Photoelectrochemical biosensor for detecting DEHP and its preparation method and application

Abstract

The present invention relates to the fields of nano-functional materials and biosensor technology, and in particular to a method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP and its application in detecting plasticizer DEHP in soil. The preparation method is to add N-graphene to a mixed solution of Zn(NO3)·6H2O and hexamethylenetetramine, and prepare it by a one-step hydrothermal method. The introduction of N-graphene not only improves the light absorption capacity and photoelectric conversion efficiency of ZnO nanorods, but also fixes the aptamer on the sensing interface through the π-π stacking effect between DNA and N-graphene. When DEHP is present, the selective binding of DEHP to the aptamer causes an increase in interface steric hindrance, resulting in a decrease in the photocurrent signal, thereby realizing DEHP analysis. The prepared sensor exhibits a wider linear range, a lower detection limit, and better selectivity and stability. The recovery rate of detecting DEHP in actual samples is 94.45% to 105.5%, and the RSD is less than 3.85%.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP, a biosensor prepared by the preparation method, and application of the biosensor in detecting DEHP in soil. Background Art

[0002] Di(2-ethylhexyl) phthalate (DEHP) is an organic compound that is often used as a plasticizer for plastic products such as polyvinyl chloride. It can increase the elasticity and toughness of plastics and is widely used in the plastics industry. DEHP in the soil mainly comes from agricultural film covering, pesticides, sewage irrigation, spray coatings and incomplete combustion of garbage. DEHP is not easy to degrade and difficult to remove after entering the soil. It will not only destroy the soil ecosystem, but also the photosynthetic system of plants. At the same time, DEHP in the soil will be absorbed into the body of crops through plant roots, and further enriched in human tissues through the food chain, affecting the synthesis and metabolism of human hormones, causing endocrine system disorders, and increasing the incidence of carcinogenesis and tumors. In addition, DEHP also damages the liver, blood and other systems of the human body. DEHP pollution has seriously affected soil quality, agricultural product quality and human health. Therefore, it is of great significance to establish a rapid, sensitive and highly selective method for detecting DEHP.

[0003] At present, the main detection methods of DEHP include chromatography, spectroscopy and electrochemical methods. Among them, gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS) in chromatography are the national standard detection methods for DEHP, which have the advantages of accuracy and reliability, but the instruments are expensive and the operation is complicated. The sensitivity and selectivity of the recently developed detection methods such as fluorescence, colorimetry, surface enhanced Raman spectroscopy need to be further improved. The electrochemical method relies on the relationship between the potential, current or resistance of the electrochemically active substances at the electrode interface and the concentration of the target to achieve quantitative detection of the target. Photoelectrochemical sensing technology is an emerging sensing method developed on the basis of electrochemical sensing technology. Photoelectrochemical sensing technology achieves quantitative analysis by causing changes in the photoelectric conversion signal of the interface due to the presence of the target. At the same time, as an emerging generation of traditional electrochemical bioanalysis, photoelectrochemical sensing technology naturally inherits the advantages of low electrochemical cost and simple equipment. In addition, since the excitation and detection signals are completely separated, the background interference can be eliminated, so the photoelectrochemical detection has higher selectivity and sensitivity, and can accurately detect the target DEHP. The key and difficulty of photoelectrochemical sensing technology lies in the design and preparation of high-performance photoactive materials. With the development of photoelectrochemistry, more and more photoactive materials have been developed. Among them, ZnO has been widely used to construct photoelectrochemical biosensors due to its high carrier concentration and good biocompatibility. However, ZnO has some shortcomings as a base material for photoelectrochemical sensors, such as weak light absorption ability and easy recombination of carriers, which leads to its low selectivity and sensitivity. Summary of the invention

