method, with cyclic voltammetry being preferred. More preferably, the cyclic voltammetry method has a scanning potential unit of 0-1.4V and a scanning speed of 0.1V / s.

[0033] In another embodiment of this implementation, the reference electrode is an Ag / AgCl electrode, and the auxiliary electrode is platinum.

[0034] In another typical embodiment of the present invention, an electrochemiluminescence sensor for detecting trimethylamine oxide is provided, the sensor comprising a signal detection system, an electrode system and a signal generation system;

[0035] The signal generation system includes a reducing agent, trimethylamine oxide, an electrolyte solution, and ruthenium tripyridine.

[0036] The electrochemiluminescence sensor provided by this invention is based on a ruthenium terpyridine electrochemiluminescence system. It indirectly detects trimethylamine oxide by using trimethylamine, generated from the reduction of trimethylamine oxide, as a co-reactant and utilizing the ECL reaction between trimethylamine and ruthenium terpyridine. Based on this invention, the electrochemiluminescence sensor enables rapid and convenient detection of trimethylamine oxide, with a detection limit of 30-1000 μM. Furthermore, to the inventors' knowledge, this is the first time that an electrochemiluminescence method has been applied to the analysis and detection of trimethylamine oxide.

[0037] In some embodiments of this implementation, the reducing agent is complexed iron Fe(II)-EDTA.

[0038] In some embodiments of this implementation, the terpyridine ruthenium is a solid electrochemiluminescent probe deposited with terpyridine ruthenium as the working electrode;

[0039] Alternatively, the terpyridine ruthenium may be added directly to the test solution.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] Example 1

[0042] (1) Preparation of solid-state electrochemiluminescence probe of ruthenium terpyridine

[0043] The glassy carbon electrode surface was successively polished with 0.3 μm and 0.05 μm alumina powders, followed by ultrasonic cleaning with ultrapure water and ethanol, and then dried. 10 μL of a 1 mg / mL single-walled carbon nanotube-0.5% Nafion solution was drop-coated onto the electrode surface and dried at 37°C for 2 h or until completely dry. The electrode was then immersed in a 1 mM terpyridine-ruthenium aqueous solution for 12 h.

[0044] (2) TMAO Reduction

[0045] Prepare a fresh 0.1M Fe(II)-EDTA aqueous solution immediately before use. The preparation method is as follows: Mix 10 mL of 0.2M FeSO4 solution (dissolved in 0.1M HCl) and 10 mL of 0.2M EDTA aqueous solution, then add 0.1 mL of glacial acetic acid. Adjust the pH of the mixture to 4.4-4.6 with 9M NaOH solution. This reducing agent must be prepared fresh before use to avoid oxidation of Fe(II). If the Fe(II)-EDTA aqueous solution turns dark yellow or brownish-red, it should be discarded and a new solution prepared.

[0046] Take an equal volume of the 0.1M Fe(II)-EDTA aqueous solution prepared above and mix it with an equal volume of 1.0mM trimethylamine oxide aqueous solution. Heat at 50℃ for 15 minutes to reduce trimethylamine oxide to trimethylamine. After standing at room temperature, perform ECL detection.

[0047] (3) Assembly of ECL detection / electrochemiluminescence sensor

[0048] The Fe(II)-EDTA-TMAO reaction solution from step (2) was added to 0.1M pH=10 phosphate buffer and then added to the ECL detection cell. A three-electrode system was used, with the terpyridine ruthenium solid-state electrochemiluminescence probe prepared in step (1) as the working electrode, the Ag / AgCl electrode as the reference electrode, and platinum as the auxiliary electrode. Cyclic voltammetry was used to generate the excitation signal, with a scanning potential range of 0 to 1.4V and a scan rate of 0.1V / s. The electrochemiluminescence signal was recorded.

[0049] like Figure 2 As shown, by controlling the concentration of trimethylamine oxide in the ECL detection cell to be 30, 50, 160, 320, 640, and 1000 μM, a linear relationship between the luminescence intensity y and the concentration of trimethylamine oxide x was established, yielding the corresponding linear regression equation y = 1.49x + 359.04. Figure 3 ).

[0050] Limit of detection: Calculated according to the formula for limit of detection. in The linear slope is given by s, where s is the standard deviation of the detection values ​​of 11 blank samples. Within the linear detection range of 30-1000 μM, the detection limit is calculated to be 26.79 μM (S / N = 3).

