A carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower and its preparation method, electrochemical / colorimetric dual-signal biosensor and its application

Abstract

The invention discloses a kind of carbon cloth in-situ growth laccase copper phosphate hybrid nanoflower and preparation method, electrochemical / colorimetric dual-signal biosensor and application, belong to the field of biosensor preparation technology, electrochemical / colorimetric dual-signal biosensor includes: using a laccase copper phosphate hybrid nanoflower material grown in situ on the carbon cloth surface by a one-pot method as an electrode, utilizing cyclic voltammetry, electrochemical detection of phenols is carried out, and the oxidation peak potential and current value are used as the qualitative and quantitative detection basis of phenols; In the electrode electrocatalytic oxidation system of phenols, 4 aminoantipyrine is added, and the absorption peak position and absorbance of the color development system are recorded, as the qualitative and quantitative detection basis of colorimetry. This method not only utilizes the specific catalytic ability of laccase to phenols, but also combines the stability of copper phosphate and the high conductivity of carbon cloth, thereby realizing the combination of colorimetric and electrochemical two detection modes, and improving the accuracy of the test result.

Description

[0001] A carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower and its preparation method, electrochemical / colorimetric dual-signal biosensor and its application Technical Field

[0002] The present invention belongs to the technical field of biosensors, and specifically relates to a carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower and a preparation method, an electrochemical / colorimetric dual-signal biosensor and its application. Background Art

[0003] Coking wastewater is an organic wastewater containing high concentrations of phenolic pollutants, which pose a serious threat to both environmental protection and human health. Conventional methods for detecting phenolic pollutants include gas chromatography, liquid chromatography, spectroscopic analysis, and electrochemical analysis. However, these methods are time-consuming, costly, require demanding experimental conditions, rely on large external equipment, and are not suitable for rapid on-site detection.

[0004] A biosensor is a device that uses biological molecules as recognition elements and can convert the specific interactions of biomolecules into measurable signals, such as electrical signals or optical signals. Electrochemical biosensors detect substrate concentrations through electrical signals and have the advantages of fast response, good selectivity, low detection limits, and ease of operation. They are currently one of the most commonly used methods for detecting phenolic pollutants. Colorimetric biosensors use color changes as signals to determine substrate concentrations and are a detection technology that can be operated on-site without the need for external instruments. However, in actual use, electrochemical biosensors have poor stability and colorimetric biosensors have low resolution. To improve the accuracy of detection results, an electrochemical / colorimetric dual-mode biosensor system can be constructed in which electrical and color signals are output synchronously and calibrated to ensure the accuracy of detection results.

[0005] In addition to signal transmission, the recognition element is a key factor in determining sensor performance. Laccase, a multi-copper oxidase that catalyzes the degradation of a wide range of aromatic compounds, is often used as a biorecognition element in biosensors for the detection of phenolic pollutants. However, the susceptibility of laccase to denaturation and inactivation has significantly limited its application in the analysis and detection of phenolic pollutants. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problems in the above-mentioned prior art and provide a carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower and a preparation method, an electrochemical / colorimetric dual-signal biosensor and its application. The biosensor generates detectable electrochemical signals (such as current and voltage changes) and optical signals (such as color changes and absorbance) by in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth to electrocatalytically oxidize phenolic substances. The dual signals are calibrated with each other to ensure the accuracy of the detection results.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth comprises the following steps:

[0009] The acid-treated carbon cloth was immersed in a CuSO4 solution, and then immersed in a PBS solution containing laccase, and allowed to react to obtain in situ grown laccase-copper phosphate hybrid nanoflowers on the carbon cloth.

[0010] Furthermore, the specific process of the carbon cloth acid treatment is: soaking the carbon cloth in nitric acid with a mass concentration of 96%-98% for 6-12 hours.

[0011] Furthermore, the concentration of the CuSO4 solution is 0.1-0.2 mol / L, and the acid-treated carbon cloth is immersed in the CuSO4 solution for 10-30 min.

[0012] Furthermore, the concentration of laccase in the PBS solution containing laccase is 0.1-0.5 mg / mL, the concentration of the PBS solution containing laccase is 50-100 mmol / L, and the pH is 6.8-7.4.

[0013] Furthermore, the temperature of the static reaction is 15-25° C., and the time is 24-72 hours.

