Dual-mode sensor for detecting ochratoxin A and application thereof

Abstract

The invention provides a dual-mode sensor for detecting ochratoxin A and application of the dual-mode sensor, and relates to the technical field of food detection. The dual-mode sensor is prepared by adopting a method comprising the following steps: depositing gold nanoparticles on the surface of an ITO (Indium Tin Oxide) electrode; (2) dispensing cDNA on the surface of the electrode obtained in the step (1), closing active sites, and then dispensing an aptamer Apt; and dropwise coating a Cu-coated MoSe2NFs-AuNPs solution on the surface of the electrode obtained in the step (2) to obtain the dual-mode sensor for detecting the ochratoxin A. The dual-mode sensor for detecting the ochratoxin A is high in sensitivity, high in accuracy, rapid in detection, simple to operate, low in cost and easy to apply.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a dual-mode sensor for detecting ochratoxin A and applications thereof. Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by fungi such as Aspergillus and Penicillium. It has teratogenic, carcinogenic, and mutagenic properties and is classified as a Class 2B carcinogen by the International Agency for Research on Cancer. OTA is frequently detected in foods, including wheat, corn, coffee, spices, beer, grapes, and animal meat products, posing a significant threat to human health. Therefore, developing a rapid, accurate, and sensitive method for detecting OTA is crucial for ensuring food safety. Although traditional methods for detecting OTA, such as high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-tandem mass spectrometry (LC-MS), have high sensitivity, these methods place high demands on equipment, sample pretreatment, and operators. Summary of the Invention

[0003] The present invention aims to provide a dual-mode sensor for detecting ochratoxin A, which has high sensitivity, high accuracy, rapid detection, simple operation, low cost and easy application.

[0004] The purpose of the present invention is achieved by adopting the following technical solutions:

[0005] A dual-mode sensor for detecting ochratoxin A was prepared using a method comprising the following steps:

[0006] (1) Depositing gold nanoparticles on the surface of the ITO electrode;

[0007] (2) applying cDNA to the surface of the electrode obtained in step (1) to block the active site, and then applying the aptamer Apt;

[0008] (3) The Cu@MoSe2NFs-AuNPs solution was drop-coated on the surface of the electrode obtained in step (2) to obtain a dual-mode sensor for detecting ochratoxin A.

[0009] In the present invention, the nucleotide sequence of the aptamer Apt is shown as SEQ ID NO: 1; the nucleotide sequence of the cDNA is shown as SEQ ID NO: 2.

[0010] In the present invention, the drop-coating concentration of cDNA in step (2) is 8-12 μM, 6-mercapto-1-ethanol is used to block the active site, and the drop-coating concentration of aptamer Apt is 8-12 μM.

[0011] In the present invention, Cu@MoSe2NFs-AuNPs were prepared by the following method:

[0012] ① Dispersing Na2MoO4·2H2O, Se powder, and NaBH4 in deionized water, adding ethanol under stirring to obtain a mixture; transferring the mixture to a reactor, and hydrothermally treating it at 180-220°C for 10-14 hours to obtain MoSe2NFs;

[0013] ② MoSe2NFs aqueous solution was mixed with CuCl2·2H2O powder under ultrasound to obtain Cu@MoSe2NFs;

[0014] ③ Disperse Cu@MoSe2NFs in deionized water, ultrasonically treat, add HAuCl4 aqueous solution, heat to boiling, add sodium citrate aqueous solution, and keep boiling for 0.8-1.2 hours under stirring; after cooling, centrifuge to obtain the precipitate, wash and dry to obtain Cu@MoSe2NFs-AuNPs.

[0015] In the present invention, the molar ratio of Na2MoO4·2H2O, Se powder and NaBH4 in step ① is 1:1-3:1-3, and the volume ratio of ethanol and deionized water is 1:0.8-1.2; 1 mmol of the mixture of Na2MoO4·2H2O, Se powder and NaBH4 is dispersed in 70-90 mL of water.

[0016] In the present invention, the mass ratio of MoSe2NFs to CuCl2·2H2O powder in step ② is 10:0.5-1.5.

[0017] In the present invention, in step ③, the mass ratio of Cu@MoSe2NFs, HAuCl4, and sodium citrate is 50:6-8:30-40.

[0018] The present invention also provides application of the dual-mode sensor in detecting ochratoxin A.

