Application of potential sensor based on polyaniline modified miniature two-electrode system to methylmalonic acid detection

Abstract

The invention belongs to the field of drug analysis and detection, and particularly relates to application of a potential sensor based on a polyaniline modified miniature two-electrode system to methylmalonic acid detection. In the potential sensor based on the polyaniline-modified miniature two-electrode system, polyaniline-loaded carbon fiber serves as a working electrode, an open-circuit potentiometric method is adopted for detecting methylmalonic acid, the sensor is particularly suitable for detecting methylmalonic acid in body fluid or excrement, pretreatment is not needed, detection is instant, rapid and accurate, the anti-interference capability is high, and the sensitivity is high. And a novel efficient and reliable analysis method is provided for detection of methylmalonic acid.

Description

Technical Field

[0001] This invention belongs to the field of drug analysis and detection, specifically relating to the application of a potential sensor based on a polyaniline-modified micro two-electrode system for the detection of methylmalonic acid. Background Technology

[0002] Methylmalonic acid (MMA) is a key blood or urine metabolic marker for diagnosing inherited metabolic diseases such as methylmalonic acidemia. Currently, its detection mainly relies on gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and genetic testing methods. Examples include CN117929564A (a method for rapid quantitative detection of methylmalonic acid in biological samples based on LC-MS), CN115508483A (a rapid LC-MS / MS method for detecting methylmalonic acid in serum samples), CN112834682A (a method for detecting methylmalonic acid content in blood), CN104991018A (a urine processing method and a method for detecting methylmalonic acid in urine using GC-MS / MS), and CN101680904A (using HIL with a zwitterionic stationary phase). While methods such as IC and mass spectrometry for the quantitative detection of methylmalonic acid and succinic acid, CN120843667A (a probe combination and detection device for detecting pathogenic genes of methylmalonic acidemia), CN120683245A (a detection kit for combined methylmalonic acidemia), and CN114457148A (a primer pair and kit for detecting gene mutations in simple methylmalonic acidemia) are highly accurate, they typically rely on large instruments, involve complex and time-consuming sample pretreatment, and are difficult to meet the needs of clinical point-of-care testing.

[0003] Potentiometric electrochemical sensors are considered a potential platform for real-time MMA detection due to their simplicity, rapid response, and ease of miniaturization. Among them, sensors based on conductive polymers such as polyaniline (PANI) have attracted attention due to their good stability and ion-electron conversion characteristics. However, existing MMA detection schemes based on potentiometric sensing principles still have limitations: First, insufficient system integration. They mostly employ traditional three-electrode systems, whose conventional reference electrodes are relatively large, restricting the overall miniaturization and portability of the sensor. Second, poor selectivity in complex physiological samples. For samples containing high concentrations of electrolytes (such as Na+),... + K + NH 4+ Real samples containing interfering substances such as urea are susceptible to ion interference, leading to reduced sensitivity and specificity of the sensor.

[0004] Therefore, how to construct a potential sensor that is simple in structure, fast in response, highly sensitive, and can be directly used for in-situ detection of complex real samples has become a technical problem that needs to be solved. The open-circuit potential method (OCPT), as a simplified potential detection strategy, offers a possible solution to this problem. Summary of the Invention

[0005] This invention provides a potential sensor based on a polyaniline-modified micro two-electrode system, which can directly detect methylmalonic acid in complex real samples such as body fluids or excrement without pretreatment. The detection is fast, accurate, has strong anti-interference ability, and high sensitivity.

[0006] The technical solution of the present invention is an application of a potential sensor based on a polyaniline-modified micro two-electrode system for the detection of methylmalonic acid. In the potential sensor based on the polyaniline-modified micro two-electrode system, carbon fiber loaded with polyaniline is used as the working electrode, and an Ag / AgCl electrode is used as the reference electrode. The open-circuit potential method is used to detect methylmalonic acid, which is particularly suitable for the quantitative detection of methylmalonic acid.

[0007] The preparation of the carbon fiber electrode loaded with polyaniline includes: using a three-electrode system and cyclic voltammetry in an electrolyte solution formed by aniline, hydrochloric acid and deionized water, aniline is oxidized in situ to polyaniline and loaded onto the surface of carbon fibers.

[0008] In the three-electrode system, carbon fiber serves as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The carbon fiber is ultrasonically cleaned with ethanol and water, respectively.