[0014] Furthermore, a nanoflower array structure was uniformly grown on the surface of the carbon cloth fiber. The nanoflower was composed of 30nm thick curled nanosheets assembled in an interlaced manner to form a nanoflower ball with a diameter of 3μm.

[0015] An electrochemical / colorimetric dual-signal biosensor comprises a three-electrode system, wherein the carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower prepared by the above method serves as a working electrode, Ag / AgCl serves as a reference electrode, and a platinum wire serves as a counter electrode.

[0016] Application of an electrochemical / colorimetric dual-signal biosensor in the detection of phenolic pollutants.

[0017] Furthermore, phenolic pollutants include phenol, m-cresol, 2, 6-dichlorophenol and hydroquinone.

[0018] Further, the following steps are included:

[0019] The carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers were used as working electrodes to perform electrochemical detection on PBS electrolyte containing phenolic pollutants;

[0020] After the electrochemical detection is completed, 4-aminoantipyrine is added to the PBS electrolyte for colorimetric measurement.

[0021] Furthermore, the concentration of PBS electrolyte was 50-100 mmol / L, pH=5.5-7.5; electrochemical detection included CV test and It test, the CV test potential window was -0.4-1.0 V, and the scan rate was 100-500 mV·s -1 ; Its test voltage is 0.19~0.68V.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The preparation method of the carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers of the present invention utilizes the principle of biomineralization to in situ grow a laccase-copper phosphate hybrid nanoflower array on the surface of the carbon cloth fiber, fundamentally solving the problems of high cost, easy inactivation, great environmental impact and difficult recycling of laccase. At the same time, the introduction of carbon cloth fibers provides a high-speed electron transmission path, effectively improving the electrochemical activity of the laccase-copper phosphate hybrid nanoflowers.

[0024] The present invention relates to an electrochemical / colorimetric dual-signal biosensor based on the above-mentioned carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower for the detection of phenolic substances. The electron exchange and transmission between the carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower electrode and the phenolic substance will cause the oxidation peak current value at a specific voltage to change. The current change value can be used as a basis for the qualitative and quantitative determination of the phenolic compound. Phenolic oxides and 4-AAP are coupled to produce quinone dyes. The rigid plane of the quinone substance causes the product color to show a significant color change, and an absorption peak appears at a specific wavelength in the ultraviolet spectrum. The changes in the colorimetry and absorbance of the product solution can be used as a basis for the qualitative and quantitative determination of the phenolic compound. The oxidation peak potential and absorption peak wavelength can be used to qualitatively identify the type of target analyte, and the current value and colorimetry / absorbance can be used to quantitatively identify the concentration of the target analyte. The dual-signal mutual calibration effectively improves the selectivity and accuracy of the enzyme hybrid nanoflower in detecting phenolic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an SEM image of the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 1;

[0026] Figure 2 The SEM images of the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 2; (a) is a low magnification, and (b) is a high magnification;

[0027] Figure 3CV curves, UV-vis spectra, and photos of the electrochemical / colorimetric dual-signal biosensor based on in situ carbon cloth-grown laccase-copper phosphate hybrid nanoflowers prepared in Example 5 for the detection of four phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone); (a) is the CV curve, (b) is the UV-vis spectrum, and (c) is a photo of the biosensor detecting four phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone); in Figure (c), phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone are shown from left to right.

[0028] Figure 4 It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor for detecting phenol at different concentrations based on in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 7; wherein, (a) is the It curve, (b) is a local enlarged view of the It curve at t = 130~575s, (c) is the relationship curve between phenol concentration and current response value, and (d) is a photo of the color response of the biosensor to different concentrations of phenol, with phenol concentrations of 0, 5μmol / L, 10μmol / L, 20μmol / L, 30μmol / L from left to right. ol / L, 40μmol / L, 50μmol / L, 60μmol / L, 70μmol / L, 80μmol / L, 90μmol / L, 100μmol / L, 110μmol / L, 120μmol / L, 130μmol / L, 140μmol / L, 150μmol / L, 160μmol / L, 170μmol / L, 180μmol / L, 190μmol / L, 200μmol / L, (e) is the UV-vis spectrum, (f) is the relationship curve between phenol concentration and absorbance;