[0004] In view of this, in order to solve the technical problems of low selectivity and low sensitivity caused by defects such as weak absorption capacity of photoactive materials and easy recombination of carriers in existing photoelectrochemical detection, on the one hand, the present invention provides a method for preparing a photoelectrochemical biosensor for detecting the plasticizer DEHP, which improves the photoelectric activity of ZnO by introducing N-graphene, a carbon-based material with good conductivity and high electron mobility. The introduction of N-graphene not only improves the light utilization rate of ZnO, but also strengthens the photoelectrochemical sensing interface based on the N-graphene / ZnO nanocomposite material, introduces Apt with selective recognition, and fixes the aptamer (Apt) on the sensing interface through the π-π stacking effect, thereby achieving highly selective, rapid and sensitive detection of DEHP.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP comprises the following steps:

[0007] Step (1), dropping a suspension of N-graphene / ZnO nanocomposite material onto the surface of a FTO electrode, and obtaining an electrode modified with N-graphene / ZnO nanocomposite material after drying;

[0008] Step (2), immobilizing Apt on the electrode surface modified by N-graphene / ZnO nanocomposite material through π-π stacking interaction with N-graphene;

[0009] Step (3), removing unbound Apt on the surface of the electrode modified with the N-graphene / ZnO nanocomposite material to obtain a photoelectrochemical biosensor.

[0010] Preferably, in step (1), the method for preparing the N-graphene / ZnO nanocomposite suspension comprises the following steps:

[0011] Step (1-1), adding N-graphene to a mixed solution of Zn(NO3)·6H2O and hexamethylenetetramine, performing a hydrothermal reaction, and washing and drying the precipitate obtained by the reaction to obtain an N-graphene / ZnO nanocomposite material;

[0012] Step (1-2), ultrasonically dispersing the N-graphene / ZnO nanocomposite material obtained in step (1-1) in ultrapure water to obtain a suspension of N-graphene / ZnO.

[0013] Preferably, step (1-1) is specifically:

[0014] First, prepare 130-170 mL of an aqueous solution containing 40-60 mmol / L of Zn(NO3)·6H2O and 40-60 mmol / L of hexamethylenetetramine;

[0015] Then, 5 to 30 mg of N-graphene is added and stirred to obtain a mixed solution;

[0016] Finally, the mixed solution is subjected to a hydrothermal reaction at a reaction temperature of 100 to 120° C. for a reaction time of 4 to 6 hours, and then cooled to room temperature. The precipitate obtained by the reaction is washed and dried to obtain a N-graphene / ZnO nanocomposite material.

[0017] Preferably, in step (1), the FTO electrode is a pretreated FTO electrode; the pretreatment method of the FTO electrode is as follows:

[0018] The FTO electrode was ultrasonically cleaned in ethanol and ultrapure water, then boiled in a NaOH solution, and then ultrasonically cleaned in ethanol and ultrapure water to complete the pretreatment of the FTO electrode.

[0019] Preferably, in step (1), the boiling time is 30 min, the ultrasonic cleaning time is 15 min, and the NaOH concentration is 0.33 mol / L.

[0020] Preferably, in step (1), the concentration of the suspension is 2.0-4.0 mg / mL, the volume of the drop is 20-40 μL, and the modified area of ​​the FTO electrode is 0.28 cm 2 .

[0021] Preferably, in step (2), the specific fixing method is:

[0022] Incubate at 4°C for 12 h, the volume of Apt added is 20 μL, and the concentration of Apt is 2.0-4.0 μmol / L.

[0023] On the other hand, the present invention further provides a photoelectrochemical biosensor for detecting plasticizer DEHP, which is prepared according to the above-mentioned preparation method of the photoelectrochemical biosensor for detecting plasticizer DEHP.

[0024] In another aspect, the present invention also provides the use of the above-mentioned photoelectrochemical biosensor for detecting plasticizer DEHP in detecting DEHP in soil.

