The determination of Ochratoxin A based on the electrochemical aptasensor by carbon aerogels and methylene blue assisted signal amplification
© The Author(s) 2018
Received: 7 November 2017
Accepted: 19 April 2018
Published: 25 April 2018
In this work, a novel aptamer-based electrochemical biosensor was developed for the determination of Ochratoxin A (OTA) by using carbon aerogels (CAs) and methylene blue (MB) as signal amplification strategy. CAs was used as carrier to load the abundant of complementary DNA (cDNA), which could enhance the hybridization between CAs-cDNA and aptamer immobilized on the electrode surface, thus provide more double-stranded DNA for MB intercalation. The current of MB on the CAs-cDNA/apt/AuE sensor was twice that on the cDNA/apt/AuE sensor, which indicated that the CAs with high surface area enabled a higher loading of the cDNA and absorbed more MB, thus realized the signal amplification strategy. The optimum experimental conditions including MB incubation time of 15 min, aptamer concentration of 4.0 μmol/L, hybridization time of 2.0 h, and OTA incubation time of 18 min were obtained. The change of peak current was linearly proportional to the OTA concentration in the range of 0.10–10 ng/mL with the actual detection limit of 1.0 × 10−4 ng/mL. The experimental results showed that the prepared CAs-cDNA/apt/AuE exhibited good specificity, acceptable reproducibility and repeatability. This sensor was applied to detect OTA in the spiked corn samples, and obtained an acceptable average recovery of 89%.
Mycotoxins are toxic contaminants produced by the secondary metabolism of fungi, mainly saprophytic molds . As one of the highly toxic mycotoxins, Ochratoxin A (OTA) secreted by Aspergillus and Penicillium has attracted much more attention because it contaminates broad range of agricultural products such as maize, wheat, rice, coffee, and peanut, then results in serious human and animal health problems including nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic activities . So, it is increasingly necessary to develop a precise, rapid and low-cost method for OTA determination in various samples. Conventional instrumental analyses such as high performance liquid chromatography, liquid chromatography tandem mass spectrometry, and fluorescence are popular because of their high sensitivity, good accuracy and reproducibility [3–6]. However, they exist some drawbacks such as sophisticated equipment, high cost and requirement of technical skills . The immunoassay methods based on antigen–antibody binding have the advantages of simple, rapid and easy to operation, and appear an useful tool for on-site detection of OTA [8–11]. However, the antibody preparation process is complex and time-consuming, high cost, and the antibody itself is unstable, immunogenic and false. So it can not be used as a final confirmation method, which hinders its wider application.
As a novel bio-recognition element, aptamers, single strand oligonucleotides, with the superiority including strong affinity, high stability, and easy modification of functional groups, have the potential designing highly sensitive, selective and structure switchable sensing assays [12–14]. Recently, aptamer-based electrochemical biosensors for OTA detection are prominent owing to their fast response, low cost, simple operation, easy to miniaturization of the instrument, and portability [15–19].
To realize the signal amplification and improve the sensitivity of the electrochemical aptasensors, nanomaterials have been chosen because their large specific surface area allows immobilizing more signal molecules on the electrode surface, and their well electronic conductivity makes the charge transfer to the electrodes easier [20–23]. Due to their favorable properties including great mesopore volume, high accessible surface area and good electrical conductivity, carbon aerogels (CAs) have attracted tremendous attention and have been extensively used as supports of precious metal for electrocatalytic reaction [24, 25], whereas have seldom been used for immobilization of biomolecules .
In this work, a novel aptamer-based electrochemical biosensor was developed for the determination of OTA by using CAs and methylene blue (MB) as signal amplification strategy. CAs was used as carrier to load the abundant of complementary DNA (cDNA), which could enhance the hybridization between CAs-cDNA and aptamer immobilized on the electrode surface, thus provide more double-stranded DNA for MB intercalation. As an electrochemical indicator, MB could intercalate both into single-stranded cDNA through the guanine bases and into double-stranded DNA, and produce a strong current signal. When OTA existed, the formation of aptamer-OTA complex changed the conformation of aptamer and prohibited the binding of cDNA-aptamer, which resulted in the release of MB from the electrode surface and produced a reduced current signal. The change of MB current signal could be used for OTA detection.
