AU2019284026B2

AU2019284026B2 - Biosensor for simultaneous detection of zearalenone and ochratoxin A , preparation and detection method thereof

Info

Publication number: AU2019284026B2
Application number: AU2019284026A
Authority: AU
Inventors: Han DU; Qi Wang; Wei Wu; Chunlei Yan; Qingli Yang; Chundi Yu; Haiyan Zhao; Yinglian Zhu
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2019-08-06
Filing date: 2019-12-20
Publication date: 2021-04-08
Anticipated expiration: 2039-12-20
Also published as: CN110261362A; AU2019284026A1; ZA201908346B; CN110261362B

Abstract

The invention displays a biosensor for simultaneous detection of zearalenone (ZEN) and ochratoxin A (OTA) , and it is a preparation and detection method thereof. The invention belongs to the technical field of detecting harmful substances. The biosensor was fabricated with ZEN aptamers, OTA aptamer and graphene oxide. The innovation of the biosensor is that it can not only detect ZEN and OTA without interfering with each other at the same time, but also has high sensitivity. Compared with other biosensors, the present invention has the following benefits: simple operation steps, low-cost and short detection time. 4/4 90 80 70 - l 512nm d 60 * 0 499nm 0 8 40 30 20 10 ZEN OTA AFBI AFM1 FB Patulin sample Figure 7

Description

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90 80 70 - l 512nm d 60 *0 499nm 0

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Figure 7

BIOSENSOR FOR SIMULTANEOUS DETECTION OF ZEARALENONE AND OCHRATOXIN A, PREPARATION AND DETECTION METHOD THEREOF

Technical Field

[0001] The invention discloses abiosensor for simultaneous detection of zearalenone (ZEN) and ochratoxin A (OTA), a preparation and detection method thereof. The invention belongs to the technical field of detecting harmful substances.

Background

[0002] Mycotoxins are toxic secondary fungal metabolites produced by filamentous fungi. Toxicity, teratogenicity, carcinogenicity and mutagenicity can occur when a certain amount of mycotoxin is ingested. ZEN is known as important nonsteroidal estrogenic mycotoxins produced by fusarium fungi species. ZEN may cause infertility, reproduction problems in animals and humans, and it has been classified as group III carcinogens by the International Agency for Research and Cancer (IARC). OTA is a secondary metabolite produced mainly by fungi such as Aspergillus and Penicillium. OTA can negatively influence the protein synthesis, increase the lipid peroxidation rate, harm the saccharide and calcium metabolisms, and paralyse the mitochondrial functions. The International Agency for Research on Cancer has classified OTA as a possible human carcinogen (group 2B) due to its toxic effects on the human body, such as nephrotoxic, hepatotoxic, immunotoxin, teratogenic, and carcinogenic effects. In recent years, mycotoxins have become a major threat to the quality and safety of crops, raw materials of food and feed. It not only seriously endangers human and animal health and life safety, but also causes huge economic losses. Therefore, the development of high sensitivity, high throughput and simultaneous identification of multiple mycotoxins has great practical significance in food safety detection.

[0003] The traditional detection methods of OTA and ZEN mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE) and enzyme-linked immunosorbent assay (ELISA). These detection approaches face challenges due to their time-consuming, high-cost and low-sensitivity.

[0004] Aptamers are (25-90 nt) single-stranded nucleic acid molecules (DNA or RNA) generated from Systematic Evolution of Ligands by Exponential Enrichment (SELEX), an in vitro selection technique. As bio-recognition elements, aptamers can recognize various target ligands with high specificity. Compared to antibodies, aptamers show higher stability to temperature, pH and ionic strength, easier synthesis and modification, longer shelf life, and lower cost. Based on the superiority, aptamers have attracted a lot of attention in construction of aptamer-based assays and sensors, referred to as apta-assays and aptasensors, respectively.

Brief Summary of the Invention

[0005] To solve the problems of tedious scheme, complex operation steps, high cost and long time of single detection method of ZEN and OTA in the prior art, the present invention provides a fluorescent aptasensor based on graphene oxide (GO) as fluorescence quencher for simultaneous detection of ZEN and OTA.