[0025] Preferably, the method for detecting DEHP in soil comprises the following steps:

[0026] The prepared photoelectrochemical biosensor was used to detect DEHP at different concentrations, and the detection solution was PBS buffer solution;

[0027] A xenon lamp was used as the excitation light source, an electrochemical workstation was used as the detection device, a three-electrode system was adopted, and Apt / N-graphene / ZnO / FTO was used as the working electrode to establish a standard curve of the correspondence between the logarithm of DEHP concentration and the photoelectrochemical signal.

[0028] The standard addition method was used to detect DEHP in soil and to evaluate the feasibility of constructing a photoelectrochemical biosensor for actual samples.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] In order to solve the above problems and improve the photoelectric conversion efficiency of ZnO nanomaterials, the composite of ZnO and carbon-based materials has become a solution. After the carbon-based materials are composited with semiconductor materials, the light absorption capacity is improved mainly by increasing the absorption intensity and reducing the reflectivity. The main reasons for the improvement of photoelectric performance are: (1) There is a Schottky barrier between ZnO and carbon-based materials, which promotes the separation of electrons and holes; (2) It accelerates the transfer of electrons and reduces the recombination of electron-hole pairs.

[0031] In the present invention, the photoelectric activity of ZnO is improved by introducing N-graphene, a carbon-based material with good conductivity and high electron mobility. The introduction of N-graphene not only improves the light utilization rate of ZnO, but also can strengthen the photoelectrochemical sensing interface based on the N-graphene / ZnO nanocomposite material, introduces Apt with selective recognition, and fixes the aptamer (Apt) on the sensing interface through the π-π stacking effect, thereby achieving highly selective, rapid and sensitive detection of DEHP.

[0032] The photoelectrochemical biosensor of the present invention has the advantages of simple operation, high sensitivity and good selectivity, and can be successfully used for the rapid and sensitive detection of DEHP in soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The schematic diagram of the N-graphene / ZnO photoelectrochemical biosensor platform for detecting DEHP of the present invention;

[0034] Figure 2 This is a result diagram of the feasibility verification of the photoelectrochemical biosensor for detecting DEHP according to Example 1 of the present invention;

[0035] Figure 3 A is the N-graphene / ZnO photoelectrochemical biosensor in Example 1 of the present invention for different concentrations

[0036] Photoelectrochemical signal of DEHP response, Figure 3 B is the relationship curve between the photoelectrochemical signal and the DEHP concentration;

[0037] Figure 4 This is an investigation on the selectivity and long-term stability of the N-graphene / ZnO photoelectrochemical biosensor in Example 1 of the present invention.

[0038] Figure 5 This is an investigation on the optimization of the conditions of the N-graphene / ZnO photoelectrochemical biosensor in the comparative example of the present invention with respect to Apt concentration, buffer solution pH, and the binding time of DEHP and Apt. DETAILED DESCRIPTION

[0039] Explanation of terms

[0040] Di(2-ethylhexyl) phthalate (DEHP) is a phthalate ester with the chemical formula C 24 H 38 O4, melting point is -50℃, boiling point is 384.9℃, density is 0.985g / cm 3 DEHP is a colorless or light yellow viscous liquid, insoluble in water, and miscible with general organic solvents;

[0041] FTO electrode is a fluorine-doped tin oxide-coated glass sheet with high transparency and conductivity; N-graphene is nitrogen-doped graphene;

[0042] An aptamer is an oligonucleotide sequence or a short polypeptide used as a biological recognition element on a sensor. The Apt sequence of DEHP in the present invention is as follows:

[0043] 5'-CTT TCT GTC CTT CCG TCA CAT CCCACG CAT TCT CCA CT-3'.

[0044] The present invention provides a method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP, comprising the following steps:

[0045] Step (1), dropping the N-graphene / ZnO nanocomposite suspension onto the surface of the FTO electrode, and obtaining an N-graphene / ZnO nanocomposite modified electrode (N-graphene / ZnO / FTO) after drying;

[0046] In step (1), the method for preparing the N-graphene / ZnO nanocomposite suspension preferably comprises the following steps:

[0047] Step (1-1), adding N-graphene to a mixed solution of Zn(NO3)·6H2O and hexamethylenetetramine, performing a hydrothermal reaction, and washing and drying the precipitate obtained by the reaction to obtain an N-graphene / ZnO nanocomposite material;

[0048] Step (1-2), ultrasonically dispersing the N-graphene / ZnO nanocomposite material obtained in step (1-1) in ultrapure water to obtain a suspension of N-graphene / ZnO.

