Carbon cloth in-situ growth laccase-copper phosphate hybrid nanoflower and preparation method thereof, electrochemical / colorimetric dual-signal biosensor and application of electrochemical / colorimetric dual-signal biosensor

Abstract

The invention discloses a carbon cloth in-situ growth laccase-copper phosphate hybrid nanoflower and a preparation method thereof, an electrochemical / colorimetric dual-signal biosensor and application, and belongs to the technical field of biosensor preparation. The electrochemical / colorimetric dual-signal biosensor is characterized in that a laccase-copper phosphate hybrid nanoflower material which grows on the surface of carbon cloth in situ by a one-pot method is used as an electrode, a cyclic voltammetry method is used for performing electrochemical detection on phenolic substances, and an oxidation peak potential and a current value are used as qualitative and quantitative detection bases of the phenolic substances; the method comprises the following steps: adding 4-aminoantipyrine into an electrode-to-phenolic substance electrocatalytic oxidation system, and recording the absorption peak position and absorbance of a color development system as a qualitative and quantitative detection basis of a colorimetric method. According to the method, the specific catalytic capability of laccase on phenolic substances is utilized, and the stability of copper phosphate and the high conductivity of the carbon cloth are combined, so that the combination of two detection modes of colorimetry and electrochemistry is realized, and the accuracy of a detection result is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower and a preparation method, an electrochemical / colorimetric dual-signal biosensor and applications. Background Art

[0002] Coking wastewater is an organic wastewater containing high concentrations of phenolic pollutants, which pose a serious threat to environmental protection and human health. Conventional detection methods for phenolic pollutants include gas chromatography, liquid chromatography, spectroscopy, electrochemical analysis, etc. However, these methods are time-consuming, costly, require harsh experimental conditions, rely on large external equipment, and are not suitable for rapid on-site detection.

[0003] A biosensor is a device that uses biological molecules as recognition elements. It can convert the specific interactions of biological molecules into measurable signals, such as electrical signals and optical signals. Electrochemical biosensors detect substrate concentrations through electrical signals. They have the advantages of fast response, good selectivity, low detection limit, and easy 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. They are a detection technology that does not require external instruments and can be operated on-site. However, in actual use, electrochemical biosensors have poor stability and colorimetric biosensors have low resolution. In order to improve the accuracy of the test results, an electrochemical / colorimetric dual-mode biosensor system can be constructed. The electrical signal and color signal are output synchronously and calibrated with each other to ensure the accuracy of the test results.

[0004] In addition to the transmission signal, the recognition element is one of the key factors that determine the performance of the sensor. Laccase is a type of multi-copper oxidase that can catalyze the degradation of a variety of aromatic compounds. Therefore, it is often used as a biological recognition element to construct biosensors for the detection of phenolic pollutants. However, due to the easy denaturation and inactivation of laccase, the application research of laccase in the analysis and detection of phenolic pollutants is greatly limited. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems in the above-mentioned prior art and to 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) through the 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.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth comprises the following steps:

[0008] 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.

[0009] 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 to 12 hours.

[0010] 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.

[0011] Furthermore, the concentration of laccase in the PBS solution containing laccase is 0.1-0.5 mg / mL, the concentration of laccase in the PBS solution containing laccase is 50-100 mmol / L, and the pH value is 6.8-7.4.

[0012] Furthermore, the temperature of the static reaction is 15 to 25° C., and the time is 24 to 72 hours.

[0013] Furthermore, a nanoflower array structure is uniformly grown on the surface of the carbon cloth fiber. The nanoflower is composed of 30nm thick curled nanosheets assembled in an interlaced manner to form a nanoflower ball with a diameter of 3μm.

[0014] 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 method described in any one of claims 1 to 5 is used as a working electrode, Ag / AgCl is a reference electrode and a platinum wire is used as a counter electrode.

[0015] Application of an electrochemical / colorimetric dual-signal biosensor in the detection of phenolic pollutants.

[0016] Furthermore, phenolic pollutants include phenol, m-cresol, 2,6-dichlorophenol and hydroquinone.

[0017] Further, the following steps are included:

[0018] The carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflowers are used as working electrodes to perform electrochemical detection on PBS electrolyte containing phenolic pollutants;

[0019] After the electrochemical detection was completed, 4-aminoantipyrine was added to the PBS electrolyte for colorimetric measurement.

