A method for detecting histamine by using an enzyme-free electrochemical sensor

Abstract

This invention relates to the field of histamine detection, and more particularly to a method for histamine detection using an enzyme-free electrochemical sensor. The method includes a catalytic electrode composed of one-dimensional CuO nanowires. The catalytic electrode, with an applied voltage of 0.55V, is placed in a detection solution at pH 13. The histamine concentration in the detection solution is determined by changes in current. This invention uses a liquid-phase oxidation method and a heat treatment calcination method to generate a one-dimensional CuO nanowire array on the Cu-foam surface. This array is then used as an electrocatalytic electrode for detecting histamine content in fermented mandarin fish. Under optimal conditions, the one-dimensional CuO nanowire array exhibits ultrasensitive sensing. Its application in detecting histamine content in fermented mandarin fish yielded accurate and effective results, demonstrating that this enzyme-free electrochemical histamine sensor is highly superior in detecting histamine content in fermented aquatic products, with low material cost and convenient operation.

Description

Technical Field

[0001] This invention relates to the field of histamine detection, and more particularly to a method for histamine detection using an enzyme-free electrochemical sensor. Background Technology

[0002] Pickled aquatic products are quite common, such as stinky mandarin fish. However, during the pickling process, some proteins in aquatic products are broken down into free histidine. Histidine then undergoes a decarboxylation reaction to produce histamine, a colorless, odorless, and non-volatile biogenic amine. Histamine is a toxic biogenic amine. Consuming fermented foods with high histamine content can cause acute poisoning symptoms or chronic diseases. Therefore, histamine poisoning is a major safety issue associated with fermented foods.

[0003] Currently available methods for histamine detection include spectrophotometry, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), gas chromatography (GC), and capillary electrophoresis. However, spectrophotometry has low sensitivity; HPLC requires expensive instruments and consumables and involves cumbersome procedures; TLC has high requirements for sample volume and thin-layer plates, which can easily lead to chromatographic quality problems. Electrochemical sensing, which has emerged in the last two decades, involves the interaction between the target substance and a bioactive substance or a non-polar modified material with molecular recognition capabilities to generate a sensing signal, which is then converted into an electrical signal by a signal converter. This method has the advantages of simplicity, accuracy, low instrument cost, and easy miniaturization, enabling rapid analysis. Currently, this method is relatively rarely used for histamine detection, mainly because of its high detection voltage and low sensitivity. These defects pose potential obstacles to the electrochemical detection of histamine content in food. Moreover, precious metals are currently mainly used as detection materials for electrochemical sensors, which are prone to leakage and poisoning, and are also costly. Summary of the Invention

[0004] The purpose of this invention is to solve the following problems existing in the prior art: the detection voltage required for detecting histamine by electrochemical sensing is high and the sensitivity is low. These defects become potential obstacles to the electrochemical detection of histamine content in food. Moreover, precious metals are currently mainly used as detection materials for electrochemical sensors. Precious metal materials are prone to leakage and poisoning and are also costly.

[0005] To address the problems existing in the prior art, the present invention provides a method for histamine detection using an enzyme-free electrochemical sensor, comprising a catalytic electrode composed of one-dimensional CuO nanowires, wherein the catalytic electrode with an applied voltage of 0.55 V is placed in a detection solution at pH 13, and the histamine concentration in the detection solution is detected by the change in current.

[0006] Preferably, the detection liquid is kept stirred and mixed when energized.

[0007] Preferably, the catalytic electrode composed of the one-dimensional CuO nanowires is prepared as follows:

[0008] Add 40 mL of Cu-foam Soak in the mixed solution for 20 minutes to generate A linear array was heat-treated in a hot N2 atmosphere for 2 hours to obtain a one-dimensional CuO nanowire (CuO / Cu-foam) array electrode.

[0009] Preferably, the The concentration of NaOH in the mixed solution is 5 mol / L and the concentration of (NH4)2S2O8 is 0.25 mol / L.

[0010] Preferably, the heat The ambient temperature is 200 ℃.

[0011] Preferably, the Cu-foam is placed in the Before mixing with the solution, clean the surface to remove impurities.