[0009] The cyclic voltammetry method scans for no less than 8 cycles in the range of +0.2 V to +1.2 V, preferably no less than 9 cycles, more preferably 10-15 cycles, and even more preferably 10-12 cycles. As one implementation method, 10 cycles are scanned.

[0010] In the electrolyte solution, the concentration of aniline is 0.08-0.35M, preferably 0.09-0.3M, more preferably 0.1-0.15M, and even more preferably 0.1M; the concentration of hydrochloric acid is 0.08-0.2M, preferably 0.09-0.15M, more preferably 0.1-0.13M, and even more preferably 0.1M.

[0011] An electrochemical detection method for methylmalonic acid includes the following steps: using a potential sensor based on a polyaniline-modified micro two-electrode system in a sample solution, with polyaniline-loaded carbon fiber as the working electrode and an Ag / AgCl electrode as the reference electrode, the method of open-circuit potential is used to detect methylmalonic acid in the sample solution by means of an external standard or an internal standard, especially the quantitative detection of methylmalonic acid in the sample solution.

[0012] The sample solution to be tested is selected from body fluids, excrement, diluents of body fluids, or diluents of excrement. Specifically, deionized water is used to dilute the body fluids or urine. The body fluids include, but are not limited to, plasma, blood, serum, tissue fluid, intrapleural fluid, intraperitoneal fluid, and cerebrospinal fluid. The excrement includes, but is not limited to, urine.

[0013] The detection limit for methylmalonic acid in the sample solution to be tested is 0.001 µmol / L, and the linear range is 0.01-800 µmol / L.

[0014] This invention employs electrochemical polymerization to construct a polyaniline polymer layer on the surface of a carbon fiber electrode, combined with open-circuit potential method (OCPT) to achieve rapid, convenient, sensitive, and in-situ detection of methylmalonic acid. The modified electrode exhibits excellent conductivity and specific recognition ability, with a detection limit as low as 0.001 µmol / L and a linear range of 0.01-800 µmol / L. This method is simple to operate, requiring only 50 seconds for a single detection, with a relative standard deviation of <0.8% and a recovery rate of 96.42%-100.34%. It can be directly used for in-situ detection of complex urine samples without complex pretreatment, providing an efficient and reliable analytical method for the convenient and accurate determination of methylmalonic acid (i.e., organic acid sepsis) in urine.

[0015] This invention has the following innovative features: 1. A methylmalonic acid potential sensor with a polyaniline-modified micro two-electrode system is constructed on the surface of a carbon fiber electrode using electrochemical polymerization technology. This sensor exhibits both high selectivity and high conductivity. Furthermore, this invention demonstrates high selectivity for methylmalonic acid and can effectively eliminate interference from urea, creatinine, inorganic ions, and other substances in urine.

[0016] 2. Optimize detection conditions and use the open circuit potential method (OCPT) to detect methylmalonic acid solutions of different concentrations, achieving rapid and highly sensitive detection.

[0017] 3. The method is simple to operate, and a single test only takes 50 seconds (relative standard deviation <0.8%, recovery rate 96.42%-100.34%), and is suitable for direct detection in complex urine environments (no complicated pretreatment required). Attached Figure Description

[0018] Figure 1 This is a photograph of the carbon fiber electrode prepared according to the present invention.

[0019] Figure 2 This is a schematic diagram illustrating the fabrication process of the methylmalonic acid electrochemical sensor based on a polyaniline-modified carbon fiber electrode according to the present invention.

[0020] Figure 3This is a SEM image of the polyaniline-modified carbon fiber electrode prepared according to the present invention.

[0021] Figure 4 The graph shows the open-circuit potential response linearity of methylmalonic acid at different concentrations in Example 1, where the concentrations of methylmalonic acid are 5µM, 10µM, 20µM, 40µM, and 50µM.

[0022] Figure 5 The graph shows the open-circuit potential response linearity of methylmalonic acid at different concentrations in Example 2, where the concentrations of methylmalonic acid are 200µM, 250µM, 300µM, 350µM, 400µM, 600µM, and 800µM.