[0020] Furthermore, the concentration of PBS electrolyte is 50-100 mmol / L, pH=5.5-7.5; electrochemical detection includes CV test and It test, the CV test potential window is -0.4-1.0 V, and the scan rate is 100-500 mV·s -1 ;It test voltage is 0.19~0.68V.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 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 carbon cloth fibers, 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 pathway, effectively improving the electrochemical activity of the laccase-copper phosphate hybrid nanoflowers.

[0023] The present invention is based on the application of the electrochemical / colorimetric dual-signal biosensor for the detection of phenolic substances based on the above-mentioned carbon cloth in-situ growth laccase-copper phosphate hybrid nanoflower. The electron exchange and transmission between the carbon cloth in-situ growth laccase-copper phosphate hybrid nanoflower electrode and the phenolic substance will cause the oxidation peak current value at a specific voltage to change, and the current change value can be used as a basis for the qualitative and quantitative analysis of phenolic compounds. Phenolic oxides and 4-AAP are coupled to produce quinone dyes. The rigid plane of quinone substances causes the product color to show obvious color changes, and an absorption peak appears at a specific wavelength of the ultraviolet spectrum. The changes in the chromaticity and absorbance of the product solution can be used as a basis for the qualitative and quantitative analysis of phenolic compounds. The oxidation peak potential and the absorption peak wavelength can be used to qualitatively identify the type of target analyte, and the current value and chromaticity / 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 the detection of phenolic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a SEM image of the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 1;

[0025] Figure 2 This is a SEM image of the in-situ grown laccase-copper phosphate hybrid nanoflower on the carbon cloth prepared in Example 2; wherein (a) is a low magnification, and (b) is a high magnification;

[0026] Figure 3CV 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 5 for the detection of four phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone); wherein (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), from left to right are phenol, m-cresol, 2,6-dichlorophenol and hydroquinone;

[0027] 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 results of phenol at different concentrations; 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 color response photo of the biosensor to different concentrations of phenol, with phenol concentrations of 0, 5 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / 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;

[0028] Figure 5It 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 results of different concentrations of m-cresol; 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 the concentration of m-cresol and the absorbance;

[0029] Figure 6 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 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 to 480s, (c) is the relationship curve between the concentration of 2,6-dichlorophenol and the current response value, and (d) is the biosensor for different concentrations of 2,6-dichlorophenol. Color response photos of phenol, 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, (f) is the relationship curve between the concentration of 2,6-dichlorophenol and the absorbance;

[0030] Figure 7It curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on the in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth prepared in Example 10 for the detection results of hydroquinone at different concentrations. Among them, (a) is the It curve, Figure (b) is a local enlarged view of the It curve at t = 160 to 480s, (c) is the relationship curve between the concentration of hydroquinone and the current response value, and (d) is a color response photo of the biosensor to different concentrations of hydroquinone. From left to right, the concentrations of hydroquinone are 0, 0.5 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / 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 the absorbance in the hydroquinone concentration range of 0 to 20μmol / L, (g) is the relationship curve between the hydroquinone concentration and the absorbance in the hydroquinone concentration range of 20 to 290μmol / L. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood.

[0032] In the present invention, laccase is immobilized on nanomaterials, thereby providing a new possibility for constructing a high-performance biosensor.

[0033] The present invention is described by specific embodiments, but the present invention 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 application.

[0034] The preparation method of the electrochemical / colorimetric dual-signal biosensor based on the in-situ growth of laccase-copper phosphate hybrid nanoflowers on carbon cloth of the present invention comprises the following steps:

[0035] (1) Preparation of laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth

[0036] The carbon cloth was cut into rectangles of 2×1 cm in size, immersed in concentrated nitric acid (mass concentration of 96%-98%) for 6-12 h, and then 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 0.1-0.2 mol / L, taken out after 10-30 min and dried; the 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 again, and allowed to stand at 15-25°C for 24-72 h, the carbon cloth was taken out and washed with ultrapure water for 3-5 times, and freeze-dried to obtain carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers.

[0037] (2) Construction of an electrochemical / colorimetric dual-signal biosensor based on in situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth and its application in the detection of phenolic substances.