[0049] In the present invention, step (1-1) may specifically be:

[0050] First, prepare 130-170 mL of an aqueous solution containing 40-60 mmol / L of Zn(NO3)·6H2O and 40-60 mmol / L of hexamethylenetetramine. For example, 130 mL of an aqueous solution containing 60 mmol / L of Zn(NO3)·6H2O and 60 mmol / L of hexamethylenetetramine can be prepared; 170 mL of an aqueous solution containing 40 mmol / L of Zn(NO3)·6H2O and 40 mmol / L of hexamethylenetetramine can be prepared; 150 mL of an aqueous solution containing 50 mmol / L of Zn(NO3)·6H2O and 50 mmol / L of hexamethylenetetramine can be prepared, etc. The specific solution can be selected according to actual needs, and no special requirements are made for this;

[0051] Next, 5 to 30 mg (for example, 5 to 25 mg, 10 to 25 mg, or 15 mg of N-graphene may be added, and no special requirement is made thereto) of N-graphene is added, and stirred at a speed of 800 r / min for 20 min at room temperature;

[0052] Finally, the mixed solution is transferred to a hydrothermal reactor for hydrothermal reaction, the reaction temperature is 100-120°C (for example, the reaction temperature can be 100°C, 110°C or 120°C, etc., no special requirements are made for this), the reaction time is 4-6h (for example, the reaction time can be 4h, 5h or 6h, no special requirements are made for this, it can be selected according to actual needs), and it is naturally cooled to room temperature. The precipitate obtained by the reaction is washed three times with ethanol and ultrapure water respectively and dried to obtain a N-graphene / ZnO nanocomposite material; the prepared N-graphene / ZnO nanocomposite material is ultrasonically dispersed in ultrapure water to obtain a N-graphene / ZnO nanocomposite suspension (the concentration is preferably 2.0-4.0mg / mL, for example, it can be 2.0mg / mL or 3.0mg / mL or 4.0mg / mL).

[0053] In the present invention, in step (1), the FTO electrode is a pretreated FTO electrode; the pretreatment method of the FTO electrode is as follows:

[0054] The FTO electrode was ultrasonically cleaned in ethanol and ultrapure water, then boiled in a NaOH solution, and then ultrasonically cleaned in ethanol and ultrapure water to complete the pretreatment of the FTO electrode.

[0055] In the pretreatment method of the FTO electrode in step (1), the preferred boiling time is 30 min, the ultrasonic cleaning time is 15 min, and the NaOH concentration is 0.33 mol / L.

[0056] In the present invention, the concentration of the suspension in step (1) is 2.0-4.0 mg / mL, the volume of the drop is 20-40 μL, and the modified area of ​​the FTO electrode is 0.28 cm 2 .

[0057] In the present invention, in step (1), it is preferred to dry in an incubator, and the drying temperature can be set according to demand, such as 37°C.

[0058] Step (2), immobilizing Apt on the electrode surface modified by N-graphene / ZnO nanocomposite material through π-π stacking interaction with N-graphene;

[0059] In the present invention, in step (2), the specific fixing method is:

[0060] Incubate at 4°C for 12 h, the volume of Apt added is 20 μL, and the concentration of Apt is 2.0-4.0 μmol / L (for example, it can be 2.0 μmol / L, 3.0 μmol / L, or 4.0 μmol / L, which can be set according to actual needs).

[0061] Step (3), removing unbound Apt on the surface of the electrode modified with the N-graphene / ZnO nanocomposite material, and obtaining a photoelectrochemical biosensor (Apt / N-graphene / ZnO / FTO).