[0023] Figure 6 In Example 3, the interfering substance NH was added. 4+ K + Na + The anti-interference results of 100µM MMA were detected under the conditions of urea (BUN) and creatinine (Cr). Detailed Implementation

[0024] Example 1 (5-50 µM) A method for preparing a polyaniline-modified carbon fiber electrode and its application in detecting methylmalonic acid includes the following steps: (1) The hydrophilic carbon cloth was pretreated by ultrasonic cleaning with ethanol and water and then dried at 50°C. Carbon filaments of approximately 5×10 mm in length were extracted from the cleaned carbon cloth, one end was wrapped with copper foil, and then dried for later use. The resulting carbon fiber electrode is shown in the image below. Figure 1 As shown.

[0025] (2) Deionized water was used as the solvent, and aniline (0.1M) and hydrochloric acid (0.1M) were used as the monomer and electrolyte, respectively. The carbon fiber electrode was immersed in the polymerization solution. Cyclic voltammetry was used, with the carbon fiber electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. Ten cycles of electropolymerization were performed between +0.2 and +1.2 V to form a polyaniline polymer film on the surface of the carbon fiber electrode. The film was then dried at 50 °C for 15 min. A polyaniline-modified carbon fiber electrode was obtained, with the structure shown below. Figure 3 As shown.

[0026] First, a standard curve for the detection of methylmalonic acid was established: a two-electrode system was used, with polyaniline-modified carbon fiber electrodes placed in 20 mL of methylmalonic acid solutions of different concentrations. The potential signal was detected using the open-circuit potential method, thus establishing a standard curve for the detection of methylmalonic acid based on its concentration versus potential signal intensity. Figure 4As shown, the concentration range of tartaric acid is 5-50 µM. The potential signal of the carbon fiber electrode increases with increasing methylmalonic acid concentration, and the linear relationship is I = 0.3435 C. 甲基丙二酸 +199.27, R 2 = 0.9852, detection limit is 0.001 µM, linear range is 5-50 µM. For example... Figure 4 As shown.

[0027] Example 2 (200-800 µM) A method for preparing a polyaniline-modified carbon fiber electrode and its application in detecting methylmalonic acid includes the following steps: (1) The hydrophilic carbon cloth was pretreated by ultrasonic cleaning with ethanol and water and then dried at 50°C. Carbon filaments of approximately 5×10 mm in length were extracted from the cleaned carbon cloth, one end was wrapped with copper foil, and then dried for later use. The resulting carbon fiber electrode is shown in the image below. Figure 1 As shown.

[0028] (2) Deionized water was used as the solvent, and aniline (0.1M) and hydrochloric acid (0.1M) were used as the monomer and electrolyte, respectively. The carbon fiber electrode was immersed in the above polymerization solution. Cyclic voltammetry was used to carry out 10 cycles of electropolymerization between +0.2 and +1.2 V to form a polyaniline polymer film on the surface of the carbon fiber electrode. Then it was dried at 50 °C for 15 min. The polyaniline-modified carbon fiber electrode was obtained, with the structure shown below. Figure 3 As shown.

[0029] First, a standard curve for the detection of methylmalonic acid was established: a two-electrode system was used, with polyaniline-modified carbon fiber electrodes placed in 20 mL of methylmalonic acid solutions of different concentrations. The potential signal was detected using the open-circuit potential method, thus establishing a standard curve for the detection of methylmalonic acid based on its concentration versus potential signal intensity. Figure 5 As shown, the concentration range of tartaric acid is 200-800 µM. The potential signal of the carbon fiber electrode increases with increasing methylmalonic acid concentration, and the linear relationship is I = 0.064 C. 甲基丙二酸 +233.04, R 2 = 0.9997, detection limit is 0.001 µM, linear range is 200-800 µmol / L. For example Figure 5 As shown.

[0030] Example 3 (Interference Resistance) The preparation of a polyaniline-modified carbon fiber electrode and its application in the detection of methylmalonic acid include the following steps: (1) The hydrophilic carbon cloth was pretreated by ultrasonic cleaning with ethanol and water and then dried at 50°C. Carbon filaments of approximately 5×10 mm in length were extracted from the cleaned carbon cloth, one end was wrapped with copper foil, and then dried for later use. The resulting carbon fiber electrode is shown in the image below. Figure 1 As shown.