[0038] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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. 100 mL of PBS electrolyte (concentration of 50-100 mmol / L, pH=5.5-7.5) containing phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, hydroquinone) was tested by CV test or It test. The test potential window was -0.4-1.0 V, and the scan rate was 100-500 mV·s -1 ;It tested voltage is 0.19~0.68V.

[0039] After the electrochemical test is completed, 150-180 μL of 0.1 mol / L 4-aminoantipyrine is added to the PBS electrolyte, and the reaction is carried out for 5-10 minutes. The color of the electrolyte is recorded by taking a photo, and UV-vis is used for colorimetric measurement with a wavelength range of 385-700 nm. A standard curve between current value, absorbance and phenolic substance concentration is established.

[0040] 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.

[0041] The electrochemical / colorimetric dual-signal biosensor of the present invention can mutually verify the detection results of the electrochemical / colorimetric dual-signal outputs, thereby improving the detection accuracy and reliability. The biosensor has good selectivity, and has large differences in potential, chromaticity and absorption peak position in the detection of four phenolic substances, namely phenol, meta-cresol, hydroquinone and dichlorophenol, which is an important advantage of the biosensor in detecting phenolic pollutants.

[0042] The present invention is described below in conjunction with specific embodiments.

[0043] Example 1

[0044] Preparation of laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth.

[0045] The carbon cloth was cut into rectangles of 2×1 cm and immersed in concentrated nitric acid for 6 hours. After the immersion, it was ultrasonically cleaned with acetone, anhydrous ethanol and ultrapure water for 3 times respectively, and then dried; the treated carbon cloth was immersed in a CuSO4 solution with a concentration of 0.1 mol / L, taken out after 10 minutes 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 hours. The carbon cloth was taken out and washed with ultrapure water for 3 times, and freeze-dried to obtain carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers.

[0046] 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.

[0047] Example 2

[0048] Preparation of laccase-copper phosphate hybrid nanoflowers grown in situ on carbon cloth.

[0049] The carbon cloth was cut into rectangles of 2×1 cm and immersed in concentrated nitric acid for 12 h. After the immersion, it was ultrasonically cleaned with acetone, anhydrous ethanol and ultrapure water for 3 times respectively, and then 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 containing 0.5 mg / mL laccase (concentration of 100 mmol / L, pH=7.4), and allowed to stand at 25°C for 72 h. The carbon cloth was taken out and washed with ultrapure water for 5 times, and freeze-dried to obtain carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers.

[0050] Figure 2 (a) and (b) are SEM images of the in-situ growth of laccase-copper phosphate hybrid nanoflowers on the carbon cloth prepared in Example 2. From the SEM images, it can be seen that a nanoflower array structure is uniformly grown on the surface of the carbon cloth fiber, and the nanoflower is a nanoflower ball with a diameter of about 3 μm assembled by interlacing nanosheets with a thickness of about 30 nm.

[0051] Example 3

[0052] 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.

[0053] An electrochemical workstation three-electrode system was used, with carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers as the working electrode, Ag / AgCl (3 mol / L KCl) as the reference electrode, and platinum wire as the counter electrode. CV tests were performed on 100 mL of PBS electrolyte (concentration of 50 mmol / L, pH = 5.5) containing phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone). The test potential window was -0.4 to 1.0 V, and the scan rate was 100 mV·s -1 After the electrochemical test, add 150 μL of 0.1 mol / L 4-aminoantipyrine to the PBS electrolyte, react for 5 minutes, take a picture to record the color of the electrolyte, and use UV-vis for measurement with a wavelength range of 385 to 700 nm to detect phenolic substances.

[0054] Example 4

[0055] 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.

[0056] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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 were performed on 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 6.5) containing phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone). The test potential window was -0.4 to 1.0 V, and the scan rate was 100 mV·s -1 After the electrochemical test, add 180 μL of 0.1 mol / L 4-aminoantipyrine to the PBS electrolyte, react for 5 minutes, take a picture to record the color of the electrolyte, and use UV-vis for measurement with a wavelength range of 385 to 700 nm to detect phenolic substances.

[0057] Example 5

[0058] 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.

[0059] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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 were performed on 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 7.5) containing phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone). The test potential window was -0.4 to 1.0 V, and the scan rate was 100 mV·s-1 After the electrochemical test, add 180 μL of 0.1 mol / L 4-aminoantipyrine to the PBS electrolyte, react for 10 min, take a picture to record the color of the electrolyte, and use UV-vis for measurement with a wavelength range of 385 to 700 nm.

