A cathode photoelectrochemical sensor for detecting fumonisin B1 and its detection method

Abstract

This invention discloses a cathodic photoelectrochemical sensor for detecting fumonisin B1. The construction method of the sensor includes the following steps: (1) preparation of CuBi2O4 / ITO electrode; (2) preparation of standard solution of analyte; (3) measurement of photocurrent; This invention utilizes H2O2 and the unsaturated Cu on the surface of CuBi2O4 material. 2+ In-situ reaction to repair Cu on the material surface 2+ By exploiting defect states, eliminating carrier recombination centers, and enhancing the cathode photocurrent, a cathodic photoelectrochemical sensing technology is constructed using a tetrahedral DNA strand substitution amplification strategy for the detection of fumonisin B1. This invention avoids complex operational steps such as DNA strand fixation and modification, and its novel detection mechanism broadens the application range of cathodic photoelectrochemical sensors.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a cathode photoelectrochemical sensor for detecting fumonisin B1 and a detection method thereof. Background Technology

[0002] Food safety is a global public health issue. Mycotoxins are secondary metabolites of fungi, and consuming food containing mycotoxins can have serious effects on humans, including teratogenicity, mutagenicity, nephrotoxicity, carcinogenicity, and even death [Chauhan, R.; Singh, J.; Sachdev, T.; Basu, T.; Malhotra, B.D. Biosens. Bioelectron. 2016, 81, 532–545]. Fumonisin B1, the most abundant and most toxic mycotoxin, is found in corn, wheat, rice, and soybeans [Bennett, JW; Klich, M. Clin. Microbiol. Rev. 2003, 16, 497-516. Stockmann-Juvala, H.; Savolainen, K. Hum. Exp. Toxicol. 2008, 27, 799-809]. It is carcinogenic to humans and can cause serious neurological disorders [Li, M.; Li, DY; Li, ZY; Hu, R.; Yang, YH; Yang, T. Biosens. Bioelectron. 2022, 209, 114-241]. Therefore, the European Commission has set maximum residue limits (MRLs) for fumonisin B1 in food [EU. Commission regulation (EC) No 1126 / 2007. 2007]. Existing methods for detecting fumonisin B1 mainly include enzyme-linked immunosorbent assay (ELISA) [Sheng, YJ; Jiang, WX; De Saeger, S.; Shen, JZ; Zhang, SX; Wang, ZHToxicon 2012, 60, 1245–1250] and high-performance liquid chromatography (HPLC) [Girolamo, AD; Pereboomde Fauw, D.; Sizoo, E.; Egmond, HPV; Gambacorta, L.; Bouten, K.; Stroka, J.; Visconti, A.; Solfrizzo, M. World Mycotoxin J. 2010. 3, 135–146]. However, ELISA involves complex procedures, while HPLC requires expensive instruments. Therefore, there is an urgent need to develop a simple, low-cost, and efficient method for the highly sensitive detection of fumonisin B1.

[0003] Cathodophotochemical sensing technology has become an emerging frontier in the field of sensing technology due to its strong resistance to photocorrosion and interference [Xu, YT; Yu, SY; Zhu, YC; Fan, GC; Han, DM; Qu, P.; Zhao, W.W. Trends Anal. Chem. 2019, 114, 81-88]. However, cathodophotochemical sensing is currently mainly limited by the photoinduced electron transfer (PET) mechanism, and its low carrier mobility [Wu, HL; Li, XB; Tung, CH; Wu, LZ. Adv. Sci. 2018, 5, 1700684] limits the use of a limited number of electron acceptor molecules as signal probes. Furthermore, no studies have reported the application of cathodophotochemical sensing for the detection of fumonisin B1. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides a cathodic photoelectrochemical sensor for detecting fumonisin B1 and a detection method thereof. This invention utilizes H2O2 and the unsaturated Cu on the surface of CuBi2O4 material. 2+ In-situ reaction to repair Cu on the material surface 2+ By exploiting defect states, eliminating carrier recombination centers, and enhancing the cathode photocurrent, a cathodic photoelectrochemical sensing technology is constructed using a tetrahedral DNA (THDs)-based strand substitution amplification strategy for the detection of fumonisin B1. This invention avoids complex operational steps such as DNA strand fixation and modification, and its novel detection mechanism broadens the application range of cathodic photoelectrochemical sensors.

