Tryptophan and 5-hydroxyindoleacetic acid detection method based on poly-L-Arg / SPCE electrochemical sensor

Abstract

The invention discloses a tryptophan and 5-hydroxyindoleacetic acid detection method based on a poly-L-Arg / SPCE electrochemical sensor. According to the present invention, the screen printed carbon electrode (SPCE) based on plasma treatment has strong hydrophilicity and strong stability, and is combined with the excellent electro-catalytic performance of the L-arginine polymer to construct the simple, convenient, rapid and high-sensitivity electrochemical sensor; the method can be used for simultaneously and quantitatively detecting the contents of Trp and 5-HIAA in the mixed solution, has higher sensitivity, wider linear range and good precision and accuracy, and provides a new idea for early diagnosis and illness monitoring of mental diseases such as depression.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical detection, and mainly relates to a tryptophan and 5-hydroxyindoleacetic acid detection method based on a poly-L-Arg / SPCE electrochemical sensor. Background Art

[0002] Tryptophan (Trp) and 5-hydroxyindoleacetic acid (5-HIAA), as key components of the tryptophan metabolic pathway, maintain brain function and neuronal regulatory mechanisms. In humans, Trp is metabolized through the kynurenine (Kyn) pathway, the 5-hydroxytryptamine (5-HT) pathway, and the microbiome-associated indole pathway. The primary metabolic pathway for Trp is the Kyn pathway, which primarily occurs in the liver, with over 95% of Trp metabolized through this pathway. Following tryptophan metabolism through the Kyn pathway, Trp and its metabolites are excreted in urine and bile (in feces), with urine being the primary route of excretion. A small amount of tryptophan (<5%) is synthesized into 5-HT via the 5-HT pathway. As the sole precursor for 5-HT synthesis, approximately 90% of Trp synthesis occurs in the distal gastrointestinal tract, with a smaller proportion occurring in the central nervous system. The key enzyme involved in the 5-HT metabolic pathway is tryptophan hydroxylase (TPH). TPH decarboxylates Trp to produce 5-HT. Within the central nervous system, 5-HT is further converted to 5-hydroxyindoleacetaldehyde by monoamine oxidase (MAO) type A. 5-hydroxyindoleacetaldehyde, a precursor of 5-HIAA, is oxidized to 5-HIAA by aldehyde dehydrogenase, which is ultimately excreted in the urine through the kidneys. Urinary Trp and 5-HIAA levels are closely associated with neurological and psychiatric disorders, such as major depressive disorder (MDD), anxiety disorders (AD), and autism spectrum disorder (ASD). Literature has demonstrated that 5-HIAA is the final metabolite of Trp via the 5-HT pathway. Therefore, measuring urinary Trp and 5-HIAA levels is crucial for the early diagnosis and monitoring of neurological and psychiatric disorders.

[0003] As the primary excretion pathway for tryptophan and its metabolites, the normal range of Trp in urine is 6.4–25.0 μmol / L, and the normal range of 5-HIAA is 17.8–58.3 μmol / L. Currently, reported methods for the simultaneous detection of tryptophan and 5-hydroxyindoleacetic acid include ultra-high-performance liquid chromatography to triple quadrupole mass spectrometry (UHPLC-MS / MS), liquid chromatography tandem mass spectrometry (LC-MS / MS), high-performance liquid chromatography-electrochemical detection (HPLC-ECD), high-performance liquid chromatography (HPLC), high-performance liquid chromatography with fluorescence detection (HPLC-FLD), and fluorescence colorimetry. Ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS / MS) and liquid chromatography-mass spectrometry (LC-MS / MS) have the advantages of high sensitivity and selectivity for the detection of Trp and 5-HIAA. However, their high instrument cost and high operator requirements limit their application in primary care settings. HPLC, HPLC-ECD, and HPLC-FLD offer advantages such as high sensitivity, good reproducibility, and a wide linear range. Furthermore, the electrochemical detector in HPLC-ECD is less expensive than other highly sensitive detectors, reducing experimental costs. However, high-performance liquid chromatography (HPLC) detection requires high sample pretreatment requirements, and HPLC is highly technically dependent, requiring specialized laboratory personnel for operation, making it difficult to widely use in routine laboratories. Furthermore, fluorescence interference during HPLC-FLD detection requires verification of results, making the detection process relatively cumbersome. Fluorescence colorimetry is relatively simple and sensitive, but suffers from poor specificity, slow reaction times, and numerous interfering factors, significantly limiting its clinical application. Therefore, a new method for the simultaneous detection of Trp and 5-HIAA that is simple, rapid, low-cost, sensitive, and specific is urgently needed.