[0060] Figure 3 The CV curves, UV-vis spectra and photos of the electrochemical / colorimetric dual-signal biosensor based on the 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 to 1.0 V, an obvious oxidation peak is observed at 0.681 V, 0.655 V, 0.626 V, and 0.191 V, respectively. The oxidation process of phenol, m-cresol, and 2,6-dichlorophenol is a single electron accompanied by a single proton transfer, so the potential value difference is small, while the potential value difference of hydroquinone is large because its catalytic oxidation process is a double electron accompanied by a double proton transfer. Figure 3 As can be seen in (b) and (c), after adding 4-aminoantipyrine, the electrolyte color and UV-visible absorption peak position for different phenolic substances are very different. The absorption peak wavelengths of quinone imine dyes produced by phenol, m-cresol, 2,6-dichlorophenol, hydroquinone and 4-aminoantipyrine are 505nm, 500nm, 518nm and 479nm respectively. The differences in oxidation peak potential value, electrolyte color and UV-visible absorption peak position indicate that the carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower electrode has good selectivity for different phenolic substances.

[0061] Example 6

[0062] 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.

[0063] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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 were performed on 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 7.5) containing phenolic substances (phenol, m-cresol, 2,6-dichlorophenol, and hydroquinone). The test potential window was -0.4 to 1.0 V, and the scan rate was 500 mV·s -1After the electrochemical test, add 180 μL of 0.1 mol / L 4-aminoantipyrine to the PBS electrolyte, react for 10 min, take a picture to record the color of the electrolyte, and use UV-vis for measurement with a wavelength range of 385 to 700 nm to detect phenolic substances.

[0064] Example 7

[0065] 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.

[0066] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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. 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 7.5) containing different concentrations of phenol (0.05-200 μmol / L) was tested with an It voltage of 0.68 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, the reaction lasted for 10 minutes, the color of the electrolyte was recorded by photographing, and UV-vis was used for measurement in the wavelength range of 385-700 nm. A standard curve, i.e. a linear relationship, was established between the current value, absorbance and phenol concentration.

[0067] 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 to 100 μmol / L, and the minimum detection limit was 16.2 nmol / L. Figure 4(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 spectra of PBS electrolytes containing different phenol concentrations, with an obvious absorption peak 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 the absorbance value and the phenol concentration, and the equation is A = 0.03C + 0.001 (R 2 =0.9995), the linear range was 5-140 μmol / L, and the minimum detection limit was 5 μmol / L.

[0068] Example 8

[0069] 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 m-cresol was detected.

[0070] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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. 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 7.5) containing different concentrations of m-cresol (0.05-215 μmol / L) was tested with an It voltage of 0.66 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, the reaction lasted for 10 min, the color of the electrolyte was recorded by photographing, and UV-vis was used for measurement in the wavelength range of 385-700 nm. A standard curve between current value, absorbance and m-cresol concentration was established.

[0071] 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 to 190 μmol / L, and the minimum detection limit was 30 nmol / L. Figure 5 (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 spectra of PBS electrolytes containing different concentrations of m-cresol, with an obvious absorption peak 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 the absorbance value and the concentration of m-cresol, and the equation is A = 6.1×10 -4 C+0.0056(R 2 =0.9949), the linear range was 5 μmol / L to 145 μmol / L, and the minimum detection limit was 5 μmol / L.

[0072] Example 9

[0073] 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.

[0074] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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. 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH=7.5) containing different concentrations of 2,6-dichlorophenol (0.05-170 μmol / L) was tested with an It voltage of 0.63 V. After the electrochemical test, 180 μL of 0.1 mol / L 4-aminoantipyrine was added to the PBS electrolyte, and the reaction lasted for 10 min. The color of the electrolyte was recorded by taking a photo, and UV-vis was used for measurement with a wavelength range of 385-700 nm. A standard curve between current value, absorbance and 2,6-dichlorophenol concentration was established.

[0075] Figure 6 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 9 for the detection of 2,6-dichlorophenol at different concentrations. Figure 6 As can be seen in (a) and (b), with the increase of 2,6-dichlorophenol concentration 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 to 100 μmol / L, and the minimum detection limit was 10 nmol / L. Figure 6 (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 spectra of PBS electrolytes containing different 2,6-dichlorophenol concentrations, with an obvious absorption peak 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.