[0006] There is disclosed herein a biosensor for simultaneously detecting zearalenone and ochratoxin A, the biosensor comprising a zearalenone aptamer with the 5' end labelled by a fluorescent group, an ochratoxin A aptamer with the 5' end labelled by a fluorescent group, and graphene oxide, wherein the nucleotide sequence of the zearalenone aptamer is 5'-CTA CCA GCT TTG AGG CTC GAT CCA GCT TAT TCA ATT ATA CCA GCT TAT TCA ATT ATA CCA GC-3', wherein the nucleotide sequence of the ochratoxin A aptamer is 5'-AGC CTC GTC TGT TCT CCC GGC GCA TGA TCA TTC GGT GGG TAA GGT GGT GGT AAC GTT GGG GAA GAC AAG CAG ACG T-3', wherein the fluorescent group of the zearalenone aptamer is labelled as Cy3, and the fluorescent group of the ochratoxin A aptamer is labelled as Alexa Fluor 488, and wherein the graphene oxide is used as a fluorescence quencher.

[0007] There is also disclosed herein a method of preparing the biosensor descrbied above, the method comprising: mixing graphene oxide with 20 L of zearalenone aptamer and 20 L of ochratoxin A aptamer; adding phosphate-buffered saline to form a mixture of a volume of 1 mL; and incubating the mixture at room temperature for 10 min to ensure that the zearalenone aptamer and the ochratoxin A aptamer are fully combined with the graphene oxide to form a zearalenone aptamer-ochratoxin A aptamer-graphene oxide complex.

[0008] The volume of graphene oxide may be 80 L.

[0009] There is further disclosed herein a method for simultaneously detecting zearalenone and ochratoxin A, the method comprising: mixing the biosensor described above with a sample to be detected, at a volume ratio of 99:1, respectively, in a water bath for 1 hour at a temperature of 45°C to form a reaction solution; scanning, using a fluorescence spectrophotometer, fluorescence emission spectra of the reaction solution at excitation wavelengths of 512 nm and 499 nm, respectively; and measuring fluorescence intensity into a standard curve to calculate the concentration of zearalenone and ochratoxin A in the sample to be detected.

[0010] The advantages of preferred embodiments the invention are as follows: 1) GO can quench about 90% of the fluorescence, creating a low background signal environment. In the presence of dual targets, the significant fluorescence signal can be restored and the fluorescence state can be changed from on to off; 2) Capacity of simultaneous detection is as low as 1 ng/mL for ZEN and OTA detection; 3) Advantage of simple operation, low cost, good specificity and high sensitivity; 4) This biosensor can simultaneously detect ZEN and OTA without interfering with each other.

Description of Figures

[0011] Figure 1: The fluorescence emission spectra of simultaneous detection ZEN and OTA by the biosensor of the invention;

[0012] Figure 2: The atomic force microscope figure of simultaneous detection ZEN and OTA by the biosensor of the invention;

[0013] Figure 3: Standard curves of ZEN and OTA standard solution at different concentrations;

[0014] Figure 4: Effect of GO volume on fluorescence background of detection platform;

[0015] Figure 5: Effect of incubation temperature onbiosensor binding ZEN and OTA;

[0016] Figure 6: The sensitivity detection of the biosensor;

[0017] Figure 7: The specificity detection of the biosensors.

Description of Embodiments

[0018] Unless otherwise specified, the terms used in the description of the invention typically have the meanings commonly appreciated by those ordinarily skilled in the art.

[0019] The invention is further detailed below in combination with embodiments and reference data. The following embodiments are only used for illustratively explaining the invention, and are not intended to limit the scope of the invention in any form.

[0020] Two aptamers and phosphate buffer saline (PBS) (containing NaCl 136.89 mM, KCl 2.67 mM, Na2HPO4 8.1 mM, KH2PO4 1.76 mM, pH = 7.4) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). GO was purchased from Xianfeng Nanomaterials Tech Co., Ltd. (Nanjing, China). ZEN aptamer and OTA aptamer were modified with the fluorescence dyes (Cy3 and Alexa Fluor 488) on their 5'-end. OTA and ZEN were purchased from Pribolab Co., Ltd. (QingDao, China, http://www.pribolab.com). All fluorescence spectra were scanned using a Hitachi F-2700 fluorescence spectrophotometer (Hitachi Ltd., Japan). The height trace images of the atomic force microscope were scanned using an SPM-9700 atomic force microscope (Shimadzu, Japan).

Embodiment

(1) The detection platform based on ZEN aptamer, OTA aptamer and GO

[0021] Pretreatment: Freeze-dried powder of the two aptamers was diluted to working concentration (1 M) in PBS, respectively. Then, aptamer was stored at 4 °C and kept it away from light with tinfoil. The suspension of GO of 500 ug/mL was diluted to 250 ug/mL with ultra-pure water. The suspension was ultrasonic for 1 h at 200W and stored at 4°C.