[0004] Plasma treatment is an emerging electrode treatment method, with only a few publications addressing its application. Plasma, an ionized gas, primarily cleans the surface of screen-printed carbon electrodes (SPCEs). During the SPCE fabrication process, some organic solvents, adhesives, and other impurities, such as air pollutants and fingerprints, remain on the surface. Plasma typically contains reactive species such as ions, electrons, and free radicals. These highly active particles interact with the electrode surface and remove impurities. Furthermore, plasma cleaning creates numerous chemically active sites on the SPCE surface, while also improving its hydrophilicity and biocompatibility. Polyarginine-modified electrode surfaces exhibit excellent electrocatalytic properties, promoting the electroactivity of analytes and enhancing electrode stability. An electrochemical method for the simultaneous detection of Trp and 5-HIAA was developed based on plasma treatment and poly-L-arginine (L-Arg) modification on SPCEs. The developed method boasts simple operation, high sensitivity, rapid response, minimal sample consumption, and low cost. Summary of the Invention

[0005] The present invention is dedicated to providing a simple, rapid and highly sensitive electrochemical detection method for Trp and 5-HIAA.

[0006] The principle of the electrochemical method for detecting Trp and 5-HIAA in the present invention is as follows: When a certain voltage is applied to the working area of ​​the prepared poly-L-Arg / SPCE electrode, the pyrrole ring in the Trp chemical structure undergoes two-electron oxidation to easily form -C=N-, thereby detecting its electrochemical behavior. The phenolic hydroxyl group on the benzene ring in the 5-HIAA chemical structure is easily oxidized on the electrode surface, converting it to a carbonyl group. Simultaneously, the pyrrole ring also undergoes oxidation to form -C=N-, resulting in electron transfer. Therefore, the electrochemical technique can be used to simultaneously and quantitatively detect Trp and 5-HIAA.

[0007] 1. The technical solutions of the present invention are as follows:

[0008] An electrochemical method for determining tryptophan and 5-hydroxyindoleacetic acid based on a poly-L-arginine modified electrode, characterized by comprising the following specific steps:

[0009] 1) Pretreatment of screen-printed carbon electrodes:

[0010] The SPCE surface was treated with a quartz plasma machine at 20.0% power to remove surface impurities and increase the chemical active sites and hydrophilicity of the electrode surface, and then sealed and stored at room temperature.

[0011] 2) Preparation of modified SPCE electrode:

[0012] 8.70 mg of L-arginine powder was weighed and added to 10.0 mL of ultrapure water to prepare a 5.00 mmol / L L-Arg standard solution. The 5.00 mmol / L L-Arg standard solution was diluted with 0.1 mol / L phosphate buffer saline (PBS) (pH 7.0) to prepare a 0.500 mmol / L L-arginine solution. 20.0 μL of the 0.500 mmol / L L-Arg solution was dropwise added to the plasma-treated SPCE working electrode surface. Cyclic voltammetry (CV) was performed over a -0.8 V to 1.0 V range for 30 cycles to electrochemically polymerize the L-arginine in the solution to form an L-arginine polymer film. The SPCE surface was then rinsed with ultrapure water with a resistivity of 18.2 MΩ·cm and dried at room temperature. This resulted in a plasma-cleaned SPCE modified with poly-L-Arg, designated poly-L-Arg / SPCE. To compare the performance of SPCE electrodes modified with different materials, poly-L-Arg modified SPCEs were prepared according to the above steps. All prepared modified electrodes were sealed and stored at room temperature.

[0013] 3) Electrochemical detection:

[0014] A 20.0 μL working solution was dripped onto the poly-L-Arg / SPCE working area. The electrode terminals were connected to an electrochemical workstation to form a closed circuit. Differential pulse voltammetry (DPV) was used for detection. DPV parameters were set as follows: operating voltage of -0.2 V to 0.6 V, pulse period of 0.5 s, pulse width of 0.2 s, and pulse amplitude of 50.0 mV. All electrochemical measurements were performed at room temperature (23.0°C ± 2.0°C).