[0029] Figure 5It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor for detecting different concentrations of m-cresol based on in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 8; wherein, (a) is the It curve, (b) is a local enlarged view of the It curve at t = 160~480s, (c) is the relationship curve between the concentration of m-cresol and the current response value, and (d) is a color response photo of the biosensor to different concentrations of m-cresol. From left to right, the concentrations of m-cresol are 0, 5 μm ol / L, 15μmol / L, 25μmol / L, 35μmol / L, 45μmol / L, 55μmol / L, 65μmol / L, 75μmol / L, 85μmol / L, 95μmol / L, 105μmol / L, 115μmol / L, 125μmol / L, 145μmol / L, 180μmol / L, 215μmol / L, (e) is the UV-vis spectrum, (f) is the relationship curve between m-cresol concentration and absorbance;

[0030] Figure 6 It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 9 for the detection results of 2,6-dichlorophenol at different concentrations; wherein, (a) is the It curve, (b) is a local enlarged view of the It curve at t = 160~480s, (c) is the relationship curve between the concentration of 2,6-dichlorophenol and the current response value, (d) is a color response photo of the biosensor to different concentrations of 2,6-dichlorophenol, from left to right, the concentrations of 2,6-dichlorophenol are 0, 0.5μmol / L, 1.5μmol / L, 2.5μmol / L, 3.5μmol / L, 4.5μmol / L, 5.5μmol / L, 10μmol / L, 20μmol / L, 30μmol / L, 40μmol / L, (e) is the UV-vis spectrum, and (f) is the color response photo of 2, The relationship curve between 6-dichlorophenol concentration and absorbance;

[0031] Figure 7The It curve, UV-vis spectrum and photos of the electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 10 for the detection of different concentrations of hydroquinone. Among them, (a) is the It curve, Figure (b) is a local enlarged view of the It curve at t = 160~480s, (c) is the relationship curve between hydroquinone concentration and current response value, and (d) is a photo of the color response of the biosensor to different concentrations of hydroquinone. From left to right, the concentrations of hydroquinone are 0, 0.5μmol / L, 2μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80 μmol / L, 100μmol / L, 120μmol / L, 140μmol / L, 170μmol / L, 230μmol / L, 290μmol / L, (e) is the UV-vis spectrum, (f) is the relationship curve between the hydroquinone concentration and absorbance in the hydroquinone concentration range of 0~20μmol / L, (g) is the relationship curve between the hydroquinone concentration and absorbance in the hydroquinone concentration range of 20~290μmol / L. DETAILED DESCRIPTION

[0032] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in a variety of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0033] The present invention provides a new possibility for constructing a high-performance biosensor by immobilizing laccase on a nanomaterial.

[0034] The present invention is described by way of specific examples, but is not limited thereto. The following further describes an electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth and its applications.

[0035] The preparation method of the electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth of the present invention comprises the following steps:

[0036] (1) Preparation of in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth

[0037] The carbon cloth was cut into rectangles of 2×1 cm and immersed in concentrated nitric acid (mass concentration of 96%-98%) for 6-12 hours. After soaking, it was ultrasonically cleaned with acetone, anhydrous ethanol and ultrapure water for 3 times each, and dried; the treated carbon cloth was immersed in a CuSO4 solution with a concentration of 0.1-0.2 mol / L, taken out after 10-30 minutes and dried; the above carbon cloth was immersed in 20 mL of PBS solution (concentration of 50-100 mmol / L, pH=6.8-7.4) containing 0.1-0.5 mg / mL laccase, and allowed to stand at 15-25°C for 24-72 hours. The carbon cloth was taken out and washed with ultrapure water for 3-5 times, and freeze-dried to obtain in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth.

[0038] (2) Construction of an electrochemical / colorimetric dual-signal biosensor based on in situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth and its application in the detection of phenolic substances.

[0039] A three-electrode electrochemical workstation system was used, with in situ carbon cloth-grown laccase-copper phosphate hybrid nanoflowers as the working electrode, Ag / AgCl (3 mol / L KCl solution) as the reference electrode, and platinum wire as the counter electrode. CV tests or It tests were performed on 100 mL of PBS electrolyte (concentration range: 50-100 mmol / L, pH = 5.5-7.5) containing phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). The test potential window was -0.4-1.0 V, and the scan rate was 100-500 mV·s. -1 ;The voltage tested by it is 0.19~0.68V.