[0062] In the present invention, step (3) may specifically be:

[0063] The electrode surface was washed with 0.01 mol / L PBS (pH 7.4) buffer solution to remove unbound Apt, and a photoelectrochemical biosensor (Apt / N-graphene / ZnO / FTO) was obtained. Thus, the N-graphene / ZnO-based photoelectrochemical biosensor for DEHP detection was prepared.

[0064] The present invention also includes:

[0065] Step (4), fixing different concentrations of DEHP on the sensing interface of the modified Apt, incubating for a period of time, and evaluating the detection performance of the prepared biosensor by photoelectrochemical testing. The specific binding of DEHP and Apt causes an increase in interface steric hindrance, resulting in a decrease in PEC signal, thereby achieving DEHP analysis.

[0066] Step (5), DEHP in soil samples was detected by standard addition method to evaluate the feasibility of constructing photoelectrochemical biosensor for complex actual samples.

[0067] On the other hand, the present invention further provides a photoelectrochemical biosensor for detecting plasticizer DEHP, which is prepared according to the above-mentioned preparation method of the photoelectrochemical biosensor for detecting plasticizer DEHP.

[0068] In another aspect, the present invention also provides the use of the above-mentioned photoelectrochemical biosensor for detecting plasticizer DEHP in detecting DEHP in soil.

[0069] In the present invention, the method for detecting DEHP in soil comprises the following steps:

[0070] The prepared photoelectrochemical biosensor was used to detect DEHP at different concentrations, and the detection solution was PBS buffer solution;

[0071] Wherein, the detection solution is preferably a 0.01 mol / L PBS buffer solution, the pH thereof is preferably 6.2 to 8.2, and the test potential is 0 V;

[0072] Specifically, it can be preferably:

[0073] 20 to 40 μL of different concentrations of DEHP (5.0×10 -11 ~1.0×10 -7 mol / L) was fixed on the sensing interface of the modified Apt. After incubation at room temperature for 10 to 60 min, the interface was washed with PBS buffer solution, and the detection performance of the prepared photoelectrochemical biosensor was evaluated using the current-time mode (IT) of the photoelectrochemical technique.

[0074] A xenon lamp was used as the excitation light source, an electrochemical workstation was used as the detection device, a three-electrode system was used, and Apt / N-graphene / ZnO / FTO was used as the working electrode to establish a standard curve of the relationship between the logarithm of DEHP concentration and the photoelectrochemical signal. The specific preferred curve is:

[0075] A 300W xenon lamp was used as the excitation light source, and a CHI660E electrochemical workstation was used as the detection device. All experiments used a three-electrode system, with platinum wire as the counter electrode, Ag / AgCl (saturated KCl solution) as the reference electrode, and Apt / N-graphene / ZnO / FTO as the working electrode; a standard curve of the relationship between the logarithm of DEHP concentration and the photoelectrochemical signal was established.

[0076] The standard addition method was used to detect DEHP in soil and to evaluate the feasibility of constructing a photoelectrochemical biosensor for actual samples.

[0077] The technical solution of the present invention is clearly described in detail below in conjunction with specific embodiments.

[0078] Example 1

[0079] The present invention first provides a highly selective photoelectrochemical biosensor for sensitive detection of DEHP, such as Figure 1 As shown, the FTO electrode is a pretreated FTO electrode, and the pretreatment method is as follows:

[0080] The FTO electrode was ultrasonically cleaned in ethanol and ultrapure water for 15 minutes, then boiled in a 0.33 mol / L NaOH solution for 30 minutes, and then ultrasonically cleaned in ethanol and ultrapure water to complete the pretreatment of the FTO electrode.