[0015] Preferably, the concentration of the L-Arg solution is 0.100 to 2.50 mmol / L, the cleaning power of the quartz plasma machine is 0.0 to 25.0%, the cleaning time of the quartz plasma machine is 0.0 to 60.0 s, the gas cleaning flow rate of the quartz plasma machine is 0.0 to 80.0 mL / min, the concentration of the PBS buffer solution is 0.1 to 0.5 mmol / L, and the pH value is 5.0 to 8.0. The differential pulse voltammetry parameters are set as follows: low potential -0.2 V, high potential 0.6 V, pulse amplitude 50.0 mV, pulse width 0.2 s, and pulse period 0.5 s.

[0016] 2. The beneficial effects of the electrochemical detection method of Trp and 5-HIAA described in the present invention are as follows:

[0017] 1) The electrochemical detection method of the present invention uses a screen-printed carbon electrode in conjunction with an electrochemical workstation to quantitatively determine Trp and 5-HIAA. The detection instrument is simple to operate, has a fast response speed, and is low in cost.

[0018] 2) The screen-printed carbon electrodes used in the electrochemical detection method of the present invention are inexpensive to manufacture, with a simple and rapid production process, enabling mass production. The SPCE electrodes are disposable, reducing the time required for polishing and grinding traditional column electrodes while also avoiding cross-contamination issues associated with repeated electrode use.

[0019] 3) The electrochemical detection method described in the present invention utilizes plasma pretreatment to remove residual organic solvents and impurities on the surface of SPCE, providing more chemically active sites on the electrode surface and increasing the surface hydrophilicity of SPCE. Furthermore, the electrocatalytic activity and stability of the poly-L-arginine membrane are incorporated to construct a simple, rapid, and highly sensitive electrochemical detection method for the simultaneous quantitative detection of Trp and 5-HIAA concentrations.

[0020] 4) The electrochemical detection method described in the present invention is expected to provide a new approach for the early diagnosis and disease monitoring of mental illnesses such as depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the electrode modification process and detection principle of the method of the present invention.

[0022] Figure 2 This is an electrical impedance characterization diagram of the electrode pretreatment and modification process of the present invention.

[0023] In the figure, curve a is the electrical impedance spectrum detected by SPCE, curve b is the electrical impedance spectrum detected by poly-L-Arg modified SPCE, curve c is the electrical impedance spectrum detected by plasma treated SPCE, and curve d is the electrical impedance spectrum detected by poly-L-Arg / SPCE.

[0024] Figure 3 This is the electrochemical response curve of measuring Trp and 5-HIAA using the method constructed by the present invention.

[0025] Figure A shows the CV curves for the determination of Trp and 5-HIAA using the developed method, and Figure B shows the DPV curves for the determination of Trp and 5-HIAA using the developed method. In Figures A and B, solid line a is the DPV curve for the determination in 0.3 mol / L PBS (pH = 6.5), solid line b is the DPV curve for the determination in 200.0 μmol / L 5-HIAA, solid line c is the DPV curve for the determination in 120.0 μmol / L Trp, and solid line d is the DPV curve for the determination in a mixed standard solution of 120.0 μmol / L Trp and 200.0 μmol / L 5-HIAA.

[0026] Figure 4 These are the DPV test results of different concentrations of Trp and 5-HIAA in the present invention.

[0027] Figure A is the DPV curve of different concentrations of Trp and 5-HIAA, Figure B is the standard curve of electrochemical detection of 5-HIAA, and Figure C is the standard curve of electrochemical detection of Trp.

[0028] Figure 5 The stability results of Trp and 5-HIAA detected by poly-L-Arg / SPCE in the present invention are shown. DETAILED DESCRIPTION

[0029] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0030] The electrochemical workstation of the present invention only takes CHI852C electrochemical workstation as an example, which was purchased from Shanghai Chenhua Instrument Co., Ltd., Trp was purchased from Shanghai Adamas Reagent Co., Ltd., 5-HIAA was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., and L-arginine was purchased from Shanghai Titan Technology Co., Ltd.