[0019] Beneficial effects: The present invention uses the electrodeposition method to prepare gold nanoflowers (AuNFs) as a sensing platform to fix complementary DNA (cDNA). The Cu@MoSe2NFs-AuNPs nanozyme with peroxidase-like activity (POD) can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to generate a blue product oxTMB. H2O2 and oxTMB are detected by electrochemical and colorimetric methods, respectively, thereby realizing dual-mode detection. When OTA is present, it specifically binds to the aptamer (Apt) fixed on the electrode through the principle of base complementary pairing, releasing the Apt modified by the Cu@MoSe2NFs-AuNPs nanozyme, reducing the nanozyme on the electrode surface, thereby realizing the detection of OTA. The dual-mode sensor of the present invention and the proposed electrochemical-colorimetric dual signal readout method provide a new way to construct an accurate, reliable and intuitive detection method, and have broad application prospects for the detection of OTA in rice and wheat samples. The dual-mode sensor of the present invention has the following characteristics: (1) AuNFs have the advantages of large specific surface area and good conductivity, forming a high-performance sensing interface that can immobilize more DNA (cDNA) and accelerate electron transfer between the electrode surface and the electroactive probe; (2) Cu@MoSe2NFs-AuNPs nanozyme with POD-like activity can catalyze the oxidation of TMB to produce the blue product oxTMB, which can be detected by electrochemical and colorimetric methods, respectively, thereby achieving dual-mode detection; (3) The combination of electrochemical and colorimetric methods can complement each other, making the analysis results more reliable and accurate. Under optimal conditions, the linear ranges of electrochemical detection and colorimetric detection of the aptamer sensor are 1 fg / mL to 100 ng / mL and 0.01 ng / mL to 100 ng / mL, respectively, with detection limits (LODs) of 0.0218 fg / mL and 0.982 pg / mL, respectively. Common single-mode detection methods, such as colorimetric or electrochemical sensors, are susceptible to environmental influences and interference from colored substrates, leading to false positives. By combining these two analytical techniques, their results can be mutually verified, thus avoiding unreliable results. The constructed dual-mode sensor was applied to the detection of OTA in real samples (rice and wheat), achieving excellent results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The characterization images of MoSe2NFs, Cu@MoSe2NFs and Cu@MoSe2NFs-AuNPs are shown. Figure 1 (A) is the SEM image of MoSe2NFs. Figure 1 (B) is the SEM image of Cu@MoSe2NFs. Figure 1 (C) is the SEM image of Cu@MoSe2NFs-AuNPs; Figure 1 (D) XRD patterns of MoSe2NFs, Cu@MoSe2NFs, and Cu@MoSe2NFs-AuNPs. The horizontal axis represents twice the diffraction angle of X-rays, and the vertical axis represents the intensity of the diffraction signal. Figure 1 (E) is the EDS spectrum of Cu@MoSe2NFs-AuNPs, the abscissa is the energy of X-rays (in keV), and the ordinate is the intensity of X-rays; Figure 1 (F) is the SEM image of AuNFs.

[0021] Figure 2 Schematic diagram of the construction principle and detection method of the dual-mode sensor of the present invention.

[0022] Figure 3 (A) DPV graphs of the dual-mode sensor of the present invention for different concentrations of OTA standards, with the abscissa being voltage (V) and the ordinate being current (μA): (a) 1 μg / mL (OTA concentration in the standard, the same below), (b) 100 ng / mL, (c) 10 ng / mL, (d) 1 ng / mL, (e) 0.1 ng / mL, (f) 10 pg / mL, (g) 1 pg / mL, (h) 0.1 pg / mL, (i) 10 fg / mL, (j) 1 fg / mL, (k) 0.1 fg / mL, and (l) 0 fg / mL; (B) Linear fitting graph of the DPV peak current change and the logarithm of the OTA concentration, with the abscissa being -1 gC OTA , C OTA The unit is g / mL, and the vertical axis is Δi p , the unit is μA; (C) Linear fitting plot of the logarithmic value and absorbance value of different OTA concentrations: (a) 1 pg / mL, (b) 10 pg / mL, (c) 100 pg / mL, (d) 1 ng / mL, (e) 10 ng / mL, (f) 100 ng / mL, (g) 1 μg / mL, the horizontal axis is -1 gC OTA , C OTA The unit is g / mL, and the vertical axis is the absorbance value.

