CN112816639B - Construction method of photoelectrochemical aptamer sensor for sensitive detection of enrofloxacin - Google Patents

Abstract

The invention provides a construction method of a photoelectrochemical sensor for sensitively detecting Enrofloxacin (ENR), which comprises the following steps: step 1, preparing bismuth oxybromide (Bi) ₄ O ₅ Br ₂ ) Nanosheets; step 2, preparing aza-bismuth oxybromide (N-Bi) ₄ O ₅ Br ₂ ) A nanocomposite; and step 3, constructing a photoelectrochemical sensor for sensitively detecting Enrofloxacin (ENR). Compared with the traditional detection method, the photoelectrochemical detection method of ENR provided by the invention has the characteristics of simpler and more flexible operation, simpler instruments and equipment, wide detection range, low detection limit, low detection cost and the like.

Description

A Construction Method of Photoelectrochemical Aptasensor for Sensitive Detection of Enrofloxacin

technical field

The invention belongs to the field of electrochemical detection, and refers to a construction method and application of a photoelectrochemical aptamer sensor for detecting enrofloxacin in lake water.

Background technique

Enrofloxacin (ENR) is a fluoroquinolone antibiotic, which is a special quinolone antibacterial drug for livestock, poultry and aquatic products, and is widely used in human activities. However, increased ENR levels found in aquatic environments can have serious health consequences for humans and livestock. The European Commission and the Chinese Ministry of Agriculture stipulate that the maximum residue limit (MRLs) of ENR and its active metabolite ciprofloxacin in animal muscle tissue is 100 μg/kg, while the US Food and Drug Administration completely prohibits its use in poultry. Some antibiotic detection methods have been developed, including high performance liquid chromatography (HPLC), liquid chromatography-electrospray ionization-tandem mass spectrometry, colorimetry, fluorescence and other research methods. Although these methods can meet the requirements of sensitivity and specificity detection, there are certain limitations in practical application. For example, chromatography can be used for qualitative and quantitative detection, and the detection results are relatively accurate and reliable, with high sensitivity and good reproducibility, but the equipment used is expensive, the operation is complicated, and professional technicians are required, so it is not suitable for large-scale samples. Processing and analysis and rapid detection in the field.

Photoelectrochemical (PEC) technology, as an emerging electroanalysis technology, has received extensive attention in many fields, such as biology, environmental science, and medicine. As a new type of analysis technology, PEC sensing technology has many advantages that cannot or are difficult to achieve on traditional electrochemical platforms. Since PEC uses two different forms of excitation and detection signals, and the background signal of this technique is low, it has higher sensitivity.

Based on nitrogen bismuth oxybromide (N-Bi ₄ O ₅ Br ₂ ) nanocomposites as photoelectric active materials, a photoelectrochemical sensing platform has not been reported for the photoelectrochemical detection of enrofloxacin in lake water.

Contents of the invention

The invention aims to provide a photoelectrochemical aptamer sensor integrating the advantages of high sensitivity, high selectivity, wide measurement range and the like. The preparation process of the sensor is simple, the cost is low, and the purpose of rapid and quantitative detection of ENR is realized.

The scheme adopted is summarized as follows: the prepared N-Bi ₄ O ₅ Br ₂ nanocomposite is used as the photoelectric active material to create an ultrasensitive photoelectrochemical sensing platform. Utilize the properties of N-Bi ₄ O ₅ Br ₂ nanocomposite to absorb and respond quickly to visible light, and play a role of signal amplification in the detection system. When the target ENR is added, the N-Bi ₄ O ₅ Br ₂ nanocomposite is excited by visible light, and the generated hole pairs oxidize ENR, and the oxidation products of ENR can effectively prevent the generated electron-hole pairs from recombining, As a result, the photocurrent response intensity is enhanced, and the relationship between the photocurrent response value and the ENR concentration is established, so as to achieve the purpose of rapid, sensitive and selective detection of the ENR content in the lake water.

The present invention is achieved through the following specific technical solutions:

A method for constructing a photoelectrochemical aptasensor for enrofloxacin (ENR) sensitive detection, comprising the steps:

Step 1, preparation of bismuth oxybromide (Bi ₄ O ₅ Br ₂ ) nanosheets by solvothermal method:

Firstly, bismuth bromide (BiBr ₃ ) was dissolved in ethylene glycol, NaOH solution was added dropwise under stirring, and then transferred into a polytetrafluoroethylene autoclave for solvothermal reaction. The solid product was recovered by filtration, washing and centrifugation, and then dried to obtain Bi ₄ O ₅ Br ₂ nanosheets.