[0031] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0032] Example 1: Preparation of the electrochemical sensor of the present invention

[0033] First, the SPCE was pretreated using a quartz plasma cleaner at 20.0% power to remove surface impurities and increase the chemically active sites and hydrophilicity of the electrode surface. The SPCE was then sealed and stored clean at room temperature. The preparation of the L-arginine polymer film of the modified electrode was slightly modified based on reference

[115] . A 5.00 mmol / L L-arginine standard solution was diluted with a 0.1 mol / L PBS solution (pH = 7.0) to prepare a 0.500 mmol / L L-arginine solution. 20.0 μL of the 0.500 mmol / L L-Arg solution was added dropwise to the plasma-treated SPCE working electrode surface. The CV method was used to scan 30 cycles in the range of -0.8 V to 1.0 V to electrochemically polymerize the L-arginine in the solution to form an L-arginine polymer film. The SPCE surface was then rinsed with ultrapure water with a resistivity of 18.2 MΩ·cm and dried naturally at room temperature to prepare a plasma-treated SPCE modified with poly-L-Arg, which was designated as poly-L-Arg / SPCE. In order to compare the performance of SPCE electrodes modified with different modification materials, poly-L-Arg modified SPCE was prepared according to the above steps. All prepared modified electrodes were sealed and stored at room temperature. SPCE pretreatment, modification process and electrochemical detection principle of Trp and 5-HIAA are as follows. Figure 1 shown.

[0034] Example 2: Electrochemical Characterization of the Electrochemical Sensor of the Present Invention

[0035] In this example, electrochemical impedance spectroscopy (EIS) was used to investigate the effects of different materials on the surface of electrodes treated or modified with different materials on the [Fe(CN)6] 3- / 4- Interface properties in solution. As a characterization technique, EIS curve is divided into two parts: semicircle and straight line. The diameter of the semicircle part of the EIS curve represents the charge transfer resistance (R ct ). If the diameter of the semicircular part of the curve is larger, it means the charge transfer rate is slower, R ct The larger the value, the smaller the R ct The straight line portion of the EIS curve represents the charge diffusion resistance. If the slope of the straight line portion is larger, it means that the R ct The smaller the value, the lower the R ct The larger the value, the higher the value. 3- / 4- The solution was used as the EIS reaction detection solution, and the characterization results were as follows Figure 2R of poly-L-Arg modified SPCE (curve b) and plasma treated SPCE (curve c) ct The values ​​are all smaller than those of bare SPCE (curve a), indicating that both poly-L-Arg modified and plasma treated electrodes have better conductivity. Compared with poly-L-Arg modified SPCE and plasma treated SPCE, the R ct The value further decreases, and the EIS spectrum of curve d is close to a straight line with a greater slope than curve c, indicating that the combined application of plasma pretreatment and poly-L-Arg modification on the SPCE surface significantly increases the chemically active sites on the modified electrode surface and significantly improves the charge transfer rate. The results show that the combined application of plasma pretreatment and L-arginine polymer on SPCE effectively improves the hydrophilicity of the electrode surface, increases the specific surface area of ​​the modified electrode, and also enhances the electrocatalytic performance and stability of the electrode surface.

[0036] Example 3: Electrochemical Detection Method of Trp and 5-HIAA

[0037] A 20.0 μL working solution was placed in the poly-L-Arg / SPCE working area. The electrode terminals were connected to an electrochemical workstation to form a closed circuit. Differential pulse voltammetry (DPV) was used for detection. The DPV parameters were: operating voltage of -0.2 V to 0.6 V, pulse period of 0.5 s, pulse width of 0.2 s, and pulse amplitude of 50.0 mV. All electrochemical measurements were performed at room temperature (23.0°C ± 2.0°C).

[0038] Example 4: Electrochemical Behavior of Trp and 5-HIAA

[0039] In this example, CV was used to study the electrochemical behavior of Trp and 5-HIAA in the working area of ​​poly-L-Arg / SPCE. The supporting electrolyte was 0.3 mol / L PBS buffer (pH = 6.5) and the scan rate was 50.0 mV / s. The CV curves of 0.3 mol / L PBS buffer solution (pH = 6.5), 200.0 μmol / L 5-HIAA solution, 120.0 μmol / L Trp solution, and a mixed solution of 200.0 μmol / L 5-HIAA and 120.0 μmol / L Trp on the modified electrode are shown in Figure 2. Figure 3As shown in Figure A, the PBS buffer solution (curve a) exhibited no significant redox signal at the modified electrode. A characteristic oxidation peak was observed for 5-HIAA (curve b) at 0.12 V, and a significant oxidation peak current was observed for Trp (curve c) at 0.47 V, indicating that 5-HIAA and Trp can generate electrochemical signals on the poly-L-Arg / SPCE. Curves b and c represent the electrochemical signals of 5-HIAA and Trp, respectively, individually at the modified electrode. When the two were mixed (curve d), the peak potentials of each were essentially identical to those observed individually, indicating that the oxidation peak potentials of 5-HIAA and Trp did not interfere with each other. Therefore, 5-HIAA and Trp can be quantitatively analyzed at their respective peak potentials.