[0012] Compared with related technologies, the histamine detection method using an enzyme-free electrochemical sensor provided by this invention has the following advantages:

[0013] This invention utilizes liquid-phase oxidation and thermal calcination to generate a one-dimensional CuO nanowire array on the Cu-foam surface. This array is then used as an electrocatalytic electrode for detecting histamine content in fermented mandarin fish. Under optimal conditions, the one-dimensional CuO nanowire array exhibits ultrasensitive sensing. When used to detect histamine content in fermented mandarin fish, accurate and effective results were obtained. This demonstrates that the enzyme-free electrochemical histamine sensor has significant advantages in detecting histamine content in fermented aquatic products, with low material cost and convenient operation. Attached Figure Description

[0014] Figure 1 For the present invention Actual product image;

[0015] Figure 2 For the present invention XRD pattern;

[0016] Figure 3 For the present invention SEM image;

[0017] Figure 4 This is a CV curve of Cu-foam and CuO / Cu-foam in the presence and absence of histamine in the present invention;

[0018] Figure 5 The CV curves and square root-current plots of CuO / Cu-foam at different scan rates are shown for this invention.

[0019] Figure 6The diagram shows the It response curves and the linear relationship between current and histamine concentration for Cu-foam and CuO / Cu-foam at different histamine concentrations according to the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0022] A method for histamine detection using an enzyme-free electrochemical sensor, taking pickled mandarin fish as an example:

[0023] Preparation of stinky mandarin fish samples: Select fresh stinky mandarin fish, remove the scales, gills and internal organs, wash them clean with water, add 3% salt, 0.6% Sichuan pepper, and 0.5% chili powder by weight of the fish meat, press them with a stone at twice the weight of the fish meat, and ferment at 10℃ for 8 days to obtain fully fermented stinky mandarin fish samples.

[0024] Sample extraction: The masses of four types of stinky mandarin fish samples (A, B, C, and D) were weighed as 3.0373 g, 2.9895 g, 2.9888 g, and 3.0384 g, respectively, and the volume was adjusted to 15 mL. The samples were then extracted using a filter membrane for 30 min each.

[0025] Sample testing: Under the conditions of optimal voltage 0.55 V and optimal pH value (pH=13), CuO modified electrode was used to inject 200 μL of extract into each sample three times under magnetic stirring, so as to obtain the histamine concentration, concentration standard deviation and histamine content in fish meat for each sample.

[0026] Recovery determination: The recovery rate of CuO-modified electrode was determined under magnetic stirring with an optimal voltage of 0.55 V and optimal pH value (pH=13) containing a 10 μM standard sample concentration.

[0027] Fabrication of one-dimensional CuO nanowire arrays

[0028] The Cu-foam (copper foam) was ultrasonically cleaned for 10 min at 150 W with 40 mL of acetone (analytical grade, 99.5% purity) and 80 mL of hydrochloric acid (analytical grade, 2 mol / L) for 150 W to remove surface impurities. After cleaning, it was placed in 40 mL of NaOH (5 mol / L) and... Soak in the mixed solution for 20 minutes to generate The linear array was heat-treated at 200 ℃ in a N2 atmosphere for 2 h to obtain a one-dimensional CuO nanowire (CuO / Cu-foam) array electrode.

[0029] The crystallographic properties, morphology and elemental composition of one-dimensional CuO nanowire arrays were characterized by XRD, SEM and EXD.

[0030] X-ray diffraction (XRD: D8 ADVANCE, Bruke) characterization conditions: Cu target material, voltage 40 kV, current 40 mA, step size 0.02 degrees, diffraction angle range 10-80 degrees;

[0031] Characterization conditions for cold field emission scanning electron microscopy (FE-SEM: Gemini SEM500, Zeiss): accelerating voltage 1 kV, working distance 8.3 mm, magnification 1000x;

[0032] X-ray scattering energy dispersive spectroscopy (EDX: OCTANE PLUS AMETEK) characterization conditions: accelerating voltage 20 kV, working distance 5.0 mm, magnification 1000x.

[0033] Electrode material performance testing

[0034] A three-electrode system was adopted, with Cu-foam-based CuO as the working electrode, saturated Ag / AgCl as the reference electrode, and Pt sheet as the counter electrode.

[0035] (1) Study on the presence or absence of histamine catalytic activity

[0036] The current changes of Cu-foam and CuO electrodes at histamine concentrations of 0 and 200 μM were investigated using colorimetry (CV). The electrolyte was 30 mL of 0.1 M NaOH solution, and the scan rate was 20 mV / s.

[0037] Within the voltage range of -0.2 to 0.7 V, the CV curves of Cu-foam and CuO electrode materials were studied under conditions with and without histamine solution. The histamine solution used was 200 mM and the electrolyte solution was 30 mL of 0.1 M NaOH solution. The scan rate was set to 20 mV / s.