Step 2. Preparation of azabismuth oxybromide N-Bi ₄ O ₅ Br ₂ nanocomposites:

The Bi ₄ O ₅ Br ₂ nanosheets were uniformly mixed with different qualities of urea, heated in a muffle furnace, and the obtained products were washed and dried with dimethyl sulfoxide and distilled water, respectively, to obtain N-Bi ₄ O with different doping contents ₅ Br ₂ composite.

Step 3. Construct a photoelectrochemical aptasensor for sensitive detection of enrofloxacin (ENR):

Disperse the N-Bi ₄ O ₅ Br ₂ composite material in N,N-dimethylformamide DMF to obtain the N-Bi ₄ O ₅ Br ₂ dispersion liquid, which is drop-coated on the ITO electrode, and then drop-coated Chitosan CS solution, after drying, drop the glutaraldehyde GA solution on the surface of the electrode, and place it at room temperature. After a period of time, rinse with PBS, and then add bovine serum albumin BSA solution dropwise to finally obtain an aptamer-modified material electrode, which is a photoelectrochemical aptasensor for enrofloxacin detection.

In step 1, the dosage ratio of _BiBr3 to NaOH solution is 1.33g:12.6mL, wherein the concentration of NaOH solution is 2mol/L; the hydrothermal reaction temperature is 140-160°C, and the reaction time is 10-12h ; The centrifugal rate is 8000-9000rmp/s, and the centrifugal time is 6-8min; the drying temperature is 50-60°C, and the drying time is 10-12h.

In step 2, the mass percentage of nitrogen in the N—Bi ₄ O ₅ Br ₂ is 10%-50%; the temperature of the muffle furnace is set at 200-220° C., and the reaction time is 3-5 hours.

In step 3, the N-Bi ₄ O ₅ Br ₂ dispersion is 2 mg/mL; the CS concentration is 0.1%; the GA concentration is 2.5%, the amount of CS solution is 10 μL, the amount of GA solution is 20 μL, and the CS The reaction time with GA is 0.5～1h;

The sequence of the ENR aptamer is: 5′-NH ₂ -CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCTCTC TGA GCC CGG GTT ATT TCA GGG GGA-3′; The reaction time is 10 h; the BSA concentration is 3%; the PBS concentration is 0.1 mol/L, pH=7.4, and the dosage is 10-20 mL.

The photoelectrochemical aptamer sensor constructed by the present invention is used to detect enrofloxacin, ENR solutions of different concentrations are dropped on the photoelectrochemical aptamer sensor used for enrofloxacin detection, and incubated for a period of time, Using ITO electrode as the working electrode, saturated calomel electrode as the reference electrode, and platinum wire as the counter electrode, through the three-electrode system of the electrochemical workstation, the photoelectric analysis is carried out in sequence according to the concentration under the irradiation of the xenon lamp light source.

Among them, the concentration of ENR is 0.1～10 ⁸ ng/mL, respectively 0.1 ng/mL, 1 ng/mL, 100 ng/mL, 10 ³ ng/mL, 10 ⁴ ng/mL, 10 ⁵ ng/mL, 10 ⁶ ng/mL mL, 10 ⁷ ng/mL and 10 ⁸ ng/mL, the drop volume is 10-20 μL; the incubation temperature is 37°C; the intensity of the xenon lamp light source is 25%-100%.

The beneficial effects of the present invention are:

The present invention prepares N-Bi ₄ O ₅ Br ₂ nanocomposites as photoelectric active materials, successfully establishes a photoelectrochemical sensing platform, and establishes a photoelectrochemical detection method for enrofloxacin in lake water. Its characteristics and advantages are expressed as follows :

(1) The present invention prepares N-Bi ₄ O ₅ Br ₂ nanocomposites as photoelectric active materials to construct photoelectrochemical aptamer sensors, which amplifies the photocurrent response signal.

(2) The present invention uses urea to further dope Bi ₄ O ₅ Br ₂ . On the one hand, the doping of semiconductor materials can play a role in signal amplification and broaden the absorption of visible light; on the other hand, using aptamers The specific recognition of ENR, combined with ENR molecules, further improves the stability and is more conducive to the transfer of electrons.