[0005] The technical solution of the present invention is as follows:

[0006] The purpose of this invention is to provide a cathodic photoelectrochemical sensor for detecting fumonisin B1. The method for constructing the sensor includes the following steps:

[0007] (1) Preparation of CuBi2O4 / ITO electrode

[0008] CuBi2O4 powder was dispersed in deionized water to form a suspension. The resulting suspension was then drop-coated onto the surface of an ITO electrode and dried to obtain a CuBi2O4 / ITO electrode.

[0009] (2) Preparation of standard solutions of analytes

[0010] a. Self-assembly of DNA tetrahedral THDs: Single-stranded DNAs: P1, P2, P3, P4, and P5 are mixed in equimolar ratio in a Tris-HCl buffer solution containing MgCl2. The mixture is then heated to 95°C and reacted for 5 minutes. After rapid cooling to 4°C, stable DNA tetrahedral THD1 is formed.

[0011] The same steps were used to prepare DNA tetrahedron THD2, which is composed of single-stranded DNA P1, P2, P3, P6, and P7.

[0012] The same steps were used to prepare DNA tetrahedron THD3, which is composed of single-stranded DNA P1, P2, P3, and P8.

[0013] b. Target recognition reaction: Fumonisin B1 and DNA tetrahedron THD1 and DNA tetrahedron THD2 of different known concentrations are mixed in reaction buffer and reacted at 37°C to carry out the target recognition reaction.

[0014] c. Strand displacement reaction: DNA tetrahedron THD3 was added, and a strand displacement reaction was carried out at 37°C to prepare biological reaction solutions of different concentrations of fumonisin B1.

[0015] d. Add hemin chloride to the biological reaction solution of fumonisin B1 at different concentrations to form G-tetramer / hemin DNase, then add single-walled carbon nanotubes, incubate the reaction and add H2O2 to prepare the standard solution of the analyte.

[0016] (3) Measurement of photocurrent

[0017] The CuBi2O4 / ITO electrode was placed in the standard solution of the analyte prepared in step (2) and incubated. Then, photoelectrochemical detection was performed to perform qualitative or quantitative analysis of fumonisin B1.

[0018] In one embodiment of the present invention, the CuBi2O4 powder is prepared by dissolving Cu(NO3)2·3H2O and Bi(NO3)3·5H2O in HNO3 solution, then adding NaOH solution and ethylene glycol to react, and after the reaction is completed, centrifuging, washing and drying are performed to obtain the CuBi2O4 powder.

[0019] In one embodiment of the present invention, the molar ratio of Cu(NO3)2·3H2O to Bi(NO3)3·5H2O is 1:2 to 6.

[0020] Preferably, the preparation method of CuBi2O4 powder is as follows: 0.5-1.5 mmol Cu(NO3)2·3H2O and 1.0-3.0 mmol Bi(NO3)3·5H2O are dissolved in 25 mL of HNO3 with a concentration of 0.1-1.0 mol / L, and the mixture is stirred continuously until the solution is clear. Then, 10-15 mL of 2.0 mol / L NaOH solution and 0.5-2 mL of ethylene glycol are added. Finally, the mixture is transferred to a reaction vessel and reacted at 140°C for 10-15 h. The final product is centrifuged, washed with deionized water, and dried at 60°C to obtain the CuBi2O4 powder.

[0021] Preferably, CuBi2O4 powder is prepared into a suspension of 1.0 mg / mL, and 30 μL of the obtained suspension is drop-coated onto the surface of the ITO electrode and allowed to air dry naturally to obtain a CuBi2O4 / ITO electrode.