[0040] After the electrochemical test, add 150-180 μL of 0.1 mol / L 4-aminoantipyrine to the PBS electrolyte and react for 5-10 minutes. Take a photo to record the color of the electrolyte and perform colorimetric measurement using UV-vis at a wavelength range of 385-700 nm. Establish a standard curve between current value, absorbance, and phenolic substance concentration.

[0041] Among them, the chronoamperometry (It) was used to test the current value of the carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower electrode under different concentrations of phenolic substances, and the test voltage was 0.19~0.68V.

[0042] The electrochemical / colorimetric dual-signal biosensor of the present invention mutually verifies the detection results of the dual-signal outputs, thereby improving detection accuracy and reliability. It also exhibits excellent selectivity, with significant differences in potential, colorimetry, and absorption peak positions for the four phenolic compounds: phenol, m-cresol, hydroquinone, and dichlorophenol. This is a key advantage of the biosensor in detecting phenolic pollutants.

[0043] The present invention is described below with reference to specific embodiments.

[0044] Example 1

[0045] Preparation of laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth.

[0046] The carbon cloth was cut into rectangles of 2×1 cm and immersed in concentrated nitric acid for 6 h. After soaking, it was ultrasonically cleaned with acetone, anhydrous ethanol and ultrapure water for 3 times respectively, and dried; the treated carbon cloth was immersed in a CuSO4 solution with a concentration of 0.1 mol / L, taken out after 10 min and dried; the above carbon cloth was immersed in 20 mL of PBS solution containing 0.1 mg / mL laccase (concentration of 50 mmol / L, pH=6.8), and allowed to stand at 15°C for 24 h. The carbon cloth was taken out and washed with ultrapure water 3 times, and freeze-dried to obtain in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth.

[0047] See also Figure 1 , it can be seen that a large number of irregular nanosheets are distributed on the surface of carbon fiber, and these nanosheets tend to form flower-like structures.

[0048] Example 2

[0049] Preparation of laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth.

[0050] The carbon cloth was cut into rectangles of 2×1 cm and immersed in concentrated nitric acid for 12 h. After soaking, it was ultrasonically cleaned with acetone, anhydrous ethanol and ultrapure water for 3 times each, and dried. The treated carbon cloth was immersed in a CuSO4 solution with a concentration of 0.2 mol / L, taken out after 30 min and dried. The above carbon cloth was immersed in 20 mL of PBS solution (concentration of 100 mmol / L, pH = 7.4) containing 0.5 mg / mL laccase again, and allowed to stand at 25 ° C for 72 h. The carbon cloth was taken out and washed with ultrapure water 5 times, and freeze-dried to obtain in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth.

[0051] Figure 2 (a) and (b) show SEM images of in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 2. The SEM images show a uniform nanoflower array structure grown on the carbon cloth fiber surface. The nanoflowers are composed of interlaced, curled nanosheets approximately 30 nm thick, forming nanoflower spheres approximately 3 μm in diameter.

[0052] Example 3

[0053] The laccase-copper phosphate hybrid nanoflowers grown in situ on the carbon cloth of Example 2 were used to construct an electrochemical / colorimetric dual-signal biosensor, and phenolic substances were detected.

[0054] A three-electrode electrochemical workstation system was used. Laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth were used as the working electrode, Ag / AgCl (3 mol / L KCl) was used as the reference electrode, and platinum wire was used as the counter electrode. CV tests were performed on 100 mL of PBS electrolyte (50 mmol / L, pH = 5.5) containing phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). The test potential window was -0.4–1.0 V, and the scan rate was 100 mV·s. -1 After the electrochemical test, 150 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte and reacted for 5 minutes. The electrolyte color was recorded by taking a photo and measured by UV-vis with a wavelength range of 385~700 nm to detect phenolic substances.

[0055] Example 4

[0056] The laccase-copper phosphate hybrid nanoflowers grown in situ on the carbon cloth of Example 2 were used to construct an electrochemical / colorimetric dual-signal biosensor, and phenolic substances were detected.