[0023] Figure 4 (A) Specificity of the dual-mode sensor of the present invention to OTA, interfering substances (AFB1, AFB2, ZEN, AFM1) and the mixture; (B) Repeatability of the dual-mode sensor of the present invention; (C) Stability of the dual-mode sensor of the present invention. DETAILED DESCRIPTION

[0024] All electrochemical measurements were performed on a CHI660E electrochemical workstation (Shanghai Huachen, China). A typical three-electrode system consisting of a saturated calomel reference electrode (SCE), an indium tin oxide (ITO) working electrode (Φ = 4 mm), and a platinum wire (Pt) counter electrode was used for all electrochemical measurements. The resulting composites were characterized by scanning electron microscopy (SEM, S4800, Japan), X-ray diffraction (XRD, XD-3, Beijing), ultraviolet and visible spectrophotometry (UV-vis, UV-1800, Japan), energy dispersive X-ray spectrometry (EDS, S4800, Japan), and zeta potential (ZC-3000, Shanghai).

[0025] Sodium molybdate dihydrate (Na2MoO4·2H2O), selenium powder (Se powder), sodium borohydride (NaBH4), cupric chloride dihydrate (CuCl2·2H2O), sodium citrate (C6H5Na3O7), and hydrogen peroxide (H2O2) were purchased from Sinopharm Chemical Reagent Co., Ltd. Chloroauric acid tetrahydrate (HAuCl4·4H2O) was purchased from Nanjing Chemical Reagent Co., Ltd.; 6-mercaptoethanol (MCH) and TMB were purchased from Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the ochratoxin A aptamer (abbreviated as Apt) is shown in SEQ ID NO:1; the nucleotide sequence of the complementary strand of the aptamer (denoted as cDNA) is shown in SEQ ID NO:2. The aforementioned oligonucleotides were all prepared by Sangon Biotech (Shanghai) Co., Ltd.; their structures are shown in Table 1.

[0026] Table 1 Oligonucleotide sequences used in the present invention

[0027] name Sequence (5′→3′) Apt <![CDATA[SH-(CH2)6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA]]> cDNA <![CDATA[SH-(CH2)6-TGT CCG ATG CTC CCT TTA CGC CAC]]>

[0028] Example 1 Preparation of a dual-mode sensor for detecting ochratoxin A

[0029] Preparation of a dual-mode sensor for detecting ochratoxin A comprises the following steps:

[0030] (1) Preparation of MoSe2NFs nanozymes

[0031] MoSe2NFs nanozymes (abbreviated as MoSe2NFs) were prepared using a hydrothermal method. The specific steps were as follows: First, 0.05mmol of Na2MoO4·2H2O, 0.1mmol of Se powder, and 0.1mmol of NaBH4 were dispersed in 20mL of deionized water. Second, 20mL of ethanol was added to the above solution while stirring to obtain a mixture. Third, the mixture was transferred to a Teflon-lined stainless steel reactor and hydrothermally treated at 200°C for 12h. Finally, the black precipitate was collected, washed three times with deionized water, then three times with ethanol, and freeze-dried to obtain the MoSe2NFs nanozyme.

[0032] (2) Preparation of Cu@MoSe2NFs

[0033] Due to the electrostatic interaction between negatively charged MoSe2NFs and positively charged copper ions, 10 mL of a 10 mg / mL MoSe2NFs aqueous solution was mixed with 10 mg of CuCl2·2H2O powder under ultrasound for 10 min. The mixture was then washed three times with deionized water, and the precipitate was collected by centrifugation and freeze-dried to obtain MoSe2NFs loaded with copper ions, which was designated as Cu@MoSe2NFs.

[0034] (3) Preparation of Cu@MoSe2NFs-AuNPs composites

[0035] First, Cu@MoSe2NFs were dispersed in deionized water to form a 10 mg / mL suspension. 10 mL of this suspension was then added to 26.7 mL of water and sonicated for 10 minutes. Next, 1.7 mL of a 24.3 mM HAuCl4·4H2O aqueous solution was added, and the solution was heated to boiling. While boiling, 1.2 mL of a 0.2 M sodium citrate aqueous solution was quickly added. The mixed solution was kept boiling for 1 hour under continuous magnetic stirring. After cooling to room temperature, the solution was centrifuged (8500 rpm for 10 minutes), and the precipitate was collected and washed three times with deionized water and three times with ethanol. Finally, the product was freeze-dried to obtain the Cu@MoSe2NFs-AuNPs composite.