[0031] (2) Deionized water was used as the solvent, and aniline (0.3M) and hydrochloric acid (0.1M) were used as the monomer and electrolyte, respectively. The carbon fiber electrode was immersed in the above polymerization solution. Cyclic voltammetry was used to carry out 10 cycles of electropolymerization between +0.2 and +1.2 V to form a polyaniline polymer film on the surface of the carbon fiber electrode. Then it was dried at 50 °C for 15 min. The polyaniline-modified carbon fiber electrode was obtained, with the structure shown below. Figure 3 As shown.

[0032] By adding physiologically relevant concentrations of the interfering substance NH 4+ K + Na + The open-circuit potential of 100 µM MMA was measured and recorded under conditions of urea (BUN) and creatinine (Cr) to investigate the anti-interference ability of the prepared PANI / CF sensor. It was found that, compared with the open-circuit potential values ​​when detecting MMA alone, the open-circuit potential value fluctuated only within the range of ≤ 2 mV after the addition of each interfering substance. This indicates that the PANI / CF sensor selectively measures MMA in urine even in the presence of common physiological interferences. The results are as follows... Figure 6 As shown.

[0033] Example 4 (Detection in urine) The preparation of a polyaniline-modified carbon fiber electrode and its application in the detection of methylmalonic acid include the following steps: (1) The hydrophilic carbon cloth was pretreated by ultrasonic cleaning with ethanol and water and then dried at 50°C. Carbon filaments of approximately 5×10 mm in length were extracted from the cleaned carbon cloth, one end was wrapped with copper foil, and then dried for later use. The resulting carbon fiber electrode is shown in the image below. Figure 1 As shown.

[0034] (2) Deionized water was used as the solvent, and aniline (0.3M) and hydrochloric acid (0.1M) were used as the monomer and electrolyte, respectively. The carbon fiber electrode was immersed in the above polymerization solution. Cyclic voltammetry was used to carry out 10 cycles of electropolymerization between +0.2 and +1.2 V to form a polyaniline polymer film on the surface of the carbon fiber electrode. Then it was dried at 50 °C for 15 min to obtain the polyaniline-modified carbon fiber electrode.

[0035] The standard addition method was used to determine the concentration of methylmalonic acid in urine. This method involves filtering the urine sample through a 0.45 µm pore size membrane, diluting the sample with deionized water, and finally adding a target methylmalonic acid of known concentration. Measurements were performed using the open-circuit potential method (OCPT), and the results are shown in Table 1.

[0036] Table 1 The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An application of a potential sensor based on a polyaniline-modified micro two-electrode system for the detection of methylmalonic acid, characterized in that, In the potential sensor based on the polyaniline-modified micro two-electrode system, carbon fiber loaded with polyaniline is used as the working electrode, and methylmalonic acid is detected by the open-circuit potential method.

2. The application according to claim 1, characterized in that, The Ag / AgCl electrode was used as a reference electrode.

3. The application according to claim 1, characterized in that, The preparation of the carbon fiber electrode loaded with polyaniline includes: using a three-electrode system and cyclic voltammetry in an electrolyte solution formed by aniline, hydrochloric acid and deionized water, aniline is oxidized in situ to polyaniline and loaded onto the surface of carbon fibers.

4. The application according to claim 3, characterized in that, In the three-electrode system, carbon fiber serves as the working electrode, Ag / AgCl electrode serves as the reference electrode, and platinum wire electrode serves as the counter electrode.

5. The application according to claim 4, characterized in that, The carbon fibers were ultrasonically cleaned with ethanol and water, respectively.

6. The application according to claim 3, characterized in that, The cyclic voltammetry method scans for no less than 8 cycles within the range of +0.2 V to +1.2 V.

7. The application according to claim 3, characterized in that, In the electrolyte solution, the concentration of aniline is 0.08-0.35M and the concentration of hydrochloric acid is 0.08-0.2M.

8. An electrochemical detection method for methylmalonic acid, characterized in that the steps include... include: In the sample solution to be tested, a potential sensor based on a polyaniline-modified micro two-electrode system was used, with polyaniline-loaded carbon fiber as the working electrode. The open-circuit potential method was used to detect methylmalonic acid in the sample solution to be tested by external or internal standard.

9. The electrochemical detection method according to claim 8, characterized in that, The Ag / AgCl electrode was used as a reference electrode.

10. The electrochemical detection method according to claim 8, characterized in that, The sample liquid to be tested is selected from any one of body fluids, excrement, dilutions of body fluids, or dilutions of excrement.

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