[0057] A three-electrode electrochemical workstation system was used. Laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth were used as the working electrode, Ag / AgCl (3 mol / L KCl solution) was used as the reference electrode, and platinum wire was used as the counter electrode. CV tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 6.5) containing phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). The test potential window was -0.4–1.0 V, and the scan rate was 100 mV·s. -1 After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte and reacted for 5 minutes. The electrolyte color was recorded by taking a photo and measured by UV-vis with a wavelength range of 385~700 nm to detect phenolic substances.

[0058] Example 5

[0059] The laccase-copper phosphate hybrid nanoflowers grown in situ on the carbon cloth of Example 2 were used to construct an electrochemical / colorimetric dual-signal biosensor, and phenolic substances were detected.

[0060] A three-electrode electrochemical workstation system was used. Laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth were used as the working electrode, Ag / AgCl (3 mol / L KCl solution) was used as the reference electrode, and platinum wire was used as the counter electrode. CV tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). The test potential window was -0.4–1.0 V, and the scan rate was 100 mV·s. -1 After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte and reacted for 10 min. The electrolyte color was recorded by taking a photo and measured using UV-vis with a wavelength range of 385~700 nm.

[0061] Figure 3 The CV curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 5 for the detection of four phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). Figure 3 As can be seen in (a), within the window range of -0.4~1.0V, a distinct oxidation peak is observed at 0.681V, 0.655V, 0.626V, and 0.191V, respectively. The oxidation processes of phenol, m-cresol, and 2,6-dichlorophenol are all single electron transfer accompanied by single proton transfer, so the potential value difference is small. However, the potential value difference of hydroquinone is large because its catalytic oxidation process is the transfer of two electrons accompanied by two protons. Figure 3 As shown in Figures (b) and (c), the addition of 4-aminoantipyrine results in significant differences in electrolyte color and UV-visible absorption peak positions for different phenolic compounds. The quinoneimine dyes produced by phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone with 4-aminoantipyrine exhibit peak absorption wavelengths of 505 nm, 500 nm, 518 nm, and 479 nm, respectively. These differences in oxidation peak potential, electrolyte color, and UV-visible absorption peak positions demonstrate the excellent selectivity of the in situ carbon cloth-grown laccase-copper phosphate hybrid nanoflower electrode for different phenolic compounds.

[0062] Example 6

[0063] The laccase-copper phosphate hybrid nanoflowers were grown in situ on the carbon cloth of Example 2 to construct an electrochemical / colorimetric dual-signal biosensor, and phenolic substances were detected.

[0064] A three-electrode electrochemical workstation system was used. Laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth were used as the working electrode, Ag / AgCl (3 mol / L KCl solution) was used as the reference electrode, and platinum wire was used as the counter electrode. CV tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing phenolic substances (phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone). The test potential window was -0.4–1.0 V, and the scan rate was 500 mV·s. -1 After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte and reacted for 10 minutes. The electrolyte color was recorded by taking a photo and measured by UV-vis with a wavelength range of 385~700 nm to detect phenolic substances.

[0065] Example 7

[0066] The laccase-copper phosphate hybrid nanoflowers were grown in situ on the carbon cloth of Example 2 to construct an electrochemical / colorimetric dual-signal biosensor, and phenol was detected.

[0067] An electrochemical workstation three-electrode system was used, with laccase-copper phosphate hybrid nanoflowers in situ grown on carbon cloth as the working electrode, Ag / AgCl (3 mol / L KCl solution) as the reference electrode, and platinum wire as the counter electrode. 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing different concentrations of phenol (0.05-200 μmol / L) was subjected to It test at a test voltage of 0.68 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, and the reaction was carried out for 10 minutes. The electrolyte color was recorded by photographing and measured using UV-vis with a wavelength range of 385-700 nm. A standard curve, i.e., a linear relationship, was established between the current value, absorbance, and phenol concentration.