(3) The signal amplification method and detection mode proposed by the present invention realize the ultra-sensitive detection of ENR. In the concentration interval of 0.1ng/mL～10 ⁶ ng/mL, the logarithm of the ENR concentration (lg C _ENR ) and The photocurrent response value showed a good linear relationship, and the detection limit could reach 0.033ng/mL.

(4) Compared with traditional detection methods, the photoelectrochemical detection method of ENR proposed in the present invention has the characteristics of more convenient and flexible operation, simpler equipment, wide detection range, low detection limit, and low detection cost.

Description of drawings

Figure 1 shows the photocurrent response (A) of the prepared materials Bi ₄ O ₅ Br ₂ (a) and N-Bi ₄ O ₅ Br ₂ (b) and 10% N-Bi ₄ O ₅ Br ₂ ( a), 20% N-Bi ₄ O ₅ Br ₂ (b), 30% N-Bi ₄ O ₅ Br ₂ (c), 40% N-Bi ₄ O ₅ Br ₂ (d) and 50% N-Bi Photocurrent response diagram of ₄ O ₅ Br ₂ (e) (B);

Fig. 2 is the corresponding relationship diagram (A) and the linear relationship diagram (B) of the ENR concentration and the photocurrent response value.

Detailed ways

The present invention is described in detail below in conjunction with examples, but the present invention is not limited to these examples.

Example 1:

(1) Preparation of bismuth oxybromide (Bi ₄ O ₅ Br ₂ ) nanosheets by solvothermal method

First, 1.33g of bismuth bromide (BiBr ₃ ) was dissolved in 50mL of ethylene glycol, and 2mol/L NaOH 12.6mL was added dropwise with stirring, then transferred to a polytetrafluoroethylene autoclave, and reacted at 140°C for 12h. After the reactor was cooled to room temperature, we washed the obtained samples three times with ethanol and deionized water, centrifuged them at 9000rmp for 8min in the laboratory, and finally dried them in an oven at 60°C for 12h. According to _{this method} , _Bi4O5Br2 nanosheets were obtained.

(2) Preparation of N-Bi ₄ O ₅ Br ₂ nanocomposites

Weigh 0.2g Bi ₄ O ₅ Br ₂ and 0.02g urea to mix, heat in a muffle furnace at 220°C for 3h, and wash the obtained compound with dimethyl sulfoxide and distilled water. Then it was dried at 60°C for 12 hours to obtain 10% N-Bi ₄ O ₅ Br ₂ .

Different amounts of urea (0.04g, 0.06g, 0.08g, and 0.1g) were prepared in the same steps to obtain N-Bi ₄ O ₅ Br ₂ composites with different contents and marked as 20% N-Bi ₄ O ₅ Br ₂ , 30% N-Bi ₄ O ₅ Br ₂ , 40% N-Bi ₄ O ₅ Br ₂ and 50% N-Bi ₄ O ₅ Br ₂ .

Figure 1(A) is the photocurrent response spectrum of Bi ₄ O ₅ Br ₂ (a) and N-Bi ₄ O ₅ Br ₂ (b) obtained in this example. It can be seen from the figure that the photocurrent response value is enlarged by 18 times, The current response signal is enhanced; Fig. 1(B) is 10% N-Bi ₄ O ₅ Br ₂ (a), 20% N-Bi ₄ O ₅ Br ₂ (b) obtained in Example 1 with different composition ratios, Photocurrent response graphs of 30% N-Bi ₄ O ₅ Br ₂ (c), 40% N-Bi ₄ O ₅ Br ₂ (d) and 50% N-Bi ₄ O ₅ Br ₂ (e), which can be viewed The photocurrent response of 30% N-Bi ₄ O ₅ Br ₂ (c) is the strongest at 0.37 μA, so we choose this proportion of N-Bi ₄ O ₅ Br ₂ as the material to build the subsequent sensing platform.

(3) Construction of photoelectrochemical aptasensors

Dissolve 2 mg of N-Bi ₄ O ₅ Br ₂ complex in 1 mL of N,N-dimethylformamide (DMF) to prepare N-Bi ₄ O ₅ Br ₂ dispersion, take 20 μL of N-Bi ₄ O ₅ Br ₂ to disperse The solution was modified on the ITO electrode, and then 10 μL of chitosan (CS) solution was drip-coated, and dried under an infrared lamp. Next, drop 20 μL of glutaraldehyde (GA) solution on the surface of the electrode, and place it at room temperature to react for 1 hour. After the reaction is completed, rinse with 0.1mol/L phosphate buffer solution (PBS) with pH=7.4 to remove excess electrode surface. the ga. A 2 μM ENR aptamer solution was prepared with PBS as a solvent, and the ENR aptamer was added dropwise on the electrode. After 12 h of reaction, it was rinsed with PBS to remove excess unadsorbed aptamer, and then 10 μL of bovine serum albumin ( BSA) solution was allowed to stand for 0.5 h to block the non-specific active sites, and finally the aptamer-modified material electrode was obtained.