[0022] In one embodiment of the present invention, in step (2), the sequences from the 5' to the 3' ends of the single-stranded DNA P1-P8 are as follows:

[0023] P1,ATG ATA CCG CCG AGA AGA GCA CAT CGT TCG ACA TTA CAA AGT CTG AATCCT TAC A;

[0024] P2, CAT AAC CTG GGA GCG TAG ATA ATG TCG AAC GAT GTG ACA GTT GAC GGACCA CTA T;

[0025] P3, TAC GCT CCC AGG TTA TGT TTG CTG TGA TGC ACC CTT CGT GTA AGG ATTCAG ACT T;

[0026] P4, ATA CCA GCT TAT TCA ATT AAT CGC ATT ACC TTA TAC CAG CTT ATT CAATTA CGT CTG CAC ATA CCA GCT TAT TCA ATT AGA TAG TAA GTG CAA TCT TTT TTC TTCTCG GCG GTA TCA TCT AAG GGT GCA TCA CAG CAA AAT AGT GGT CCG TCA ACT;

[0027] P5, AAT GTT CAG TGA GCG TAA TTG AAT AAG CTG GTA TAA GGT AAT GCG ATT;

[0028] P6, TTC AGT GAG CTT TTT CTT CTC GGC GGT ATC ATC TAA GGG TGC ATC ACAGCA AAA TAG TGG TCC GTC AAC T;

[0029] P7, CAT CCC GCC CAA CCC GCT CAC TGA ACA TT;

[0030] P8, AAT GTT CAG TGA GCG GGT TGG GCG GGA TGG GTT TTT CTT CTC GGC GGTATC ATC TAA GGG TGC ATC ACA GCA AAA TAG TGG TCC GTC AAC T.

[0031] In one embodiment of the present invention, the single-stranded DNA P4 contains a fumonisin B1 aptamer sequence, which is: 5'-ATA CCA GCT TAT TCA ATT AAT CGC ATT ACC TTA TAC CAG CTT ATTCAA TTA CGT CTG CAC ATA CCA GCT TAT TCA ATT AGA TAG TAA GTG CAA TCT-3'.

[0032] In one embodiment of the present invention, the method for preparing the standard solution of the analyte is as follows:

[0033] a. Self-assembly of DNA tetrahedral THDs: Single-stranded DNAs: P1, P2, P3, P4, and P5 are mixed in equimolar ratio in a Tris-HCl buffer solution (20 mmol / L, pH = 7.4) containing 50 mmol / L MgCl2. The mixture is then heated to 95°C and reacted for 5 min. After rapid cooling to 4°C, stable THD1 is formed.

[0034] The same steps were used to prepare DNA tetrahedron THD2, which is composed of single-stranded DNA P1, P2, P3, P6, and P7.

[0035] The same steps were used to prepare DNA tetrahedron THD3, which is composed of single-stranded DNA P1, P2, P3, and P8.

[0036] b. Target recognition reaction: 10 μL of different known concentrations of fumonisin B1, 0.5–2.0 μmol / L DNA tetrahedron THD1, and 0.5–2.0 μmol / L DNA tetrahedron THD2 were mixed in 30 μL of reaction buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2.0 mmol / L MgSO4, 10 mmol / L (NH4)2SO4, pH = 8.8) and reacted at 37°C for 115 min to carry out the target recognition reaction.

[0037] c. Strand displacement reaction: Add 10 μL of 0.5–2.0 μmol / L DNA tetrahedron THD3 and carry out the strand displacement reaction at 37°C for 45–80 min to prepare biological reaction solutions of different concentrations of fumonisin B1.

[0038] d. Add 10 μL of 1.5 μmol / L hemin to the biological reaction solution of fumonisin B1 at different concentrations, react at room temperature for 50 min to form G-tetramer / hemin DNase, then add 10 μL of 100 μg / mL single-walled carbon nanotubes, incubate for 30 min to adsorb excess free hemin, and centrifuge the mixture (12000 rpm) for 10 min to remove the single-walled carbon nanotubes that adsorb excess free hemin; add 10 μL of H2O2 and react for 10 min to obtain the standard solution of the analyte.