[0068] Figure 4 It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 7 for the detection of phenol at different concentrations. Figure 4 As can be seen in (a) and (b), with the increase of phenol concentration from 0 to 160 μmol / L, the current response value gradually increases; Figure 4 (c) shows that there is a clear linear relationship between the current response value and the phenol concentration, and the equation is Y = 0.0024X + 0.0055 (R 2 =0.9983), the linear range was 50 nmol / L ~100 μmol / L, and the minimum detection limit was 16.2 nmol / L. Figure 4Middle (d) is a photograph of PBS electrolyte with different phenol concentrations. From left to right, the concentrations are 0μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 30μmol / L, 40μmol / L, 50μmol / L, 60μmol / L, 70μmol / L, 80μmol / L, 90μmol / L, 100μmol / L, 110μmol / L, 120μmol / L, 130μmol / L, 140μmol / L, 150μmol / L, 160μmol / L, 170μmol / L, 180μmol / L, 190μmol / L, and 200μmol / L. With the increase of phenol concentration, the electrolyte gradually changes from colorless to pink, and the higher the concentration, the darker the color. Figure 4 (e) is the UV-vis spectrum of PBS electrolyte containing different phenol concentrations. An obvious absorption peak appears at λ=505nm, and the absorbance value increases with the increase of phenol concentration; Figure 4 (f) shows that there is a clear linear relationship between absorbance and phenol concentration, and the equation is A=0.03C+0.001(R 2 =0.9995), the linear range was 5~140μmol / L, and the minimum detection limit was 5μmol / L.

[0069] Example 8

[0070] The laccase-copper phosphate hybrid nanoflowers were grown in situ on the carbon cloth of Example 2 to construct an electrochemical / colorimetric dual-signal biosensor, and m-cresol was detected.

[0071] An electrochemical workstation three-electrode system was used, with laccase-copper phosphate hybrid nanoflowers in situ grown on carbon cloth as the working electrode, Ag / AgCl (3 mol / L KCl solution) as the reference electrode, and platinum wire as the counter electrode. It tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing different concentrations of m-cresol (0.05-215 μmol / L) at a test voltage of 0.66 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, and the reaction was carried out for 10 minutes. The electrolyte color was recorded and measured using UV-vis with a wavelength range of 385-700 nm. A standard curve between current value, absorbance, and m-cresol concentration was established.

[0072] Figure 5 It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers prepared in Example 8 for the detection of different concentrations of m-cresol. Figure 5As can be seen in (a) and (b), with the increase of m-cresol concentration from 0 to 210 μmol / L, the current response value gradually increases; Figure 5 (c) shows that there is a clear linear relationship between the current response value and the concentration of m-cresol, and the equation is Y = 0.0011X-0.0058 (R 2 =0.9974), the linear range was 90 nmol / L ~190 μmol / L, and the minimum detection limit was 30 nmol / L. Figure 5 Middle (d) is a photograph of PBS electrolyte with different m-cresol concentrations. From left to right, the concentrations are 0μmol / L, 5μmol / L, 15μmol / L, 25μmol / L, 35μmol / L, 45μmol / L, 55μmol / L, 65μmol / L, 75μmol / L, 85μmol / L, 95μmol / L, 105μmol / L, 115μmol / L, 125μmol / L, 145μmol / L, 180μmol / L, and 215μmol / L. With the increase of m-cresol concentration, the electrolyte gradually changes from colorless to dark pink, and the higher the concentration, the darker the color. Figure 5 (e) is the UV-vis spectrum of PBS electrolyte containing different concentrations of m-cresol. An obvious absorption peak appears at λ = 500nm, and the absorbance value increases with the increase of m-cresol concentration. Figure 5 (f) shows that there is a clear linear relationship between absorbance and m-cresol concentration, and the equation is A=6.1×10 -4 C+0.0056(R 2 =0.9949), the linear range was 5μmol / L~145μmol / L, and the minimum detection limit was 5μmol / L.

[0073] Example 9

[0074] The laccase-copper phosphate hybrid nanoflowers grown in situ on the carbon cloth of Example 2 were used to construct an electrochemical / colorimetric dual-signal biosensor, and 2, 6-dichlorophenol was detected.

[0075] An electrochemical workstation three-electrode system was used, with in situ carbon cloth-grown laccase-copper phosphate hybrid nanoflowers as the working electrode, Ag / AgCl (3 mol / L KCl solution) as the reference electrode, and platinum wire as the counter electrode. It tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing different concentrations of 2, 6-dichlorophenol (0.05-170 μmol / L) at a test voltage of 0.63 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte. The reaction was allowed to proceed for 10 min, and the electrolyte color was recorded and measured using UV-visible light in the wavelength range of 385-700 nm. A standard curve was established between current value, absorbance, and 2, 6-dichlorophenol concentration.