Thereafter, different concentrations of ENR solutions were dropped onto the aptamer-modified material electrode and incubated at 37°C for a period of time. Using ITO electrode as the working electrode, saturated calomel electrode as the reference electrode, and platinum wire as the counter electrode, through the three-electrode system of the electrochemical workstation, the photoelectric analysis is carried out in sequence according to the concentration under the irradiation of the xenon lamp light source.

Fig. 2(A) is the corresponding relationship between the ENR concentration (a→j:0.1→10 ⁸ ng/mL) obtained in this example and the photocurrent response value, and Fig. 2(B) is its linear relationship diagram. From the figure It can be seen that with the increase of ENR concentration, the photocurrent response of N-Bi ₄ O ₅ Br ₂ gradually increases, and there is a good linear relationship between the photocurrent and the ENR concentration (R ² =0.991), the linear equation is I=0.01391+0.00456 lg[C _ENR (ng mL ⁻¹ )]. As shown in Figure 2(B), in the concentration range of 0.1ng/mL～10 ⁶ ng/mL, the logarithmic value of ENR concentration (lg C _ENR ) and the photocurrent response value showed a good linear relationship, and the detection limit could reach 0.033 ng/mL.

Example 2:

(1) Preparation of bismuth oxybromide (Bi ₄ O ₅ Br ₂ ) nanosheets by solvothermal method

First, 2.66g of bismuth bromide (BiBr ₃ ) was dissolved in 50mL of ethylene glycol, and 2mol/L NaOH25.2mL was added dropwise with stirring, then transferred to a polytetrafluoroethylene autoclave, and reacted at 140°C for 12h. After the reactor was cooled to room temperature, we washed the obtained samples three times with ethanol and deionized water, centrifuged them at 9000rmp for 8min in the laboratory, and finally dried them in an oven at 60°C for 12h. According to _{this method} , _Bi4O5Br2 nanosheets were obtained.

(2) Preparation of N-Bi ₄ O ₅ Br ₂ nanocomposites

Weigh 0.4g Bi ₄ O ₅ Br ₂ and 0.04g urea to mix, heat in a muffle furnace at 220°C for 3h, and wash the obtained compound with dimethyl sulfoxide and distilled water. Then it was dried at 60°C for 12 hours to obtain 10% N-Bi ₄ O ₅ Br ₂ .

Different amounts of urea (0.08g, 0.12g, 0.16g and 0.2g) were prepared in the same steps to obtain different contents of N-Bi ₄ O ₅ Br ₂ composites and marked as 20% N-Bi ₄ O ₅ Br ₂ , 30% N-Bi ₄ O ₅ Br ₂ , 40% N-Bi ₄ O ₅ Br ₂ and 50% N-Bi ₄ O ₅ Br ₂ .

Step (3) is the same as step (3) in Example 1.

Example 3:

(1) Preparation of bismuth oxybromide (Bi ₄ O ₅ Br ₂ ) nanosheets by solvothermal method

First, 1.33g of bismuth bromide (BiBr ₃ ) was dissolved in 50mL of ethylene glycol, and 2mol/L NaOH 12.6mL was added dropwise with stirring, then transferred to a polytetrafluoroethylene autoclave, and reacted at 160°C for 10h. After the reactor was cooled to room temperature, we washed the obtained samples three times with ethanol and deionized water, centrifuged them at 9000rmp for 8min in the laboratory, and finally dried them in an oven at 60°C for 12h. According to _{this method} , _Bi4O5Br2 nanosheets were obtained.

(2) Preparation of N-Bi ₄ O ₅ Br ₂ nanocomposites

Weigh 0.2g Bi ₄ O ₅ Br ₂ and 0.02g urea to mix, heat in a muffle furnace at 200°C for 5h, and wash the obtained compound with dimethyl sulfoxide and distilled water. Then it was dried at 60°C for 12 hours to obtain 10% N-Bi ₄ O ₅ Br ₂ . Different amounts of urea (0.04g, 0.06g, 0.08g and 0.1g) were prepared in the same steps to obtain different contents of N-Bi ₄ O ₅ Br ₂ composites and marked as 10% N-Bi ₄ O ₅ Br ₂ , 20% N-Bi ₄ O ₅ Br ₂ , 30% N-Bi ₄ O ₅ Br ₂ , 40% N-Bi ₄ O ₅ Br ₂ and 50% N-Bi ₄ O ₅ Br ₂ .