[0039] In one embodiment of the present invention, the molar ratio of THD1, THD2, and THD3 is 1:1:1.

[0040] In one embodiment of the present invention, in step (3), the CuBi2O4 / ITO electrode is the working electrode, the Ag / AgCl electrode is the reference electrode, and the Pt electrode is the counter electrode in photoelectrochemical detection.

[0041] In one embodiment of the present invention, a potential of -0.1V is applied relative to the Ag / AgCl reference electrode, the buffer medium is a Tris-HCl buffer solution, and the excitation light source is an LED light source in the 410-420nm wavelength band.

[0042] The purpose of this invention is to provide a method for detecting fumonisin B1 using the aforementioned cathode photoelectrochemical sensor, comprising the following steps:

[0043] The CuBi2O4 / ITO electrode was incubated in the standard solution of the analyte, and then photoelectrochemical detection was performed. The intensity of the generated photoelectric signal was used to plot the working curve. The sample solution of the analyte was used instead of the standard solution for detection.

[0044] In one embodiment of the present invention, the method for detecting fumonisin B1 using a cathode photoelectrochemical sensor is as follows:

[0045] The CuBi₂O₄ / ITO electrode was incubated in the standard solution of the analyte for 1–5 min, and then washed with Tris-HCl buffer (0.1 mol / L, pH = 7.4) before photoelectrochemical experiments were performed.

[0046] The working electrode used in the photoelectrochemical experiment was a CuBi2O4 modified ITO electrode, the reference electrode and the counter electrode were an Ag / AgCl electrode and a Pt electrode, respectively, the excitation light source was an LED light source in the 410-420 nm wavelength range, the applied potential was -0.1 V (relative to the Ag / AgCl reference electrode), and the buffer medium was a Tris-HCl buffer solution (0.1 mol / L, pH = 7.4).

[0047] In one embodiment of the present invention, in step (3), the working curve formed by the linear relationship between the concentration of fumonisin B1 and ΔI is: ΔI=68.93Log[fumonisin B1]+257.31.

[0048] In one embodiment of the present invention, the detection limit of the sensor is 0.17 pg / mL (S / N = 3).

[0049] The beneficial technical effects of this invention are as follows:

[0050] This invention utilizes H2O2 and the unsaturated Cu on the surface of CuBi2O4 material 2+ Combined, repairing surface Cu 2+ By exploiting defect states and eliminating carrier recombination centers, an enhanced cathodic photocurrent is obtained, leading to the construction of a cathodic photoelectrochemical sensor based on a THD chain substitution amplification strategy for the detection of fumonisin B1. In the presence of fumonisin B1, it binds to the aptamer sequence in THD1, releasing single-stranded DNA and inducing a subsequent chain substitution reaction. This chain substitution reaction effectively prevents the formation of G-quadrimers / hemin, thus inhibiting the catalytic reduction by H2O2. H2O2 can repair surface defect states in the CuBi2O4 material, eliminating carrier recombination centers and generating an enhanced cathodic photocurrent, thereby enabling the detection of fumonisin B1. Attached Figure Description

[0051] Figure 1 (A) Photocurrent spectrum and (B) Raman spectrum of CuBi2O4 before and after the reaction with H2O2.

[0052] Figure 2 (A) Gel electrophoresis image; (B) Circular dichroism spectrum.

[0053] Figure 3(A) Photocurrent plots of fumonisin B1 at different concentrations (0, 0.0005, 0.001, 0.005, 0.05, 0.5, 10.0, 100.0 and 800.0 ng / mL from left to right); (B) Linear relationship between the concentration of fumonisin B1 and ΔI.