[0036] The morphology and composition of Cu@MoSe2NFs-AuNPs were characterized by SEM, XRD and EDS. Figure 1 (A) is the SEM image of MoSe2NFs. It can be seen from the image that MoSe2NFs is a flower-like structure composed of a large number of nanosheets. In order to determine whether the microscopic morphology parameters of MoSe2NFs loaded with copper ions change, the nanoparticles were tested by SEM, such as Figure 1(B) shows the SEM image of MoSe2NFs loaded with copper ions. It can be seen that the surface of MoSe2NFs becomes somewhat rough after loading with copper ions. Figure 1 (C) It was found that AuNPs were evenly distributed on Cu@MoSe2NFs. Figure 1 (D) is the XRD pattern of MoSe2NFs, Cu@MoSe2NFs, and Cu@MoSe2NFs-AuNPs. The MoSe2NFs curve has diffraction peaks at 12.8°, 27.5°, and 44.8°, corresponding to crystal planes (002), (004), and (105), respectively, and is consistent with the standard card (JCPDS No.29-0914), indicating the successful preparation of MoSe2NFs. The XRD comparison of MoSe2NFs before and after copper ion modification shows that the diffraction peaks of MoSe2NFs and MoSe2NFs after copper ion loading are consistent with the characteristic diffraction peak positions at 12.8°, 27.5°, and 44.8° at 2θ, and the peak intensities of the two before and after loading are basically the same. This indicates that the structure of MoSe2NFs has not been changed during the copper ion loading process. The Cu@MoSe2NFs-AuNPs composite material curve shows additional peaks at 38.1°, 64.5°, and 77.6°, corresponding to the (111), (220), and (311) crystal planes of AuNPs, respectively, and consistent with the standard card (JCPDS No. 04-0784), indicating the successful combination of AuNPs and Cu@MoSe2NFs-AuNPs. The peak at 2θ = 21° is the diffraction peak of cristobalite, i.e., the base peak. Figure 1 (E) is the EDS analysis result of Cu@MoSe2NFs-AuNPs. From the analysis in the figure, it can be seen that the composite material is composed of Mo, Se, Cu and Au elements.

[0037] (4) Construction of aptamer sensors

[0038] The method for constructing an aptamer sensor comprises the following steps:

[0039] ① First, the ITO electrode (active area diameter of the working electrode Φ = 4 mm) was ultrasonically treated in acetone, ethanol and pure water for 10 minutes in sequence to clean the electrode.

[0040] ②Then, the cleaned ITO electrode was used as the working electrode, saturated calomel as the reference electrode (SCE), and platinum wire (Pt) as the counter electrode was immersed in an aqueous solution containing 0.5mM HAuCl4·4H2O. At a potential of 0.3V, electrodeposition was performed for 1800s to deposit gold nanoparticles on the surface of the ITO electrode to obtain AuNFs-ITO.

[0041] ③ Then, 5 μL of 1 M TCEP (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution was added to 10 μL of 10 μM cDNA aqueous solution, and then drop-coated on the AuNFs-ITO surface, allowing the cDNA to be successfully adsorbed to the electrode surface through the Au-S bond.

[0042] ④ Subsequently, 1 mL of 1 mM MCH (6-mercapto-1-ethanol) aqueous solution was drop-coated on the surface of the electrode and incubated at room temperature for 2 h to block the unbound active sites.

[0043] ⑤ Add 5 μL of 1 M TCEP aqueous solution to 10 μL of 10 μM Apt aqueous solution, then drop-coat it on the electrode surface after blocking the active sites and incubate at room temperature for 120 min.

[0044] ⑥Finally, 10 μL of a 0.8 mg / mL Cu@MoSe2NFs-AuNPs aqueous solution was drop-coated on the electrode surface and incubated overnight at room temperature. Cu@MoSe2NFs-AuNPs and Apt were bound through the Au-S bond to obtain a dual-mode sensor for detecting ochratoxin A, which is referred to as the dual-mode sensor of the present invention.