[0040] In this example, DPV was used to further investigate the electrochemical behavior of 5-HIAA and Trp in the poly-L-Arg / SPCE working area. Figure 3 As shown in Figure B. The PBS buffer solution (curve a) has no significant electrochemical response on poly-L-Arg / SPCE. 5-HIAA (curve b) shows a characteristic oxidation peak at 0.08V, and Trp (curve c) produces a clear oxidation peak at 0.42V, indicating that 5-HIAA and Trp are electrochemically active on the modified SPCE and can be analyzed by electrochemical technology. The DPV oxidation peaks of the two after mixing (curve d) are basically consistent with the DPV peak potentials when detected separately (curves b and c). The results show that the oxidation peak potentials of the two after mixing do not interfere with each other. The DPV curve results are consistent with the above-mentioned CV test results. Considering the detection sensitivity issue, this study subsequently used DPV technology to conduct quantitative analysis of 5-HIAA and Trp.

[0041] Example 5: Standard curve of Trp and 5-HIAA

[0042] This example is about the linear relationship between the electrochemical detection method for quantitative oxidation peak current I and the concentration c of Trp and 5-HIAA. The effect of a mixed solution of Trp and 5-HIAA at a certain concentration ratio (taking 3:5 as an example) on the electrochemical signal was investigated. 1.50-150.0 μmol / L Trp solution and 2.50-250.0 μmol / L 5-HIAA solution were mixed at a concentration ratio of 3:5 and then DPV quantitative detection was performed. The results are shown in FIG. Figure 4 When the Trp concentration is between 1.50 and 150.0 μmol / L, 5-HIAA has a good linear relationship with its oxidation peak current value in the concentration range of 2.50 to 250.0 μmol / L, as shown in Figure 2. Figure 4 B. The linear regression equation is I=0.0199c+0.1414(R 2=0.9959), LOD was 0.30 μmol / L (S / N=3), and LOQ was 1.01 μmol / L. When the concentration of 5-HIAA was between 2.50 and 250.0 μmol / L, there was a good linear relationship between the Trp and its oxidation peak current value in the concentration range of 1.50 to 150.0 μmol / L. The results are as follows Figure 4 C. The linear regression equation is I=0.0258c+0.0343(R 2 =0.9967), LOD was 0.18 μmol / L (S / N=3), and LOQ was 0.59 μmol / L.

[0043] Example 6: Precision of electrochemical detection of Trp and 5-HIAA

[0044] This example investigates the precision of the electrochemical detection method of the present invention for determining the concentrations of Trp and 5-HIAA. Low, medium and high concentrations of Trp and 5-HIAA were added to the urine matrix to prepare a mixed solution. The low, medium and high concentrations of Trp in the mixed solution were 3.00 μmol / L, 60.00 μmol / L and 120.0 μmol / L, respectively; the low, medium and high concentrations of 5-HIAA were 5.00 μmol / L, 100.0 μmol / L and 200.0 μmol / L, respectively. Under optimal conditions, a reproducibility experiment was performed on the mixed solution of Trp and 5-HIAA by the DPV method. The results were measured in parallel 5 times a day and continuously for 5 days. The intra-day and inter-day RSDs of the low, medium and high concentrations of Trp and 5-HIAA were calculated, respectively. The results are shown in Table 1. The intra-day and inter-day RSDs for Trp in the mixed solution were lower than 3.51% and 6.55%, respectively, and the intra-day and inter-day RSDs for 5-HIAA were lower than 7.08% and 5.10%, respectively. These results demonstrate that the electrochemical sensor has good reproducibility for simultaneous detection of Trp and 5-HIAA in urine matrix.