[0022] 80 L GO nanosheets were homogeneously mixed with 20 L APT Iand 20 L APT2 at room temperature for 10 min. The final volume was 1 ml by adding PBS. It is worth noting that ZEN and OTA aptamers need to be combined with graphene oxide in the mixing process. The formed ZEN aptamer-OTA atamer-GO complex was the platform for simultaneous detection of ZEN and OTA.

[0023] Herein, The sequence of ZEN aptamer was 5'-Cy3-CTA CCA GCT TTG AGG CTC GAT CCA GCT TAT TCA ATT ATA CCA GCT TAT TCA ATT ATA CCA GC-3'; The sequence of OTA aptamer was 5'-Alexa Fluor 488-AGC CTC GTC TGT TCT CCC GGC GCA TGA TCA TTC GGT GGG TAA GGT GGT GGT AAC GTT GGG GAA GAC AAG CAG ACG T-3'; GO was used as fluorescence quencher.

(2) Specific recognition of aptamers for targets

[0024] The ZEN&OTA aptamer-GO complexes (990 L) prepared by the above method were mixed with sample (10 L) at a volume ratio of 99:1. In order to mix the biosensor with the sample fully, the biosensor was bathed in water for 1 hour at 45 °C. The interaction forces of the aptamer target were more powerful than those of the GO aptamer. Subsequently, the aptamer target was released from GO, recovering the prominent fluorescence of ZEN apatmers and OTA aptamers. The fluorescence intensity of the solution to be tested was measured by scanning the fluorescence emission spectrum at the excitation wavelength of 512 nm and 499nm, respectively. The target concentration and the restored value of fluorescence intensity have a good proportional relationship.

[0025] ZEN aptamer and OTA aptamer have strong fluorescence. Upon the addition of GO, ZEN aptamer and OTA aptamer were adsorbed by GO due to the presence of oxygen containing functional groups and conjugated structures on the GO surface. In addition, the fluorescence of the aptamers was quenched by fluorescence resonance energy transfer (FRET), forming a low background signal environment. In the presence of the dual targets, the aptamer target was released from GO and formed ZEN aptamer-ZEN complexes and OTA aptamer-OTA complexes, recovering the prominent fluorescence of ZEN apatmers and OTA aptamers. The maximum emission wavelength of ZEN aptamer is 560-570 nm, and that of OTA aptamer is 520-540 nm. At the excitation wavelength of the fluorescence spectrum, the two have different maximum emission wavelengths and do not interfere with each other (Figure 1).

[0026] The precipitate of GO, ZEN & OTA aptamer-GO and ZEN & OTA aptamer-GO ZEN&OTA were scanned by SPM-9700 AFM to determine the height change of the samples. As shown in Figure 2, the average height of the GO sheets was approximately 1.28 0.01 nm (Figure 2A). Then, the height of the aptamer-GO complexes was 2.2 0.008 nm (Figure 2B). The increased thickness demonstrated that aptamers were successfully adsorbed on the surface of GO. Finally, when the GO-APT complex was incubated with targets, its thickness significantly decreased to and 1.65 0.006 nm (Figure 2C). This phenomenon confirmed that the aptamer was released from the surface of GO because the interaction forces of the aptamer target were stronger than those of the GO aptamer.

[0027] According to the above steps, different concentrations of ZEN and OTA standard solution were detected, and the fluorescence intensity values of each sample were recorded. The standard curve was constructed by measuring the fluorescence intensity and the known sample concentration. Finally, the fluorescence intensity of samples with unknown concentration was brought into the standard curve to calculate the concentration of ZEN and OTA in the samples to be measured.

[0028] As shown in Figure 3, the regression equation of ZEN was y = 8.9712 ln(x)+ 84.532, R2= 0.9986 and that of OTA was y = 13.352 ln(x) + 88.132, R2 = 0.9886. Hence, a conclusion was drawn that the target concentration and the restored value of fluorescence intensity have a good proportional relationship.

Effect of GO concentration on fluorescencebackground of detection platform

[0029] 1 M ZEN & OTA aptamers were incubated with different volume of GO. The volume of GO is set as 40 L, 60 L, 80 L,1I00L and 120 [L, corresponding to samples1 6 respectively. As shown in Figure 4, With the increase of GO volume, the fluorescence intensity of ZEN aptamer and OTA aptamer decreased gradually. When the volume of GO reaches 80 L, the fluorescence intensity tends to be stable. Therefore, the optimal volume of GO is 80 L.