[0045] It should be noted that in order to remove nonspecific binding molecules, the electrode sensing interface was rinsed with PBS buffer (pH 7.0, 0.1 M) after completing each of the above steps.

[0046] The gold nanoflowers (AuNFs) on the AuNFs-ITO surface obtained in step ② were characterized by SEM. Figure 1 (F) The image shows that the surface of AuNFs is uneven and has a flower-like shape, indicating that it has a large specific surface area and can load more DNA.

[0047] The construction principle of the dual-mode sensor of the present invention is shown in Figure 2 .

[0048] Example 2 Application of the dual-mode sensor of the present invention

[0049] The method for detecting a sample using the dual-mode sensor of the present invention comprises the following steps:

[0050] (1) Create a standard curve

[0051] Using methanol as the solvent, a 1 mg / mL OTA solution was accurately prepared, and methanol was used for gradient dilution to obtain standard solutions with concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 10 pg / mL, 1 pg / mL, 0.1 pg / mL, 10 fg / mL, 1 fg / mL, and 0.1 fg / mL, respectively. Each concentration standard solution was tested using the following method: 20 μL of OTA standard solution was drop-coated on the surface of the dual-mode sensor of the present invention and incubated at 37 ° C for 2 h. OTA specifically binds to Apt fixed on the electrode by the base complementary pairing principle, thereby releasing Apt modified by Cu@MoSe2NFs-AuNPs nanozyme (abbreviated as Cu@MoSe2NFs-AuNPs-Apt). The dual-mode sensor of the present invention was then rinsed with PBS buffer (pH 7.0, 0.1 M), and the rinse solution was centrifuged to obtain a precipitate. The complex of OTA and Cu@MoSe2NFs-AuNPs-Apt was obtained and dispersed in 600 μL of acetic acid-sodium acetate buffer (pH 5.0, 0.2 M) as the test solution. The test solution was detected by colorimetry, and the washed dual-mode sensor of the present invention was subjected to electrochemical measurement.

[0052] ① Colorimetric detection

[0053] 600 μL of the test solution containing the complex of OTA and Cu@MoSe2NFs-AuNPs-Apt was mixed with 50 μL of a solution containing 5 mM H2O2 and 20 mM TMB (solvent: 0.2 M acetic acid-sodium acetate buffer, pH 5.0) and incubated in the dark for 10 minutes. Because the Cu@MoSe2NFs-AuNPs in the complex possess peroxidase-like activity (POD), they catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to produce the blue product oxTMB, allowing the change in color intensity to be observed visually. To quantitatively analyze OTA, the absorbance of the colored product oxTMB in each well was measured at 652 nm using a microplate reader, and the data were recorded.

[0054] As the OTA concentration (C OTA ) increases, the color of oxTMB changes from light to blue ( Figure 3 C), and the absorbance value (y) gradually increased, achieving the quantitative analysis of OTA, and y and lg C OTA (Logarithm of OTA concentration, C OTA The unit is g / mL) has a good linear relationship, and the linear regression equation obtained by fitting is y=-0.0222lg C OTA +0.273(R 2=0.991), the LOD (limit of detection) was 0.982 pg / mL, and the linear detection range was 0.01 ng / mL to 100 ng / mL.

[0055] ②Electrochemical measurement

[0056] The washed dual-mode sensor was then subjected to differential pulse voltammetry (DPV) for quantitative analysis of OTA standards at various concentrations. Electrochemical testing was performed on a CHI660E electrochemical workstation (Shanghai Brilliance, China), using DPV as the testing method. Saturated calomel was used as the reference electrode (SCE), the washed dual-mode sensor as the working electrode, and a platinum wire (Pt) as the counter electrode in PBS buffer (pH 7.0, 0.1 M) containing 5 mM H₂O₂. DPV detection was performed over a sweep potential range of -0.1 to 0.5 V, with a pulse amplitude of 50 mV and a pulse width of 50 ms.

[0057] like Figure 3 As shown in (A), with C OTA As the voltage increases, the oxidation peak response current corresponding to 0.25V gradually decreases. The change in DPV peak current Δi is calculated by the following formula: p ,

[0058] Δi p =|i p -i0|,

[0059] Among them, i p For different C OTA The peak current signal obtained by the above DPV detection of the dual-mode sensor of the standard product is obtained, i0 is the peak current signal obtained by the blank sample (methanol, C OTA The peak current signal obtained by the above DPV detection was obtained by using a dual-mode sensor instead of a standard product (0).