[0038] Within a voltage range of -0.2 to 0.7 V, the CV curves of two electrode materials, Cu-foam and CuO, were studied in 30 mL of 0.1 M NaOH electrolyte solution containing 30 μL of 200 mM histamine solution. The scan rates were set to 0.005 mV / s, 0.01 mV / s, 0.02 mV / s, 0.03 mV / s, 0.05 mV / s, 0.08 mV / s, and 0.1 mV / s, respectively.

[0039] (2) Determination of the It curve (chronoamperometry)

[0040] Under optimal experimental conditions, the current-time (It) response curve of the CuO-modified electrode in histamine solution was determined by chronoamperometry, with histamine concentrations of 0.5 μM, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 500 μM, and the electrolyte being 30 mL of 0.1 M NaOH solution.

[0041] Under optimal voltage (0.55 V) and optimal pH (pH=13), the It curves of Cu-foam and CuO modified electrodes were measured in a 0.1 M NaOH electrolyte solution containing histamine under magnetic stirring. The histamine concentrations used were 0.5 μM, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 500 μM, respectively. The sensitivity, linear range, response time, and detection limit of the electrochemical sensor were thus determined.

[0042] (3) Sample testing and recovery determination

[0043] Under optimal conditions, 200 μL of extract (fermented mandarin fish sample) was added to the electrolyte, and the recovery rate was determined using 10 μM standard solution.

[0044] Material structure and morphology characterization

[0045] Made of reddish-brown Cu as a base ( Figure 1 a) Introducing oxidants (NH4)S2O8 and NaOH, blue-green Cu(OH)2 / Cu-foam is generated via liquid-phase oxidation. Figure 1 b), and then after heat treatment in a nitrogen atmosphere, a black one-dimensional CuO nanowire array was synthesized ( Figure 1 c) The successful preparation of the CuO catalytic electrode can be predicted from the color change of the actual object.

[0046] XRD analysis

[0047] From XRD patterns ( Figure 2As can be seen, Cu-foam exhibits three distinct diffraction peaks, corresponding to the (111), (200), and (220) crystal planes, respectively. After liquid-phase oxidation, the Cu-foam surface forms Cu(OH)2 / Cu-foam (…). Figure 2 This is attributed to the appearance of diffraction peaks on the (020), (021), (002), (111), and (130) crystal planes in the Cu(OH)2 phase. Further observation shows that the diffraction peaks of Cu-foam still exist, indicating that the Cu-foam phase retains its main framework structure after liquid-phase oxidation. After heat treatment, ( Figure 2 The diffraction peaks of Cu(OH)2 disappeared, and at the same time, diffraction signals of CuO phase (-111) and (111) crystal planes appeared. This indicates that heat treatment caused Cu(OH)2 phase to be completely transformed into CuO phase. This result shows that by using this method, the precursor Cu is transformed into intermediate Cu(OH)2 / Cu-foam, and finally CuO array is obtained.

[0048] SEM analysis

[0049] Depend on( Figure 3 The morphological changes of Cu-foam, Cu(OH)2 / Cu-foam, and CuO / Cu-foam can be observed, such as ( Figure 3 -a1) Cu-foam achieves a smooth Cu surface after the removal of the surface organic phase and oxide layer; Figure 3 -a2), Cu-foam, after liquid-phase oxidation, yields a Cu(OH)2 nanowire array with a surface of several micrometers in length and 200-300 nanometers in width; such as ( Figure 3 As shown in -a3), after heat treatment in a nitrogen atmosphere, the CuO phase structure in CuO / Cu-foam remains a nanowire array, maintaining the structural integrity of the electrode. We also performed EDX elemental analysis, which showed that the ratios of copper to oxygen in the interfacial phases Cu-foam, Cu(OH)2 / Cu-foam, and CuO / Cu-foam were 1:0, 1:2, and 1:1, respectively, consistent with the XRD results. From the perspective of elemental analysis, this indicates that the CuO catalytic electrode was successfully synthesized.

[0050] Determining whether an electrode has catalytic activity against histamine

[0051] Figure 4 The CV curves for Cu-foam and CuO / Cu-foam are shown in 0.1 M NaOH solution, at a scan rate of 20 mV / s, with and without histamine. Figure 4As shown in (a) and (b), compared to the condition without histamine, the oxidation current value increased significantly when the histamine concentration was 200 μM. This indicates that both Cu-foam and CuO / Cu-foam played an electrocatalytic role in the oxidation of histamine. Further observation is needed. Figure 4 As shown in (a) and (b), after adding histamine solution of the same concentration, the current value of CuO / Cu-foam is much larger than that of Cu-foam. This indicates that CuO / Cu-foam electrode material has a stronger catalytic oxidation ability for histamine than Cu-foam electrode material, and is more suitable as a catalytic electrode for electrochemical oxidation of histamine.