[0045] Table 1 Precision test (n=5)

[0046]

[0047] Example 7: Recovery of Trp and 5-HIAA by electrochemical detection

[0048] This embodiment uses a recovery experiment to evaluate the accuracy of the experimental method. Low, medium and high concentrations of Trp and 5-HIAA were added to the urine matrix to prepare a mixed solution. The low, medium and high concentrations of Trp in the mixed solution were 3.00μmol / L, 60.00μmol / L and 120.0μmol / L, respectively; the low, medium and high concentrations of 5-HIAA were 5.00μmol / L, 100.0μmol / L and 200.0μmol / L, respectively. Under optimal conditions, DPV technology was used to detect Trp and 5-HIAA in low, medium and high concentration mixed solutions on poly-L-Arg / SPCE in parallel 5 times, and a recovery experiment was performed. The relative recovery was calculated using the following formula, and the results are shown in Table 2.

[0049]

[0050] The results showed that the average recovery of Trp in the mixed solution was 95.2%-106.6%, with an RSD lower than 5.52%; the average recovery of 5-HIAA was 91.0%-102.2%, with an RSD lower than 7.68%, indicating that the electrochemical sensor can be used to simultaneously detect the concentrations of Trp and 5-HIAA in urine matrix with good accuracy.

[0051] Table 2 Recovery test results (n=5)

[0052]

[0053]

[0054] Example 8:

[0055] To verify the practical application potential of the electrochemical sensor constructed in this study in biological samples, this study systematically evaluated the effect of ascorbic acid, a common coexisting component in urine matrix, on the determination of Trp and 5-HIAA, referring to the EP7-A interference test standard issued by the Clinical and Laboratory Standards Institute (CLSI). C is defined as the concentration of 5-HIAA or Trp in the mixed solution before the addition of interfering substances, and X T It is defined as the concentration of Trp or 5-HIAA in the mixed solution after adding the interfering substance. The interference value is expressed as (X T -X C). The 95% confidence interval calculation formula was used for calculation. If the interference value detected by this method is less than 1.96S, it means that the interfering substance has no significant interference with the detection of Trp or 5-HIAA, which is represented by N; if the interference value detected is greater than 1.96S, it means that it has significant interference with the detection of Trp or 5-HIAA, which is represented by I. 0.0-1000 μmol / L ascorbic acid solution was added to a mixed solution containing 15.0 μmol / L Trp and 25.0 μmol / L 5-HIAA, and the concentrations of Trp and 5-HIAA in the mixed solution before and after the addition of different interfering substances were detected. The results are shown in Table 3. When the ascorbic acid concentration does not exceed 50.0 μmol / L, it will not interfere with the detection of Trp in the mixed solution by this experimental method; when the ascorbic acid concentration is lower than 600.0 μmol / L, it will not interfere with the detection of 5-HIAA in the mixed solution by this experimental method. The ascorbic acid concentration in normal human urine over a 24-hour period is generally below 151.5 μmol / L. Physiologically, consuming excessive amounts of vitamin C-rich foods or taking vitamin C supplements can lead to elevated urinary ascorbic acid levels. Pathologically, urinary ascorbic acid levels can also be elevated in patients with renal diseases (such as nephrotic syndrome) or endocrine disorders (such as hyperthyroidism). Therefore, these factors should be eliminated during measurement to maintain a stable urinary ascorbic acid concentration. Interference testing results showed that the experimental method developed in this study did not interfere with the simultaneous detection of Trp and 5-HIAA in normal human urine when the ascorbic acid concentration was controlled to no more than 50.0 μmol / L. In summary, under normal conditions (when the ascorbic acid concentration was controlled to no more than 50.0 μmol / L), ascorbic acid did not significantly interfere with the detection of urinary Trp and 5-HIAA concentrations, demonstrating that the constructed electrochemical sensor has a certain degree of anti-interference ability for the determination of urinary Trp and 5-HIAA.

[0056] Table 3 Interference test results

[0057]

[0058]

[0059] Example 9:

[0060] In order to investigate the stability of the constructed sensor, a batch of SPCE modified with poly-L-Arg after plasma treatment was prepared in the same experimental environment and under the same experimental conditions. The prepared sensors were sealed and stored at room temperature. The poly-L-Arg / SPCE stored for different times was used to detect the same concentration of Trp and 5-HIAA standard mixed solution, and the stability of the electrochemical sensor was evaluated by the change of the detection peak current value of the two over time. Three pieces of poly-L-Arg / SPCE were taken every 4 days, and the DPV technology was used to measure the 120.0μmol / LTrp and 200.0μmol / L 5-HIAA standard mixed solution. The test results were compared with the results on day 0. The results are as follows: Figure 5 As shown in the figure. After 12 days of storage, the oxidation peak current for Trp detection decreased by 9.01% compared to day 0, with a coefficient of variation of 1.38%. The oxidation peak current for 5-HIAA detection decreased by 4.27% compared to day 0, with a coefficient of variation of 1.46%. After 16 days of storage, the oxidation peak current for Trp detection decreased by 13.32% compared to day 0, with a coefficient of variation of 1.11%. The oxidation peak current for 5-HIAA detection decreased by 6.78% compared to day 0, with a coefficient of variation of 0.80%. Although the electrochemical sensor did not show a significant decrease in 5-HIAA detection after 16 days of storage compared to day 0, the oxidation peak current for Trp detection decreased by more than 10%. These results demonstrate that the electrochemical sensor constructed in this study exhibits good stability, and 12 days of storage has little effect on the electrochemical detection peak current of a standard mixed solution of Trp and 5-HIAA, demonstrating its high stability and excellent practicality.

Claims

1. A method for detecting tryptophan and 5-hydroxyindoleacetic acid based on a poly-L-Arg / SPCE electrochemical sensor, characterized in that: The specific steps include: 1) Pretreatment of screen-printed carbon electrodes: The surface of the screen-printed carbon electrode (SPCE) was treated with a quartz plasma machine at 20.0% power to remove surface impurities and increase the chemically active sites and hydrophilicity of the electrode surface. The SPCE was then sealed and stored at room temperature. 2) Preparation of modified SPCE electrode: Weigh 8.70 mg of L-arginine (L-Arg) powder and add 10.0 mL of ultrapure water to prepare a 5.00 mmol / L L-Arg standard solution. Dilute the 5.00 mmol / L L-Arg standard solution with 0.1 mol / L phosphate buffer (PBS) (pH = 7.0) to prepare a 0.500 mmol / L L-Arg solution. 20.0 μL of 0.500 mmol / L L-Arg solution was added dropwise to the plasma-treated SPCE working electrode surface. Cyclic voltammetry (CV) was performed over a range of -0.8 V to 1.0 V for 30 cycles to electrochemically polymerize the L-Arg in the solution to form a polymer film. The SPCE surface was then rinsed with ultrapure water with a resistivity of 18.2 MΩ·cm and naturally dried at room temperature to prepare plasma-treated SPCE modified with poly-L-Arg, designated poly-L-Arg / SPCE. To compare the performance of SPCE modified with different electrode modification materials, poly-L-Arg-modified SPCEs were prepared according to the above steps. All prepared modified electrodes were sealed and stored at room temperature. 3) Electrochemical detection: A total of 20.0 μL of the working solution was dripped onto the working area of ​​the poly-L-Arg / SPCE modified electrode. The end of the electrode was connected to an electrochemical workstation to form a closed circuit. Differential pulse voltammetry (DPV) was used for detection. The DPV detection parameters were as follows: operating voltage of -0.2 V to 0.6 V, pulse period of 0.5 s, pulse width of 0.2 s, and pulse amplitude of 50.0 mV. All electrochemical detection processes were carried out at room temperature (23.0°C ± 2.0°C).

2. The pretreatment method according to claim 1, characterized in that The cleaning power of the quartz plasma machine in step 1 is 0.0-25.0%.

3. The pretreatment method according to claim 1, characterized in that The quartz plasma machine cleaning time in step 1 is 0.0 to 60.0 seconds.

4. The pretreatment method according to claim 1, characterized in that The quartz plasma machine gas cleaning flow rate in step 1 is 0.0 to 80.0 mL / min.

5. The electrode modification method according to claim 1, characterized in that The concentration of the L-Arg solution in step 2 is 0.100-2.50 mmol / L, and the solution added to the polymerization system is 0.1 mol / L PBS solution (pH=7.0).

6. The electrochemical detection method according to claim 1, characterized in that: The concentration ratio of tryptophan to 5-hydroxyindoleacetic acid in the working solution of step 3 is 3:5, and the detection system is a 0.1-0.5 mol / L PBS buffer solution with a pH value of 6.5-8.

0.

7. The electrochemical detection method according to claim 1, characterized in that: The differential pulse voltammetry parameters in step 3 are set as follows: low potential -0.2 V to high potential 0.6 V, pulse amplitude 50.0 mV, pulse width 0.2 s, and pulse period 0.5 s.

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