[0054] Figure 4 For photoelectrochemical sensors, consider (A) stability and (B) selectivity. Detailed Implementation

[0055] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0056] The photocurrent before and after the reaction of CuBi2O4 with H2O2 is as follows: Figure 1 As shown in Figure A, it can be seen from the figure that H2O2 can enhance the cathode photocurrent of CuBi2O4; compare the Raman spectra of CuBi2O4 before and after the reaction with H2O2 ( Figure 1 B) It was found that CuBi₂O₄ after reacting with H₂O₂ showed a peak density of 833 cm⁻¹. -1 A new characteristic peak appeared at this location, which is attributed to the stretching vibration mode of the OO bond in H2O2, indicating that H2O2 is bound to the surface of CuBi2O4 material.

[0057] Using gel electrophoresis ( Figure 2 A) and circular dichroism spectrum ( Figure 2 B) Verifying the feasibility of the photoelectrochemical sensor. Bands 1-7 represent the self-assembly process of THDs, while bands 8 and 9 represent the cases with and without the target substance, respectively. The appearance of a new band in the low molecular weight region in band 9 indicates the successful construction of the sensor.

[0058] Example 1:

[0059] A cathodic photoelectrochemical sensor for detecting fumonisin B1, the method for constructing the sensor includes the following steps:

[0060] (1) Preparation of CuBi2O4 / ITO electrode

[0061] First, 0.5 mmol of Cu(NO3)2·3H2O and 1.0 mmol of Bi(NO3)3·5H2O were dissolved in 25 mL of 0.3 mol / L HNO3, and the solution was stirred continuously until clear. Next, 10 mL of 2.0 mol / L NaOH solution and 0.5 mL of ethylene glycol were added to the above solution. Finally, the mixture was transferred to a reaction vessel and reacted at 140 °C for 12 h. The final product was centrifuged, washed with deionized water, and dried at 60 °C to obtain CuBi2O4 powder. A 1.0 mg / mL suspension of the obtained CuBi2O4 powder was prepared, and 30 μL was dropped onto a pretreated ITO electrode. After air drying, the CuBi2O4 / ITO electrode was obtained.

[0062] (2) Preparation of standard solutions of analytes

[0063] a. Self-assembly of DNA tetrahedral THDs: Single-stranded DNAs: P1, P2, P3, P4, and P5 were mixed in equimolar ratio in a Tris-HCl buffer solution containing 50 mmol / L MgCl2 (20 mmol / L, pH = 7.4). The mixture was then heated to 95°C and reacted for 5 min. After rapid cooling to 4°C, stable THD1 was formed.

[0064] The same steps were used to prepare DNA tetrahedron THD2, which is composed of single-stranded DNA P1, P2, P3, P6, and P7.

[0065] The same steps were used to prepare DNA tetrahedron THD3, which is composed of single-stranded DNA P1, P2, P3, and P8.

[0066] b. Target recognition reaction: 10 μL of different known concentrations of fumonisin B1, 0.8 μmol / L of DNA tetrahedron THD1 and DNA tetrahedron THD2 were mixed in 30 μL of reaction buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2.0 mmol / L MgSO4, 10 mmol / L (NH4)2SO4, pH=8.8) and reacted at 37℃ for 115 min to carry out the target recognition reaction.

[0067] c. Strand displacement reaction: Add 10 μL of 0.8 μmol / L DNA tetrahedron THD3 rich in G base sequence and incubate at 37℃ for 75 min to carry out the strand displacement reaction to obtain biological reaction solutions of different concentrations of fumonisin B1.

[0068] d. Add 10 μL of 1.5 μmol / L hemin to the biological reaction solutions of fumonisin B1 at different concentrations, and react at room temperature for 50 min to form G-tetramer / hemin DNase. Then add 10 μL of 100 μg / mL single-walled carbon nanotubes to the above solution, incubate for 30 min to adsorb excess free hemin, and centrifuge the mixture (12000 rpm) for 10 min to remove single-walled carbon nanotubes that have adsorbed excess free hemin. Subsequently, add 10 μL of 5.0 mmol / L H2O2 to the solution and react for another 10 min to prepare the standard solution of the analyte.