[0052] Research on the control of histamine electrochemical oxidation process

[0053] Figure 5 The CV curves of CuO / Cu-foam in 30 mL of 0.1 M NaOH solution containing 30 μL of 200 μM histamine at different scan rates are shown. Figure 5 As clearly seen in (a), the current value increases with increasing scan rate, and the electrode potentials of the oxidation and reduction peaks shift towards positive and negative potentials, respectively, indicating that the catalytic oxidation of histamine at this electrode is essentially a quasi-reversible process. To further investigate whether this catalytic oxidation process is controlled by adsorption or diffusion, i.e., to determine the exponential value of the relationship between scan rate and oxidation current, [further details are needed]. Figure 5 As can be seen in (b) above, there is a good linear relationship between the oxidation current value and the square root of the scan rate (R). 2 =0.994), which indicates that the catalytic oxidation process of histamine by the electrode material is mainly controlled by the diffusion rate of histamine in the solution, thus providing a good electrochemical kinetic support for the chronoamperometry method for measuring histamine concentration.

[0054] Chronoamperometry for studying the catalytic oxidation behavior of histamine by electrodes

[0055]

[0056] Table 1 Performance parameters of Cu-foam and CuO / Cu-foam sensors

[0057] like Figure 6 As shown in (a) and (b), under optimal conditions (0.55 V – much lower than the practical voltage of carbon materials and noble metal electrodes, 0.1 M NaOH), the It curves of Cu-foam and CuO / Cu-foam electrodes at different histamine concentrations were measured. Observation Figure 6(a) It can be seen that with the continuous addition of histamine standard solutions of different concentrations, the oxidation current value shows a step increase and quickly reaches equilibrium. The response time of Cu-foam is 2 s, while the response time of CuO / Cu-foam is 10 s. Linear fitting ( Figure 6 (a) and (b) show that the oxidation current of the Cu-foam electrode exhibits a good linear relationship with histamine concentration in the range of 0.5 μM to 1195 μM, and the linear regression equation is as follows: Sensitivity is The detection limit is The oxidation current of the CuO / Cu-foam electrode showed a good linear relationship with histamine concentration in the range of 0.5 μM to 246 μM, and its linear regression equation was as follows: Sensitivity is The detection limit is Table 1 compares the characteristic parameters of the two electrodes. Regarding the core parameter of sensitivity, the CuO / Cu-foam enzyme-free electrochemical histamine sensor exhibits 31 times the sensitivity of the Cu-foam electrode, indicating superior detection performance.

[0058] This experiment used a CuO / Cu-foam-based enzyme-free sensor to detect histamine content in fermented mandarin fish. As shown in Table 2, the histamine content in different mandarin fish samples was 16.0495 mg / kg, 15.1515 mg / kg, 15.5414 mg / kg, and 14.2652 mg / kg, respectively, with recoveries ranging from 104.68% to 106.79%. Further research revealed a high degree of consistency between these results and those obtained by high-performance liquid chromatography (HPLC), indicating that the CuO / Cu-foam-based enzyme-free electrochemical sensing method for detecting histamine content in fermented mandarin fish is reliable, effective, and accurate.

[0059]

[0060] Table 2. Histamine content and recovery rate in fermented mandarin fish.

[0061] This invention utilizes a liquid-phase oxidation method and a heat treatment calcination method to generate a one-dimensional CuO nanowire array on the surface of Cu-foam. This array is then used as an electrocatalytic electrode for detecting histamine content in fermented mandarin fish. Studies have shown that under optimal conditions, the one-dimensional CuO nanowire array exhibits ultrasensitive sensitivity, with a ratio significantly higher than that of Cu-foam, reaching 31 times its sensitivity. Its application in detecting histamine content in fermented mandarin fish yielded accurate and effective results. This demonstrates the superior performance of this enzyme-free electrochemical histamine sensor in detecting histamine content in fermented mandarin fish and provides potential application value for the detection of histamine in fermented fish.