[0076] Figure 6 The It curve, UV-vis spectrum and photos of the electrochemical / colorimetric dual-signal biosensor based on carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers prepared in Example 9 for the detection of different concentrations of 2,6-dichlorophenol. Figure 6 As shown in (a) and (b), as the concentration of 2, 6-dichlorophenol increases from 0 to 170 μmol / L, the current response value gradually increases; Figure 6 (c) shows that there is a clear linear relationship between the current response value and the concentration of 2, 6-dichlorophenol, and the equation is Y=0.0042X-0.0046 (R 2 =0.9919), the linear range was 30 nmol / L ~100 μmol / L, and the minimum detection limit was 10 nmol / L. Figure 6 Middle (d) is a photograph of PBS electrolyte with different 2, 6-dichlorophenol concentrations. From left to right, the concentrations are 0μmol / L, 0.5μmol / L, 1.5μmol / L, 2.5μmol / L, 3.5μmol / L, 4.5μmol / L, 5.5μmol / L, 10μmol / L, 20μmol / L, 30μmol / L, and 40μmol / L. As the concentration of 2, 6-dichlorophenol increases, the electrolyte gradually changes from colorless to pink-purple, and the higher the concentration, the darker the color. Figure 6 (e) is the UV-vis spectrum of PBS electrolyte containing different 2, 6-dichlorophenol concentrations. An obvious absorption peak appears at λ = 518nm, and the absorbance value increases with the increase of 2, 6-dichlorophenol concentration; Figure 6 (f) shows that there is a clear linear relationship between the absorbance value and the concentration of 2, 6-dichlorophenol, and the equation is A=0.0181C+0.015(R 2=0.9981), the linear range was 0.5μmol / L~10μmol / L, and the minimum detection limit was 0.5μmol / L.

[0077] Example 10

[0078] The laccase-copper phosphate hybrid nanoflowers were grown in situ on the carbon cloth of Example 2 to construct an electrochemical / colorimetric dual-signal biosensor, and hydroquinone was detected.

[0079] An electrochemical workstation three-electrode system was used, with laccase-copper phosphate hybrid nanoflowers in situ grown on carbon cloth as the working electrode, Ag / AgCl (3 mol / L KCl solution) as the reference electrode, and platinum wire as the counter electrode. It tests were performed on 100 mL of PBS electrolyte (100 mmol / L, pH = 7.5) containing different concentrations of hydroquinone (0.05-290 μmol / L) at a test voltage of 0.19 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, and the reaction was carried out for 10 minutes. The electrolyte color was recorded and measured using UV-vis at a wavelength range of 385-700 nm. A standard curve between current value, absorbance, and hydroquinone concentration was established.

[0080] Figure 7 It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 10 for the detection of hydroquinone at different concentrations. Figure 7 As can be seen in (a) and (b), with the increase of hydroquinone concentration from 0 to 220 μmol / L, the current response value gradually increases; Figure 7 (c) shows that there is a clear linear relationship between the current response value and the concentration of hydroquinone, and the equation is Y = 0.0016X-0.0036 (R 2 =0.9989), the linear range was 60 nmol / L~200 μmol / L, and the minimum detection limit was 20 nmol / L. Figure 7 Middle (d) is a photograph of PBS electrolyte with different hydroquinone concentrations. From left to right, the concentrations are 0, 0.5μmol / L, 2μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L, 100μmol / L, 120μmol / L, 140μmol / L, 170μmol / L, 230μmol / L, and 290μmol / L. As the concentration of hydroquinone increases, the electrolyte gradually changes from colorless to dark red, and the higher the concentration, the darker the color. Figure 7(e) is the UV-vis spectrum of PBS electrolyte containing different hydroquinone concentrations. A clear absorption peak appears at λ = 513 ~ 494nm. With the increase of hydroquinone concentration, the absorption peak shifts to the blue and the absorbance value gradually increases. Figure 7 (f) and (g) show that there are two clear linear relationships between the absorbance value and the concentration of hydroquinone, equation 1 is A=0.0043In(C)+0.0052(R 2 =0.9626), the linear range is 0.5 μmol / L~20 μmol / L, and equation 2 is A=5.03×10 -4 C+0.008(R 2 =0.9955), the linear range was 20 μmol / L~170 μmol / L, and the minimum detection limit was 0.5 μmol / L.