[0060] The change of DPV peak current Δi p Different from C OTA The relationship between Figure 3 As shown in (B), in the range of 1 fg / mL to 100 ng / mL, Δi p Different from C OTA Logarithm of the value (log C OTA ) is linearly related, and the linear regression equation obtained by fitting is Δi p =-6.216lgC OTA +101.9(R 2 =0.9922), where lg C OTA is the logarithmic value of OTA concentration, C OTA The unit is g / mL, and the LOD is 0.0218fg / mL.

[0061] (2) Method for detecting samples using the dual-mode sensor of the present invention

[0062] The solid sample to be tested was crushed with a high-speed universal grinder and passed through a 20-mesh sieve to obtain a powdered sample to be tested. 5 g of the powdered sample to be tested was weighed and dissolved in 25 mL of methanol, centrifuged after vibration, and the supernatant was collected. The supernatant was then diluted with 30 mL of PBS buffer (pH 7.0, 0.1 M). The mixed solution was filtered through a 0.22 μm filter membrane, and the filtrate was collected as a sample extract for standby use. OTA standard solution was applied dropwise to the surface of the dual-mode sensor of the present invention instead of the sample extract, incubated at 37 ° C for 2 h, and then the dual-mode sensor was rinsed with PBS buffer (pH 7.0, 0.1 M). The rinse solution was centrifuged and precipitated to obtain a complex of OTA and Cu@MoSe2NFs-AuNPs-Apt, which was dispersed in 600 μL of acetic acid-sodium acetate buffer (pH 5.0, 0.2 M) as a test solution. The test solution is detected by colorimetry, and the washed dual-mode sensor of the present invention is subjected to electrochemical measurement, the specific method is the same as in Example 1. The absorbance obtained by colorimetry is substituted into the standard curve y = -0.02221 gC OTA +0.273, calculated C OTA Substitute the change in DPV peak current obtained from electrochemical measurement into the standard curve Δi p =-6.216lgC OTA +101.9, we get C OTA By combining the above two analysis techniques, the results can be mutually verified to avoid unreliable results. Figure 2 .

[0063] Example 3 Application and performance of the dual-mode sensor of the present invention

[0064] (1) Actual sample testing

[0065] The practical application capability of the dual-mode sensor of the present invention was demonstrated using rice and wheat samples purchased from a supermarket. First, the samples were pretreated according to GB5009.96-2016. The rice and wheat were each pulverized using a high-speed universal grinder and passed through a 20-mesh sieve to obtain a powdered sample. 5 g of the powdered sample was weighed and dissolved in 25 mL of methanol. After shaking, the supernatant was collected by centrifugation. The supernatant was then diluted with 30 mL of PBS buffer (pH 7.0, 0.1 M). The mixed solution was filtered through a 0.22 μm filter membrane, and the filtrate was collected as rice extract and wheat extract for later use. The dual-mode sensor of the present invention was used to detect rice and wheat extracts supplemented with different final concentrations of OTA (0.5, 1, 2.5, 5, and 10 ng / mL) using the standard addition method, with three replicates for each concentration. The electrochemical measurement results are shown in Table 3, with recoveries ranging from 97.3% to 106.6% and RSDs ranging from 2.31% to 4.33%, demonstrating the accuracy of the dual-mode sensor of the present invention.

[0066] Table 3 Results of the dual-mode sensor of the present invention for measuring OTA in rice and wheat samples (electrochemical measurement)

[0067]

[0068] To validate the dual-mode sensor's application in real-world sample testing, the standard addition method was used to test rice and wheat extracts spiked with different final concentrations of OTA (1 and 10 ng / mL). The results from the dual-mode sensor (including colorimetric and electrochemical methods) were compared with those from high-performance liquid chromatography (HPLC). The results, shown in Table 4, demonstrate a higher degree of consistency between the electrochemical data and the HPLC analysis. Compared to colorimetric methods, the electrochemical method exhibits significant advantages in terms of detectable concentration range, LOD, and data accuracy.