[0062] High Performance Liquid Chromatography - Construction of Histamine Standard Curve

[0063] 1) Configuration of standard products

[0064] Accurately weigh 0.1 g of histamine standard into a 10 mL volumetric flask, and dilute to the mark with ultrapure water to prepare a 10 mg / mL histamine standard solution. Protect from light and store at 4 °C. Dilute 0.1 mL of the histamine standard solution into a 10 mL volumetric flask with ultrapure water. Use fractional dilution to obtain histamine standard solutions with mass concentrations of 0.5, 1.0, 5.0, 10.0, and 20.0 μg / mL, respectively. Prepare fresh solutions immediately before use.

[0065] 2) Derivatives of standard products

[0066] Add 300 μL of histamine standard to a 2 mL centrifuge tube, add 40 μL of 0.1 mol / L NaOH solution, adjust the pH to 9.5, then add 600 μL of 10 mg / mL Dns-Cl solution, stir well, and place in a constant temperature bath at 40 ℃ for 20 min. After the reaction is complete, add 100 μL of ammonia water, mix well, and let stand for 30 min. Make up the volume to 1.5 mL with acetonitrile, stir well, and filter the supernatant through a 0.22 μm organic needle filter at 4 ℃ and 10000 r / min.

[0067] Determination of histamine content in samples

[0068] 1) Sample pretreatment

[0069] Weigh 5.0672 g, 5.0952 g, 5.1020 g, and 5.0421 g of ABCD mandarin fish samples using an electronic balance. Place them in a 50 mL centrifuge tube, add 15 mL of 5% trichloroacetic acid, then crush the fish meat and centrifuge at 4 ℃ and 6000 r / min for 15 min. Transfer the supernatant to a 25 mL brown volumetric flask. Add 10 mL of 5% trichloroacetic acid to the centrifuge tube, repeat once, and combine the supernatants from both tests. Place the mixture in a volumetric flask, dilute to the mark with 5% trichloroacetic acid, and filter the supernatant through a 0.22 μm organic needle filter.

[0070] 2) Sample derivatization

[0071] Add 300 μL of the sample to a 2 mL centrifuge tube, add 40 μL of 2 mol / L NaOH solution, adjust the pH to 9.5, then add 600 μL of 10 mg / mL Dns-Cl solution, stir well, and place in a constant temperature bath at 40 ℃ for 20 min. After the reaction is complete, add 100 μL of ammonia water, mix well, and let stand for 30 min. Make up the volume to 1.5 mL with acetonitrile, stir well, and filter the supernatant through a 0.22 μm organic needle filter at 4 ℃ and 10,000 r / min.

[0072] Chromatographic conditions

[0073] Chromatographic column: X-Bridge C18 column (150 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (70+30); flow rate: 1 mL / min; column temperature: 35 ℃; injection volume: 20 μL; detection wavelength: 254 nm.

[0074] Calculation formula

[0075]

[0076] In the formula:

[0077] X - Amount of histamine in the stinky mandarin fish, mg / Kg; C - Mass concentration of histamine in the sample solution, mg / L;

[0078] V - Volume of sample after dilution, mL; f - Dilution factor; m - Mass of sample, g.

[0079] Table 3. Data obtained by high performance liquid chromatography.

[0080]

Claims

1. A method for histamine detection using an enzyme-free electrochemical sensor, comprising a catalytic electrode composed of one-dimensional CuO nanowires, wherein the catalytic electrode with an applied voltage of 0.55 V is placed in a detection solution at pH 13, and the histamine concentration in the detection solution is reflected by the change in current.

2. The method for histamine detection using an enzyme-free electrochemical sensor according to claim 1, characterized in that, The detection solution is kept stirred and mixed when energized.

3. The method for histamine detection using an enzyme-free electrochemical sensor according to claim 1, characterized in that, The method for preparing the catalytic electrode composed of the one-dimensional CuO nanowires is as follows: Cu-foam was immersed in a mixed solution of NaOH and (NH4)2S2O8 for 20 min to generate a Cu(OH)2 linear array. After heat treatment under a hot N2 atmosphere for 2 h, a one-dimensional CuO nanowire array electrode was obtained.

4. The method for histamine detection using an enzyme-free electrochemical sensor according to claim 3, characterized in that, The NaOH and (NH4)2S2O8 mixed solution has a NaOH concentration of 5 mol / L and a (NH4)2S2O8 concentration of 0.25 mol / L.

5. The method for histamine detection using an enzyme-free electrochemical sensor according to claim 3, characterized in that, The temperature of the hot N2 atmosphere is 200 ℃.

6. The method for histamine detection using an enzyme-free electrochemical sensor according to claim 3, characterized in that, The Cu-foam is cleaned before being placed in the mixed solution of NaOH and (NH4)2S2O8 to remove surface impurities.

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