[0081] Table 1 shows the detection results of phenol, m-cresol, 2,6-dichlorophenol and hydroquinone in coal tar wastewater by the electrochemical / colorimetric dual-signal biosensor based on in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 10.

[0082] Table 1

[0083]

[0084] As shown in Table 1, the biosensor was validated using the standard addition method. Coal tar wastewater was pre-dephenolized, and then standard concentrations of phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone were added for electrochemical and colorimetric detection. The electrochemical biosensor demonstrated recoveries of 104.51%, 106.36%, 102.30%, and 113.30% for phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone in the coal tar wastewater, respectively, with relative standard deviations of 0.63%, 0.67%, 0.34%, and 1.43%. The colorimetric biosensor demonstrated recoveries of 108.68%, 111.76%, 104.30%, and 88.05% for phenol, m-cresol, 2, 6-dichlorophenol, and hydroquinone in the coal tar wastewater, respectively, with relative standard deviations of 1.37%, 1.52%, 0.98%, and 4.20%. The test results show that the constructed electrochemical / colorimetric dual-signal biosensor has good accuracy and precision for the detection of phenol, m-cresol, 2, 6-dichlorophenol and hydroquinone in coal char wastewater.

[0085] The present invention first uses a one-pot method to in situ grow a laccase-copper phosphate hybrid nanoflower material on a carbon cloth surface. Second, using this material as an electrode, cyclic voltammetry is used to electrochemically detect phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone). The oxidation peak potential and current values ​​serve as the basis for qualitative and quantitative detection of the phenolic substances. Then, 4-aminoantipyrine is added to the electrocatalytic oxidation system of the phenolic substances at the electrode. The color of the system solution is observed, and the absorption peak position and absorbance of the color development system are recorded using an ultraviolet spectrophotometer as the basis for qualitative and quantitative detection by colorimetry. This method not only utilizes the specific catalytic ability of laccase for phenolic substances, but also combines the stability of copper phosphate and the high conductivity of carbon cloth, thereby achieving a combination of colorimetric and electrochemical detection modes, improving the accuracy of the detection results.

[0086] The above description is merely a description of the preferred embodiment of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiment, and variations in the specific structure are permitted. Any variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth, characterized in that: The following steps are involved: The acid-treated carbon cloth was immersed in a CuSO4 solution, and then immersed in a PBS solution containing laccase, and allowed to react to obtain in situ grown laccase-copper phosphate hybrid nanoflowers on the carbon cloth; The specific process of carbon cloth acid treatment is as follows: soak the carbon cloth in nitric acid with a mass concentration of 96%-98% for 6-12 hours; The concentration of the CuSO4 solution is 0.1~0.2mol / L, and the acid-treated carbon cloth is immersed in the CuSO4 solution for 10~30min; The PBS solution containing laccase has a laccase concentration of 0.1-0.5 mg / mL, a laccase concentration of 50-100 m mol / L, and a pH of 6.8-7.

4.

2. The method for preparing laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth according to claim 1, characterized in that: The reaction temperature is 15~25℃ and the reaction time is 24~72h.

3. A carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower prepared by the method according to any one of claims 1-2, characterized in that: A nanoflower array structure is uniformly grown on the surface of the carbon cloth fiber. The nanoflower is composed of 30 nm thick curled nanosheets assembled in an interlaced manner to form a nanoflower ball with a diameter of 3 μm.

4. An electrochemical / colorimetric dual-signal biosensor, characterized in that: The invention comprises a three-electrode system, wherein the carbon cloth in situ grown laccase-copper phosphate hybrid nanoflower prepared by the method according to any one of claims 1-2 is used as a working electrode, Ag / AgCl is used as a reference electrode and a platinum wire is used as a counter electrode.

5. Use of the electrochemical / colorimetric dual-signal biosensor according to claim 4 in the detection of phenolic substances.

6. Use of the electrochemical / colorimetric dual-signal biosensor according to claim 5, characterized in that: Phenolic substances include phenol, m-cresol, 2, 6-dichlorophenol and hydroquinone.

7. Use of the electrochemical / colorimetric dual-signal biosensor according to claim 5, characterized in that: The following steps are involved: The carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers were used as working electrodes to perform electrochemical detection on PBS electrolyte containing phenolic substances; After the electrochemical detection is completed, 4-aminoantipyrine is added to the PBS electrolyte for colorimetric measurement.

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