[0051] Reliability: Whiteleg shrimp were used as the actual sample, and the trimethylamine oxide content was determined using the method in Example 1. First, the whiteleg shrimp were shelled and washed. 10g of shrimp meat was weighed and homogenized in 50mL of 5% trichloroacetic acid solution. The homogenate mixture was then centrifuged (8000rpm, 15min) and filtered (0.45μm filter membrane). The supernatant was taken and diluted 10 times with ultrapure water to obtain the test solution. Three equal portions of the test solution were taken, and the trimethylamine oxide content was determined. Then, 300μM, 500μM, and 700μM trimethylamine oxide were added to the solutions respectively, and the recovery rates were determined and calculated. As shown in Table 1, the recoveries were 96.90%, 105.10%, and 98.57%, respectively.

[0052] Table 1. Results of Trimethylamine Oxide Recovery in Shrimp Meat

[0053]

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting trimethylamine oxide based on electrochemiluminescence, characterized in that, Includes the following steps: (1) Trimethylamine oxide is reduced to trimethylamine using a reducing agent, and the reaction solution containing the reducing agent and trimethylamine oxide is added to the electrolyte solution; (2) The solid electrochemiluminescence probe of terpyridine ruthenium is used as the working electrode. The working electrode, reference electrode and auxiliary electrode are installed in the device containing the solution obtained in step (1). The electrochemiluminescence signal detection system is connected. The electroluminescence signal is generated by the excitation of the electrical signal. The electrochemiluminescence signal is recorded. The linear relationship between the luminescence intensity and the concentration of trimethylamine oxide is established, and the corresponding linear regression equation is obtained.

2. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The method for reducing trimethylamine oxide is as follows: mix an equal volume of 0.05-0.15 M reducing agent aqueous solution with trimethylamine oxide aqueous solution, and heat at 45-55℃ for 5-20 minutes to reduce trimethylamine oxide to trimethylamine.

3. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The reducing agent is complexed iron Fe(II)-EDTA.

4. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The electrolyte solution includes a buffer solution with a pH of 7-12.

5. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 4, characterized in that, The buffer solutions include phosphate buffer and carbonate buffer.

6. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The preparation method of the ruthenium terpyridine solid-state electrochemiluminescence probe is as follows: Polish the surface of the glassy carbon electrode, then ultrasonically clean it with ultrapure water and ethanol in sequence, and blow it dry; drop a single-walled carbon nanotube-0.5% Nafion solution onto the electrode surface, dry it, and then immerse it in a terpyridine-ruthenium aqueous solution to obtain the final product.

7. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 6, characterized in that, The polishing process involves sequentially polishing the surface of the glassy carbon electrode with 0.3 μm and 0.05 μm alumina powders.

8. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 6, characterized in that, The concentration of single-walled carbon nanotubes in 0.5% Nafion solution is 0.5-2.0 mg / mL, and the volume is 5-15 μL.

9. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 6, characterized in that, The concentration of the terpyridine ruthenium aqueous solution is 0.5-10 mM.

10. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 6, characterized in that, Immersion time in terpyridine ruthenium aqueous solution is 5-24 h.

11. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The methods for generating the electrical signal that excites the electrochemiluminescence signal include: cyclic voltammetry, linear scanning voltammetry, potentiostatic method, and potential step pulse method.

12. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 11, characterized in that, The method for generating the electrochemiluminescence signal is cyclic voltammetry.

13. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 12, characterized in that, The cyclic voltammetry method is as follows: the scanning potential unit is 0-1.4V, and the scanning speed is 0.1V / s.

14. The method for detecting trimethylamine oxide based on electrochemiluminescence as described in claim 1, characterized in that, The reference electrode is an Ag / AgCl electrode, and the auxiliary electrode is platinum.

15. An electrochemiluminescence sensor for detecting trimethylamine oxide, characterized in that, The sensor includes a signal detection system, an electrode system, and a signal generation system; The signal generation system includes a reducing agent, trimethylamine oxide, an electrolyte solution, and ruthenium tripyridine.

16. The electrochemiluminescence sensor as described in claim 15, characterized in that, The reducing agent is complexed iron Fe(II)-EDTA.

17. The electrochemiluminescence sensor as described in claim 15, characterized in that, The terpyridine ruthenium is a solid electrochemiluminescent probe deposited with terpyridine ruthenium as the working electrode.

18. The electrochemiluminescence sensor as described in claim 15, characterized in that, The terpyridine ruthenium was added directly to the test solution.

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