[0069] (3) Measurement of photocurrent

[0070] The CuBi₂O₄ / ITO electrode was incubated in the analyte standard solution prepared in step (2) for 2 min, and then washed with Tris-HCl buffer (0.1 mol / L, pH = 7.4) before photoelectrochemical detection was performed to qualitatively or quantitatively analyze fumonisin B1. The working electrode used in the photoelectrochemical experiment was a CuBi₂O₄ modified ITO electrode, the reference electrode and the counter electrode were an Ag / AgCl electrode and a Pt electrode, respectively, the excitation light source was an LED light source in the 410-420 nm wavelength range, the applied potential was -0.1 V (relative to the Ag / AgCl reference electrode), and the buffer medium was Tris-HCl buffer solution (0.1 mol / L, pH = 7.4).

[0071] In this example, the photocurrents of different concentrations of fumonisin B1 (from left to right: 0, 0.0005, 0.001, 0.005, 0.05, 0.5, 10.0, 100.0, and 800.0 ng / mL) are as follows: Figure 3 As shown in Figure A; the linear relationship between the concentration of fumonisin B1 and ΔI is as follows: Figure 3 As shown in B, the working curve is: ΔI=68.93Log[fumonisin B1]+257.31; the detection limit is 0.17pg / mL (S / N=3).

[0072] The test results for the stability and selectivity of this method are as follows: Figure 4 As shown, the photocurrent did not change significantly after multiple excitations by the light source, indicating that the method has good stability. Furthermore, apart from the target compound fumonisin B1, other interfering substances failed to alter the photocurrent of the CuBi₂O₄ / ITO electrode, demonstrating the good selectivity of this method.

[0073] Example 2:

[0074] A cathodic photoelectrochemical sensor for detecting fumonisin B1, the method for constructing the sensor includes the following steps:

[0075] (1) Preparation of CuBi2O4 / ITO electrode

[0076] First, 1.0 mmol of Cu(NO3)2·3H2O and 2.0 mmol of Bi(NO3)3·5H2O were dissolved in 25 mL of 1.0 mol / L HNO3, and the solution was stirred continuously until clear. Next, 15 mL of 2.0 mol / L NaOH solution and 1 mL of ethylene glycol were added to the above solution. Finally, the mixture was transferred to a reaction vessel and reacted at 140 °C for 10 h. The final product was centrifuged, washed with deionized water, and dried at 60 °C to obtain CuBi2O4 powder. A 1.0 mg / mL suspension of the obtained CuBi2O4 powder was prepared, and 30 μL was dropped onto a pretreated ITO electrode. After air drying, the CuBi2O4 / ITO electrode was obtained.

[0077] (2) Preparation of standard solutions of analytes

[0078] a. Self-assembly of DNA tetrahedral THDs: Single-stranded DNAs: P1, P2, P3, P4, and P5 were mixed in equimolar ratio in a Tris-HCl buffer solution (20 mmol / L, pH = 7.4) containing 50 mmol / L MgCl2. The mixture was then heated to 95°C and reacted for 5 min. After rapid cooling to 4°C, stable DNA tetrahedral THD1 was formed.

[0079] The same steps were used to prepare DNA tetrahedron THD2, which is composed of single-stranded DNA P1, P2, P3, P6, and P7.

[0080] The same steps were used to prepare DNA tetrahedron THD3, which is composed of single-stranded DNA P1, P2, P3, and P8.

[0081] b. Target recognition reaction: 10 μL of different known concentrations of fumonisin B1, 1.5 μmol / L of DNA tetrahedron THD1 and DNA tetrahedron THD2 were mixed in 30 μL of reaction buffer (20 mmol / L Tris-HCl, 10 mmol / L KCl, 2.0 mmol / L MgSO4, 10 mmol / L (NH4)2SO4, pH=8.8) and reacted at 37℃ for 115 min to carry out the target recognition reaction.

[0082] c. Strand displacement reaction: Add 10 μL of 1.5 μmol / L DNA tetrahedron THD3 rich in G base sequence and incubate at 37℃ for 60 min to carry out the strand displacement reaction to obtain biological reaction solutions of different concentrations of fumonisin B1.