[0081] The following steps are involved:

[0082] 1) Prepare 150mL of an aqueous solution containing 50mmol / L of Zn(NO3)·6H2O and 50mmol / L of hexamethylenetetramine; then add 15mg of N-graphene and stir at 800r / min for 20min at room temperature; finally transfer the mixed solution to a hydrothermal reactor for hydrothermal reaction at a temperature of 100°C for 6h and cool naturally to room temperature. Wash the precipitate obtained by the reaction three times with ethanol and ultrapure water respectively and dry it to obtain an N-graphene / ZnO nanocomposite material;

[0083] 2) The obtained nanocomposite material was dispersed in water to obtain a suspension with a concentration of 3.0 mg / mL. The suspension was then dripped onto the pretreated FTO electrode. The volume of the dripped suspension was 20 μL, and the modified area of ​​the FTO electrode was 0.28 cm 2 .

[0084] 3) The FTO electrode to which the suspension was added was placed in an incubator at 37° C. to dry, thereby obtaining an electrode modified with N-graphene / ZnO nanocomposite material (N-graphene / ZnO / FTO).

[0085] 4) Apt was fixed on the electrode surface modified with N-graphene / ZnO nanocomposite material through π-π stacking interaction with N-graphene and incubated at 4°C for 12 h. The volume of Apt added was 20 μL and the concentration of Apt was 2.5 μmol / L.

[0086] 5) After Apt was modified on the electrode, the interface was cleaned with a PBS (pH 7.4) buffer solution with a concentration of 0.01 mol / L to obtain a photoelectrochemical biosensor (Apt / N-graphene / ZnO / FTO). At this point, the preparation of the photoelectrochemical biosensor for DEHP detection was completed.

[0087] Example 2

[0088] The feasibility of the photoelectrochemical biosensor prepared in Example 1 for detecting DEHP was verified:

[0089] like Figure 2 As shown in (A), a weak photocurrent response was detected on the bare FTO electrode (curve a). Then, the N-graphene / ZnO nanocomposite was modified on the FTO electrode, and the photocurrent was significantly enhanced (curve b, 1.273 μA). The π-π stacking effect between the aromatic ring and the base in N-graphene was further used to fix Apt, and the interface steric hindrance increased, and the photocurrent decreased (curve c, 0.8489 μA). When DEHP (1×10 -9 mol / L), the specific binding of Apt and DEHP formed a DEHP-Apt complex with greater steric hindrance, and the photocurrent decreased (curve d, 0.5669μA); when the DEHP concentration increased (1×10 -7 mol / L), the photocurrent further decreased (curve e, 0.2684 μA). The above results show that the constructed photoelectrochemical biosensor can be used for DEHP detection.

[0090] EIS was further used to investigate the assembly and detection process of the photoelectrochemical biosensor. Figure 2 As shown in (B), compared with bare FTO (curve a, 42.64Ω), the charge transfer resistance (R et ) increased to 111.6Ω (curve b), indicating that the N-graphene / ZnO nanocomposite was successfully modified to the electrode interface. After the introduction of Apt, the interface steric hindrance increased, and R et When DEHP is present, Apt forms a complex with DEHP with greater steric hindrance, R et increases (curve d, 144.8Ω). As the DEHP concentration increases, R et It further increases (curve e, 179.3Ω), and the above results are consistent with the trend of photocurrent response change.

[0091] Example 3

[0092] The photoelectrochemical test method was used to evaluate the detection performance of the photoelectrochemical biosensor prepared in Example 1 for DEHP:

[0093] like Figure 3 As shown in (A), as the DEHP concentration increases, the photocurrent response gradually decreases. -11 ~1.0×10 -7 In the range of mol / L, the photocurrent and DEHP concentration (C DEHP ) has a good linear relationship, such as Figure 3 As shown in (B); the linear equation is I = -0.155lgC DEHP -0.847, linear correlation coefficient (R 2 ) was 0.997, and the detection limit was 1.4×10 -11 mol / L.