Step (3) is the same as step (3) in Example 1.

Claims (8)

1. A construction method of a photoelectrochemical aptamer sensor for sensitive detection of enrofloxacin is characterized by comprising the following steps:

step 1, preparing bismuth oxybromide Bi by solvothermal method ₄ O ₅ Br ₂ Nanosheet:

first, bismuth bromide BiBr ₃ Dissolving in glycol, dropwise adding NaOH solution while stirring, transferring into a polytetrafluoroethylene autoclave for solvothermal reaction, filtering, washing, centrifuging to recover a solid product, and drying to obtain Bi ₄ O ₅ Br ₂ Nanosheets;

step 2, calcining method for preparing N-Bi of aza-oxybromide ₄ O ₅ Br ₂ Nano-composite:

firstly, bi prepared in step 1 is added ₄ O ₅ Br ₂ The nano-sheets are respectively and uniformly mixed with urea with different mass, the mixture is heated in a muffle furnace, the obtained product is respectively washed and dried by dimethyl sulfoxide and distilled water, and N-Bi with different doping contents is obtained ₄ O ₅ Br ₂ A composite material;

step 3, constructing a photoelectric aptamer sensor for sensitively detecting enrofloxacin ENR:

the N-Bi prepared in the step 2 ₄ O ₅ Br ₂ Dispersing the composite material in N, N-dimethylformamide DMF to obtain N-Bi ₄ O ₅ Br ₂ Dispersing liquid, namely dripping the dispersing liquid on an ITO electrode, dripping chitosan CS solution, drying, dripping glutaraldehyde GA solution on the surface of the electrode, placing the electrode at room temperature, reacting, and leaching with phosphoric acid buffer solution PBS; dripping ENR aptamer on an electrode, reacting for a period of time, leaching with PBS, dripping bovine serum albumin BSA solution to obtain an aptamer modified material electrode, namely a photoelectrochemical electrode for detecting enrofloxacinAn aptamer sensor.

2. The method according to claim 1, wherein in step 1, the BiBr is ₃ The dosage ratio of the NaOH solution to the NaOH solution is 1.33g:12.6mL, wherein the concentration of the NaOH solution is 2mol/L; the temperature of the solvothermal reaction is 140-160 ℃, and the reaction time is 10-12 h; the centrifugation speed is 8000-9000 rmp/s, and the centrifugation time is 6-8 min; the drying temperature is 50-60 ℃, and the drying time is 10-12 h.

3. The method according to claim 1, wherein in step 2, the N-Bi ₄ O ₅ Br ₂ The mass percentage content of the medium nitrogen is 10-50%; the muffle furnace is set at 200-220 ℃ and the reaction time is 3-5 h.

4. The method according to claim 1, wherein in step 3, the N-Bi ₄ O ₅ Br ₂ The dispersion is 2mg/mL; the CS concentration is 0.1%; the GA concentration is 2.5%, the dosage of the CS solution is 10 mu L, the dosage of the GA solution is 20 mu L, and the reaction time of the CS and the GA is 0.5-1 h.

5. The construction method according to claim 1, wherein in step 3, the ENR aptamer sequence is: 5' -NH ₂ -CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3'; the ENR aptamer concentration is 3 mu M, the dripping amount is 20 mu L, and the reaction time is 10h; the BSA concentration is 3%; the PBS concentration is 0.1mol/L, the pH =7.4, and the dosage is 10-20 mL.

6. Use of the photoelectrochemical aptamer sensor constructed by the construction method according to any one of claims 1 to 5 for detecting enrofloxacin.

7. The use according to claim 6, characterized in that ENR solutions with different concentrations are dropped on a photoelectrochemical aptamer sensor for enrofloxacin detection and incubated for a period of time, an ITO electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and photoelectric analysis is sequentially carried out according to the concentrations under the irradiation of a xenon light source through an electrochemical workstation three-electrode system.

8. Use according to claim 7, wherein the ENR concentrations are 0.1 to 10, respectively ⁸ ng/mL, the dropping amount is 10-20 mu L; the incubation temperature was 37 ℃; the intensity of the xenon lamp light source is 25-100%.

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