[0083] d. Add 10 μL of 1.5 μmol / L hemin to the biological reaction solutions of fumonisin B1 at different concentrations, and react at room temperature for 50 min to form G-tetramer / hemin DNase. Then add 10 μL of 100 μg / mL single-walled carbon nanotubes to the above solution, incubate for 30 min to adsorb excess free hemin, and centrifuge the mixture (12000 rpm) for 10 min to remove single-walled carbon nanotubes that have adsorbed excess free hemin. Subsequently, add 10 μL of 6.0 mmol / L H2O2 to the solution and react for another 10 min to prepare the standard solution of the analyte.

[0084] (3) Measurement of photocurrent

[0085] The CuBi₂O₄ / ITO electrode was incubated in the analyte standard solution prepared in step (2) for 1 min, and then washed with Tris-HCl buffer (0.1 mol / L, pH = 7.4) before photoelectrochemical detection was performed to qualitatively or quantitatively analyze fumonisin B1. The working electrode used in the photoelectrochemical experiment was a CuBi₂O₄ modified ITO electrode, the reference electrode and the counter electrode were an Ag / AgCl electrode and a Pt electrode, respectively, the excitation light source was an LED light source in the 410-420 nm wavelength range, the applied potential was -0.1 V (relative to the Ag / AgCl reference electrode), and the buffer medium was Tris-HCl buffer solution (0.1 mol / L, pH = 7.4).

[0086] Example 3: Detection of fumonisin B1 in maize

[0087] 4 g of fresh corn flour was dissolved in 8 mL of methanol-water (3:1 v / v) and ultrasonically dispersed at room temperature for 20 min. The mixture was then centrifuged at 12000 rpm for 10 min, and the supernatant was diluted 10-fold with Tris-HCl buffer (0.1 mol / L, pH 7.4). Different concentrations of fumonisin B1 solution were then added to the corn sample to prepare the test sample.

[0088] The sensor prepared in Example 1 was used to detect the sample to be tested. The detection results of fumonisin B1 in corn are shown in Table 1.

[0089] Table 1

[0090]

[0091] As shown in Table 1, the recovery rate of the tested corn flour samples ranged from 103.3% to 109.0%, and the relative standard deviation was between 1.9% and 2.3%, indicating that this method can be used for the analysis of actual samples.