[0069] Table 3 Comparison of OTA detection results in rice and wheat samples using the dual-mode sensor of the present invention and HPLC

[0070]

[0071] (2) Detection performance

[0072] In order to verify the specificity of the dual-mode sensor of the present invention, according to the method in the title (1) of this example, other fungal toxins with a final concentration of 0.1 ng / mL were added to the rice and wheat extracts, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin M1 (AFM1), zearalenone (ZEN), OTA and a mixture of the above five substances (the concentration of each substance was 0.1 ng / mL), and then the dual-mode sensor of the present invention was used for detection. Figure 4 In (A), it can be seen that the dual-mode sensor of the present invention produces a significant electrochemical signal in response to OTA, while all other parameters remain unchanged. A visible electrochemical response was also observed in a mixture containing 0.1 ng / mL OTA, but this response was not significantly different from the electrochemical response to the same OTA concentration. These results demonstrate the high specificity of the constructed aptasensor.

[0073] Reproducibility is crucial for practical applications of sensors. Five independent experiments were conducted using the dual-mode sensor of the present invention with 0.1 ng / mL of OTA, and the DPV peak current was recorded and the change value was calculated. The results are shown in Figure 2. Figure 4 B. The relative standard deviation (RSD) of the peak current is 2.34%, indicating that the dual-mode sensor of the present invention has good reproducibility.

[0074] Finally, the stability of the dual-mode sensor of the present invention was further investigated. The dual-mode sensor of the present invention was stored at 4°C for two weeks. The 0.1 ng / mL OTA solution was tested every seven days. The DPV peak current was recorded and the change value was calculated. The results are shown in Figure 2. Figure 4 C. After 14 days, 94.4% of the current response was still maintained. These results indicate that the dual-mode sensor of the present invention has good stability.

Claims

1. A dual-mode sensor for detecting ochratoxin A, characterized in that The dual-mode sensor is prepared by a method comprising the following steps: (1) Depositing gold nanoparticles on the surface of the ITO electrode; (2) applying cDNA to the surface of the electrode obtained in step (1) to block the active site, and then applying the aptamer Apt; (3) The Cu@MoSe2NFs-AuNPs solution was drop-coated on the surface of the electrode obtained in step (2) to obtain a dual-mode sensor for detecting ochratoxin A.

2. The dual-mode sensor according to claim 1, characterized in that The nucleotide sequence of the aptamer Apt is shown in SEQ ID NO: 1; the nucleotide sequence of the cDNA is shown in SEQ ID NO:

2.

3. The dual-mode sensor according to claim 1 or 2, characterized in that In step (2), the drop-coating concentration of cDNA is 8-12 μM, 6-mercapto-1-ethanol is used to block the active site, and the drop-coating concentration of aptamer Apt is 8-12 μM.

4. The dual-mode sensor according to claim 3, characterized in that Cu@MoSe2NFs-AuNPs were prepared by the following method: ① Dispersing Na2MoO4·2H2O, Se powder, and NaBH4 in deionized water, adding ethanol under stirring to obtain a mixture; transferring the mixture to a reactor, and hydrothermally treating it at 180-220°C for 10-14 hours to obtain MoSe2NFs; ② MoSe2NFs aqueous solution was mixed with CuCl2·2H2O powder under ultrasound to obtain Cu@MoSe2NFs. ③ Disperse Cu@MoSe2NFs in deionized water, ultrasonically treat, add HAuCl4 aqueous solution, heat to boiling, add sodium citrate aqueous solution, and keep boiling for 0.8-1.2 hours under stirring; after cooling, centrifuge to obtain the precipitate, wash and dry to obtain Cu@MoSe2NFs-AuNPs.

5. The dual-mode sensor according to claim 4, characterized in that In step ①, the molar ratio of Na2MoO4·2H2O, Se powder and NaBH4 is 1:1-3:1-3, and the volume ratio of ethanol and deionized water is 1:0.8-1.2; 1 mmol of the mixture of Na2MoO4·2H2O, Se powder and NaBH4 is dispersed in 70-90 mL of water.

6. The dual-mode sensor according to claim 5, characterized in that In step ②, the mass ratio of MoSe2NFs to CuCl2·2H2O powder is 10:0.5-1.

5.

7. The dual-mode sensor according to claim 6, characterized in that In step ③, the mass ratio of Cu@MoSe2NFs, HAuCl4, and sodium citrate is 50:6-8:30-40.

8. Use of the dual-mode sensor according to claim 1 in detecting ochratoxin A.

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