[0094] Example 4

[0095] Anti-interference analysis of the photoelectrochemical biosensor prepared in Example 1 in the detection of DEHP:

[0096] like Figure 4 As shown in (A), compared with 1.0×10 -9 mol / L DEHP, 10 times the concentration of interference (1.0×10 -8 The introduction of 10 mol / L did not cause a significant change in the photocurrent, but only when DEHP was present did the photocurrent change significantly. The above results prove that the constructed photoelectrochemical biosensor has high selectivity for the analysis of DEHP. The long-term stability of the sensor was further investigated, and the target was detected using the biosensor stored at 4°C. Figure 4 As shown in (B), the photocurrent response of the sensor was observed for 7 consecutive days without obvious attenuation, and the RSD was 0.701%, indicating that the developed photoelectrochemical biosensor has good stability.

[0097] Comparative Example 1

[0098] In order to obtain the best analytical performance of the photoelectrochemical biosensor, the Apt concentration was optimized:

[0099] like Figure 5 As shown in (A), the photocurrent decreases with the increase of Apt concentration in the range of 0.5-3.0 μmol / L. When the Apt concentration increases to 2.0 μmol / L, the photocurrent tends to stabilize, which may be because the amount of Apt loaded on the electrode interface reaches saturation. Therefore, the Apt concentration of 2.5 μmol / L was selected as the optimal concentration for constructing the photoelectrochemical biosensor.

[0100] Comparative Example 2

[0101] In order to obtain the best analytical performance of the photoelectrochemical biosensor, the pH value of the buffer solution during the sensor detection process was optimized:

[0102] like Figure 5As shown in (B), the photocurrent response increases in the pH range of 6.2 to 7.4. In the pH range of 7.4 to 8.2, the photocurrent response decreases, and the photocurrent response is the largest at pH 7.4. The possible reason is that at lower pH conditions, excess H + The ZnO was dissolved and, under weak alkaline conditions, ZnO was slightly corroded, resulting in a decrease in the photocurrent. Therefore, the optimal pH value of the buffer was selected to be 7.4.

[0103] Comparative Example 3

[0104] In order to obtain the best analytical performance of the photoelectrochemical biosensor, the binding time of DEHP and Apt was optimized:

[0105] like Figure 5 As shown in (C), within the range of 10 to 60 minutes, as the binding time increases, the amount of DEHP specifically recognized by the interface increases, resulting in a gradual decrease in the photocurrent signal. After 40 minutes, the photocurrent tends to stabilize, and the DEHP captured by the interface Apt has reached saturation. Therefore, this work selects 50 minutes as the optimal binding time for DEHP and Apt.

[0106] Comparative Examples 1-3 are all verification screenings carried out on the basis of Example 1.

[0107] Example 5

[0108] The sensors and detection methods prepared in Example 1 and the comparative example were used to detect DEHP in soil samples taken from a greenhouse of Jiangsu University (Zhenjiang, China) using a standard addition method.

[0109] The specific steps of sample pretreatment were as follows: the soil samples were air-dried at room temperature, ground, and mixed evenly; sieved with 60-mesh stainless steel, sealed in a brown glass bottle, and stored at -20°C; ultrasonically extracted the sample soil three times with 20 mL of acetone / n-hexane mixed solvent (1:1, V / V), centrifuged at 6000 rpm for 10 min, and the supernatant was extracted; finally, the sample impurities were filtered with a 0.22 μm aqueous filter membrane.

[0110] The pre-treated samples were used for detection and analysis. As shown in Table 1, no DEHP was detected in the soil using the photoelectrochemical sensor and gas chromatography-mass spectrometry. In the spiked samples, the recovery rate of DEHP detected by the constructed photoelectrochemical biosensor was 94.45% to 105.5%, and the RSD was less than 3.85%; the recovery rate of DEHP detected by gas chromatography-mass spectrometry was 105.5% to 107.1%. Compared with the national standard detection method, the photoelectrochemical sensor developed in this work has comparable detection performance and a lower detection limit, which indicates that the constructed photoelectrochemical biosensor has potential application value in detecting DEHP in actual samples.