[0076] Example 10

[0077] 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.

[0078] An electrochemical workstation three-electrode system was used, with carbon cloth in situ 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. 100 mL of PBS electrolyte (concentration of 100 mmol / L, pH = 7.5) containing different concentrations of hydroquinone (0.05-290 μmol / L) was tested with an It 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, the reaction lasted for 10 minutes, the color of the electrolyte was recorded by photographing, and UV-vis was used for measurement in the wavelength range of 385-700 nm. A standard curve between current value, absorbance and hydroquinone concentration was established.

[0079] 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 to 200 μmol / L, and the minimum detection limit was 20 nmol / L. Figure 7 (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 spectra of PBS electrolytes containing different hydroquinone concentrations, with an obvious absorption peak at λ=513-494nm. As the concentration of hydroquinone increases, the absorption peak blue-shifts 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 to 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 to 170 μmol / L, and the minimum detection limit was 0.5 μmol / L.

[0080] 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 carbon cloth in situ grown laccase-copper phosphate hybrid nanoflowers prepared in Example 10.

[0081] Table 1

[0082]

[0083]

[0084] As can be seen from Table 1, the biosensor was validated by the standard addition method. The coal tar wastewater was dephenolized in advance, and then standard concentrations of phenol, m-cresol, 2,6-dichlorophenol and hydroquinone were added for electrochemical and colorimetric detection. As can be seen from the table, the recovery rates of phenol, m-cresol, 2,6-dichlorophenol and hydroquinone in coal tar wastewater by electrochemical signal biosensor are 104.51%, 106.36%, 102.30% and 113.30%, respectively, and the relative standard deviations are 0.63%, 0.67%, 0.34% and 1.43%; the recovery rates of phenol, m-cresol, 2,6-dichlorophenol and hydroquinone in coal tar wastewater by colorimetric signal biosensor are 108.68%, 111.76%, 104.30% and 88.05%, and the relative standard deviations are 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 in the detection of phenol, m-cresol, 2,6-dichlorophenol and hydroquinone in coal tar wastewater.

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

[0086] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. However, all changes made within the protection scope of the independent claims of the present invention are within the protection scope of the present invention.

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

2. The method for preparing the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth according to claim 1, characterized in that: The specific process of the carbon cloth acid treatment is: soak the carbon cloth in nitric acid with a mass concentration of 96%-98% for 6 to 12 hours.

3. The method for preparing the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth according to claim 1, characterized in that: 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 minutes.

4. The method for preparing the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth according to claim 1, characterized in that: The concentration of laccase in the PBS solution containing laccase is 0.1-0.5 mg / mL, the concentration of laccase in the PBS solution containing laccase is 50-100 mmol / L, and the pH value is 6.8-7.

4.

5. The method for preparing the in-situ grown laccase-copper phosphate hybrid nanoflowers on carbon cloth according to claim 1, characterized in that: The temperature of the static reaction is 15-25°C and the time is 24-72 hours.

6. A carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower prepared by the method according to any one of claims 1 to 5, characterized in that: A nanoflower array structure is uniformly grown on the surface of the carbon cloth fiber. The nanoflower is a nanoflower ball with a diameter of 3 μm, which is assembled by interlacing curled nanosheets with a thickness of 30 nm.

7. An electrochemical / colorimetric dual-signal biosensor, characterized in that: It comprises a three-electrode system, wherein the carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflower prepared by the method described in any one of claims 1 to 5 is used as a working electrode, Ag / AgCl is a reference electrode and a platinum wire is a counter electrode.

8. Use of the electrochemical / colorimetric dual-signal biosensor as claimed in claim 7 in the detection of phenolic substances.

9. Use of the electrochemical / colorimetric dual-signal biosensor according to claim 8, characterized in that: Phenolic substances include phenol, m-cresol, 2,6-dichlorophenol and hydroquinone.

10. Use of the electrochemical / colorimetric dual-signal biosensor according to claim 8, characterized in that: The following steps are involved: The carbon cloth in-situ grown laccase-copper phosphate hybrid nanoflowers are used as working electrodes to perform electrochemical detection on PBS electrolyte containing phenolic substances; After the electrochemical detection was completed, 4-aminoantipyrine was added to the PBS electrolyte for colorimetric measurement.

Images