Claims

1. A cathode photoelectrochemical sensor for detecting fumonisin B1, characterized in that, The method for constructing the sensor includes the following steps: (1) Preparation of CuBi2O4 / ITO electrode CuBi2O4 powder was dispersed in deionized water to form a suspension. The resulting suspension was then drop-coated onto the surface of an ITO electrode and dried to obtain a CuBi2O4 / ITO electrode. (2) Preparation of standard solutions of analytes a. Self-assembly of DNA tetrahedral THDs: Single-stranded DNAs: P1, P2, P3, P4, and P5 are mixed in equimolar ratio in a Tris-HCl buffer solution containing MgCl2. The mixture is then heated to 95°C and reacted for 5 minutes. After rapid cooling to 4°C, stable DNA tetrahedral THD1 is formed. The same steps were used to prepare DNA tetrahedron THD2, which is composed of single-stranded DNA P1, P2, P3, P6, and P7. The same steps were used to prepare DNA tetrahedron THD3, which is composed of single-stranded DNA P1, P2, P3, and P8. b. Target recognition reaction: Fumonisin B1 and DNA tetrahedron THD1 and DNA tetrahedron THD2 of different known concentrations are mixed in reaction buffer and reacted at 37°C to carry out the target recognition reaction. c. Strand displacement reaction: DNA tetrahedron THD3 was added, and a strand displacement reaction was carried out at 37°C to prepare biological reaction solutions of different concentrations of fumonisin B1. d. Add hemin chloride to the biological reaction solution of fumonisin B1 at different concentrations to form G-tetramer / hemin DNase, then add single-walled carbon nanotubes, incubate the reaction and add H2O2 to prepare the standard solution of the analyte. (3) Measurement of photocurrent The CuBi2O4 / ITO electrode was placed in the standard solution of the analyte prepared in step (2) and incubated. Then, photoelectrochemical detection was performed to perform qualitative or quantitative analysis of fumonisin B1. In step (2), the sequences from the 5' to the 3' ends of the single-stranded DNA P1-P8 are as follows: P1,ATG ATA CCG CCG AGA AGA GCA CAT CGT TCG ACA TTA CAA AGT CTG AAT CCTTAC A; P2, CAT AAC CTG GGA GCG TAG ATA ATG TCG AAC GAT GTG ACA GTT GAC GGA CCACTA T; P3, TAC GCT CCC AGG TTA TGT TTG CTG TGA TGC ACC CTT CGT GTA AGG ATT CAGACT T; P4, ATA CCA GCT TAT TCA ATT AAT CGC ATT ACC TTA TAC CAG CTT ATT CAA TTACGT CTG CAC ATA CCA GCT TATTCA ATT AGA TAG TAA GTG CAA TCT TTT TTC TTC TCGGCGGTA TCA TCT AAG GGT GCA TCA CAG CAA AAT AGT GGTCCG TCA ACT; P5, AAT GTT CAG TGA GCG TAA TTG AAT AAG CTG GTA TAA GGT AAT GCG ATT; P6, TTC AGT GAG CTT TTT CTT CTC GGC GGT ATC ATC TAA GGG TGC ATC ACA GCAAAA TAG TGG TCC GTC AAC T; P7, CAT CCC GCC CAA CCC GCT CAC TGA ACA TT; P8, AAT GTT CAG TGA GCG GGT TGG GCG GGA TGG GTT TTT CTT CTC GGC GGT ATCATC TAA GGG TGC ATC ACA GCA AAA TAG TGG TCC GTC AAC T; The molar ratio of THD1, THD2, and THD3 is 1:1:1; The single-stranded DNA P4 contains the fumonisin B1 aptamer sequence, which is: 5'-ATA CCAGCT TAT TCA ATT AAT CGC ATT ACC TTA TAC CAG CTT ATT CAA TTA CGT CTG CAC ATACCA GCT TAT TCA ATT AGA TAG TAA GTG CAA TCT-3'.

2. The cathode photoelectrochemical sensor according to claim 1, characterized in that, The preparation method of CuBi2O4 powder is as follows: Cu(NO3)2·3H2O and Bi(NO3)3·5H2O are dissolved in HNO3 solution, and then NaOH solution and ethylene glycol are added to react. After the reaction is completed, the mixture is centrifuged, washed and dried to obtain CuBi2O4 powder.

3. The cathode photoelectrochemical sensor according to claim 2, characterized in that, The molar ratio of Cu(NO3)2·3H2O to Bi(NO3)3·5H2O is 1:2 to 6.

4. The cathode photoelectrochemical sensor according to claim 1, characterized in that, In step (3), the CuBi2O4 / ITO electrode is the working electrode, the Ag / AgCl electrode is the reference electrode, and the Pt electrode is the counter electrode in photoelectrochemical detection.

5. The cathode photoelectrochemical sensor according to claim 4, characterized in that, The potential applied relative to the Ag / AgCl reference electrode is -0.1V, the buffer medium is Tris-HCl buffer solution, and the excitation source is an LED light source in the 410-420nm wavelength range.

6. A method for detecting fumonisin B1 using the cathode photoelectrochemical sensor described in claim 1, characterized in that, The method is as follows: The CuBi2O4 / ITO electrode was incubated in the standard solution of the analyte, and then photoelectrochemical detection was performed. The intensity of the generated photoelectric signal was used to plot the working curve. The sample solution of the analyte was used instead of the standard solution for detection.

7. The method for detecting fumonisin B1 according to claim 6, characterized in that, The working curve showing the linear relationship between the concentration of fumonisin B1 and ΔI is: ΔI = 68.93Log(concentration of fumonisin B1) + 257.31; the limit of detection is 0.17 pg / mL, and S / N = 3.

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