Materials and chemicals
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), methylene blue (MB) were purchased by Macklin Biochemical Co., Ltd. (Shanghai, China). All oligonucleotides were synthesized by Sangong Biotech (Shanghai, China) Co., Ltd., and their base sequences were: complementary DNA (cDNA): 5′-NH2-GGA GGA GGA GGA GGA GGA GGA GGA GGA GGA GGA GGA GGA TGT CCG ATG CTC CCT TTA CGC CTC-3′; OTA aptamer (apt): 5′-HS-GAT CGG GTG TGGGTG GCG TAA AGG GAG CAT CGG ACA-3′. 50 mM, pH7.4 Tris–HCl was prepared by 0.20 M NaCl and 1.0 mM EDTA and adjusting the pH with 0.10 M HCl. All other chemicals were of analytical-reagent grade.
All the electrochemical experiments were performed on a CHI 660E Electrochemical Workstation (Shanghai Chenhua Instrument Corporation, China). A three-electrode system was comprised of Au electrode (AuE) as working electrode, platinum wire as auxiliary electrode, and Ag/AgCl as reference electrode. Scanning electron microscopy (SEM) was performed using a JEOL JSM7100F SEM facility (Jeol, Japan).
Preparation of the CAs-cDNA/apt/AuE sensor for OTA detection
The AuE was polished with 0.30 and 0.050 μm gamma alumina powder successively and then rinsed with ultrapure water and dried by nitrogen. The AuE was activated by scanning cyclic voltammogram (CV) with 0.50 M H2SO4.
CAs were synthesized by the sol–gel polymerization of resorcinol (R) and formaldehyde (F) in an aqueous solution according to the method described elsewhere . cDNA (25 μL, 100 μM) was put into 500 μL of CAs suspension, then 250 μL of EDC and NHS was separately added into the solution, the mixture was incubated overnight at 37 °C. Next, the above solution was incubated with NaCl (50 μL, 2.0 M) for 24 h and centrifugated at 12,000 rpm to remove the unbound cDNA. The precipitate was repeatedly rinsed and redispersed in 5.0 mL Tris–HCl solution to obtain the CAs-cDNA.
Results and discussion
Characterization of the prepared CAs
Electrochemical characterization of the CAs-cDNA/apt/AuE sensor
Electrochemical behavior of MB on the different sensors
The detection mechanism of OTA based on the CAs-cDNA/apt/AuE sensor
The optimization of the important factors
Analytical performance of the CAs-cDNA/apt/AuE sensor
The specificity of the CAs-cDNA/apt/AuE sensor
Reproducibility and repeatability of the CAs-cDNA/apt/AuE sensor
The reproducibility of the developed CAs-cDNA/apt/AuE sensor was evaluated with inter-assay precision. The five CAs-cDNA/apt/AuE sensors were tested for DPV with same OTA concentration under the same experimental conditions. A relative standard deviation (RSD) of 6.7% was calculated, indicating a good reproducibility of the developed aptasensor. The intra-assay precision of the CAs-cDNA/apt/AuE sensor was evaluated by five repetitive measurements with one electrode and RSD of 7.3% was obtained, indicating that the prepared aptasensor had acceptable repeatability. The prepared CAs-cDNA/apt/AuE was stored at 4 °C when not in use. After a 20-day storage period, the sensor retained 93% of its initial current response, providing the acceptable stability.
The application of the aptasensor to corn sample
The detection of OTA in the spiked corn sample
Spiked concentration (ng/mL)
Theoretical value C (ng/mL)
Average recovery %
In this work, a novel CAs-cDNA/apt/AuE sensor was developed to detect OTA using CAs and MB assisted signal amplification. The CAs could load the abundant cDNA and absorb more MB, so the peak current of MB on the CAs-cDNA/apt/AuE sensor was higher than that on the cDNA/apt/AuE sensor. Under the optimized experimental conditions, the developed aptasensor could detect OTA at the level of 1.0 × 10−4 ng/mL, and exhibited good specificity against ZEA and AFB1. This sensor was also applied to detect OTA in the spiked corn samples, and an acceptable average recovery of 89% was obtained. By changing the aptamers for different target molecules, this strategy has potential prospect for detecting other targets in the convenient field monitoring.
MW planed and supervised the whole work, and revised the manuscript. WYZ carried out the experiments and drafted the manuscript. Both authors read and approved the final manuscript.
This study was funded by the Natural Science Foundation of Henan Province (182300410188), the Fundamental Research Funds for the Henan Provincial Colleges and Universities in Henan University of Technology (2016RCJH04).
The authors declare that they have no competing interests.
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