Effect of incubation temperature on biosensor binding ZEN and OTA

[0030] At room temperature, the fluorescence intensity did not recover significantly after adding the dual target. The interaction of GO and the aptamer was weakened by increasing the temperature, and the APT target desorbed. Thus, to enhance the fluorescence restoration, increasing the temperature is an ideal method. The selected temperature range is 30-50°C. The fluorescence intensity restoration increased with increasing temperature (30°C, 35°C, °C, 45°C, and 50°C) in general when there was no target (blue bars in Figure 5). This confirmed the previous theory that high temperature accelerates the release of the aptamer from the GO surface. However, the phenomenon was different when targets were added to the GO-APT compound. The fluorescence signal increased regularly below 45°C (red bars in Figure 5). When the temperature exceeded 45°C, the fluorescence intensity restoration was weakened. Thus, to enhance the fluorescence restoration, increasing the temperature is an ideal method. At the same time, excessively high temperatures will destroy the binding between the aptamer and the target. Thus, 45°C is the critical point of the fluorescence intensity restoration.

The sensitivity and specificity detection of the biosensor

[0031] As shown in Figure 6, the aptasensor exhibited LOD of 1 ng/mL for ZEN and 1 ng/mL for OTA in linear concentration range of 1-500ng/mL.

[0032] The specificity of the fluorescence aptasensor was further checked using other possible interfering mycotoxins, such as aflatoxin B I(AFB1), aflatoxin M1 (AFM1), fumonisin B1 (FB1), and patulin. ZEN, OTA, AFB1, AFM1, FB1 and patulin were added separately to the GO-APT1&2 complex at a concentration of 100 ng/mL each. The value of the fluorescence recovery is shown in Figure 7. The fluorescence recovery value of ZEN at 512 nm was at least three times higher than that of other mycotoxins. Under the excitation wavelength of 499 nm, the fluorescence recovery value of OTA is about six times that of other mycotoxins. The results strongly illustrated that APT1&2 possesses a higher specificity for ZEN and OTA, respectively, than other mycotoxins.

[0033] The embodiments are only preferred ones of the invention, and are not intended to limit the invention in any form. Any skilled in the art can transform or modify the technical contents disclosed below to obtain equivalent embodiments. Any simple modifications or equivalent transformations to the following embodiments according to the technical essence of the invention without deviating from the contents of the technical solutions of the invention should also fall within the protection scope of the technical solutions of the invention.

Claims

CLAIMS:

1. A biosensor for simultaneously detecting zearalenone and ochratoxin A, the biosensor comprising a zearalenone aptamer with the 5' end labelled by a fluorescent group, an ochratoxin A aptamer with the 5' end labelled by a fluorescent group, and graphene oxide, wherein the nucleotide sequence of the zearalenone aptamer is 5'-CTA CCA GCT TTG AGG CTC GAT CCA GCT TAT TCA ATT ATA CCA GCT TAT TCA ATT ATA CCA GC-3', wherein the nucleotide sequence of the ochratoxin A aptamer is 5'-AGC CTC GTC TGT TCT CCC GGC GCA TGA TCA TTC GGT GGG TAA GGT GGT GGT AAC GTT GGG GAA GAC AAG CAG ACG T-3', wherein the fluorescent group of the zearalenone aptamer is labelled as Cy3, and the fluorescent group of the ochratoxin A aptamer is labelled as Alexa Fluor 488, and wherein the graphene oxide is used as a fluorescence quencher.

2. A method of preparing the biosensor of claim 1, the method comprising: mixing graphene oxide with 20 L of zearalenone aptamer and 20 L of ochratoxin A aptamer; adding phosphate-buffered saline to form a mixture of a volume of 1 mL; and incubating the mixture at room temperature for 10 min to ensure that the zearalenone aptamer and the ochratoxin A aptamer are fully combined with the graphene oxide to form a zearalenone aptamer-ochratoxin A aptamer-graphene oxide complex.

3. The method of claim 2, wherein the volume of graphene oxide is 80 L.

4. A method for simultaneously detecting zearalenone and ochratoxin A, the method comprising: mixing the biosensor of claim 1 with a sample to be detected, at a volume ratio of 99:1, respectively, and incubating in a water bath for 1 hour at a temperature of 45°C to form a reaction solution; scanning, using a fluorescence spectrophotometer, fluorescence emission spectra of the reaction solution at excitation wavelengths of 512 nm and 499 nm, respectively; and measuring fluorescence intensity into a standard curve to calculate the concentration of zearalenone and ochratoxin A in the sample to be detected.