[0111] Table 1 Detection results of DEHP in soil using sensors constructed in Example 1 and Comparative Example

[0112]

[0113] Note: “-”: not detected, “ / ”: not tested in experiments

[0114] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail in this specification with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP, characterized in that: The steps include: Step (1), dropping a suspension of N-graphene / ZnO nanocomposite material onto the surface of a FTO electrode, and obtaining an electrode modified with N-graphene / ZnO nanocomposite material after drying; Step (2), immobilizing Apt on the electrode surface modified by N-graphene / ZnO nanocomposite material through π-π stacking interaction with N-graphene; Step (3), removing unbound Apt on the surface of the electrode modified with the N-graphene / ZnO nanocomposite material to obtain a photoelectrochemical biosensor; In step (1), the method for preparing the N-graphene / ZnO nanocomposite suspension comprises the following steps: Step (1-1), adding N-graphene to a mixed solution of Zn(NO3)·6H2O and hexamethylenetetramine, performing a hydrothermal reaction, and washing and drying the precipitate obtained by the reaction to obtain an N-graphene / ZnO nanocomposite material; Step (1-2), ultrasonically dispersing the N-graphene / ZnO nanocomposite material obtained in step (1-1) in ultrapure water to obtain a suspension of N-graphene / ZnO.

2. The method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP according to claim 1, characterized in that: Step (1-1) is specifically: First, prepare 130-170 mL of an aqueous solution containing 40-60 mmol / L of Zn(NO3)·6H2O and 40-60 mmol / L of hexamethylenetetramine; Then, 5 to 30 mg of N-graphene is added and stirred to obtain a mixed solution; Finally, the mixed solution is subjected to a hydrothermal reaction at a reaction temperature of 100 to 120° C. for a reaction time of 4 to 6 hours, and then cooled to room temperature. The precipitate obtained by the reaction is washed and dried to obtain a N-graphene / ZnO nanocomposite material.

3. The method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP according to claim 1, characterized in that: In step (1), the FTO electrode is a pretreated FTO electrode; the pretreatment method of the FTO electrode is as follows: The FTO electrode was ultrasonically cleaned in ethanol and ultrapure water, then boiled in a NaOH solution, and then ultrasonically cleaned in ethanol and ultrapure water to complete the pretreatment of the FTO electrode.

4. The method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP according to claim 3, characterized in that: In step (1), the boiling time is 30 min, the ultrasonic cleaning time is 15 min, and the NaOH concentration is 0.33 mol / L.

5. The method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP according to claim 1, characterized in that: In step (1), the concentration of the suspension is 2.0-4.0 mg / mL, the volume of the drop is 20-40 μL, and the modified area of ​​the FTO electrode is 0.28 cm 2 .

6. A method for preparing a photoelectrochemical biosensor for detecting plasticizer DEHP according to any one of claims 1 to 5, characterized in that: In step (2), the specific fixing method is: Incubate at 4°C for 12 h, the volume of Apt added is 20 μL, and the concentration of Apt is 2.0-4.0 μmol / L.

7. A photoelectrochemical biosensor for detecting plasticizer DEHP, characterized in that: The photoelectrochemical biosensor for detecting the plasticizer DEHP is prepared according to any one of claims 1-6.

8. Use of the photoelectrochemical biosensor for detecting plasticizer DEHP as claimed in claim 7 in detecting DEHP in soil.

9. The use according to claim 8, characterized in that: The method for detecting DEHP in soil includes the following steps: The prepared photoelectrochemical biosensor was used to detect DEHP at different concentrations, and the detection solution was PBS buffer solution; A xenon lamp was used as the excitation light source, an electrochemical workstation was used as the detection device, a three-electrode system was used, and Apt / N-graphene / ZnO / FTO was used as the working electrode to establish a standard curve of the relationship between the logarithm of DEHP concentration and the photoelectrochemical signal. The standard addition method was used to detect DEHP in soil and to evaluate the feasibility of constructing a photoelectrochemical biosensor for actual samples.

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