A novel electrochemical aptasensor for fumonisin B1 determination using DNA and exonuclease-I as signal amplification strategy
In this work, using DNA and exonuclease-I (Exo-I) as signal amplification strategy, a novel and facile electrochemical aptasensor was constructed for fumonisin B1 (FB1) detection. The G-rich complementary DNA (cDNA) was immobilized onto the electrode surface. Then, aptamer of FB1 was hybridized with cDNA to form double-stranded DNA. In the absence of FB1, double-stranded DNA and G-rich cDNA on the electrode surface promoted effectively methylene blue (MB) enrichment and amplified the initial electrochemical response. In the presence of FB1, the combination of aptamer and FB1 led to the release of aptamer from the electrode surface and the expose of 3′ end of single-stranded cDNA. When Exo-I was added onto the electrode surface, the single-stranded cDNA was degraded in the 3′–5′ direction. The decrease of double-stranded DNA and G-rich cDNA resulted in the less access of MB to the electrode surface, which decreased the electrochemical signal. The experimental conditions including incubation time of FB1, the amount of Exo-I and incubation time of Exo-I were optimized. Under the optimal conditions, the linear relationship between the change of peak current and the logarithmic concentration of FB1 was observed in the range of 1.0 × 10−3–1000 ng mL−1 with a low limit of detection of 0.15 pg mL−1. The experimental results showed that the prepared aptasensor had acceptable specificity, reproducibility, repeatability and stability. Therefore, this proposed aptasensor has a potential application in the food safety detection.
KeywordsElectrochemical aptasensor Fumonisin B1 Exonuclease-I G-rich DNA Methylene blue
aptamer of FB1
differential pulse voltammetry
electrochemical impedance spectroscopy
the gold electrode
the charge transfer resistance
the difference of peak current
limit of detection
relative standard deviation
As the metabolic product of Fusarium moniliforme Sheld, fumonisin B1 (FB1) is a kind of the most toxic and prevalent fumonisins . FB1 can contaminate various food and feedstuff such as corn, wheat, rice, peanut, beer, and animal feed. A large number of studies have reported that FB1 can cause serious diseases such as horse white matter softening, nephrotoxicity, hepatotoxicity and liver cancer [2, 3]. Therefore, it is necessary to monitor FB1 for food safety and human health.
Among the various methods for FB1 detection [4, 5, 6, 7], the electrochemical aptasensor has attracted widespread attention due to their low cost, simple operation, high selectivity and affinity, chemical stability, and easy storage [8, 9]. Recently, with the advantages including effective amplification strategy, easy design, simple operation and rapid reaction, the nuclease-based electrochemical aptasensor has become research focus [10, 11]. Among the different nucleases, exonuclease I (Exo-I) has attracted increasing attention, owing to its structure-sensitive digestion for the single-stranded DNA in the direction of 3′ to 5′, low cost, good specificity and buffer compatibility [12, 13, 14]. As a kind of electrochemical signal probe, methylene blue (MB) can highly interact with G-rich single-stranded DNA and double-stranded DNA, and is therefore suitable for the application in electrochemical aptasensor [15, 16].
Herein, based on MB, Exo-I, aptamer of FB1 (Apt) and G-rich cDNA, a novel signal-off sensor was firstly designed for the electrochemical detection of FB1. The existing double-stranded DNA on the electrode surface, came from the hybridization of Apt and G-rich cDNA, enriched abundant MB and amplified the initial electrochemical response. In the presence of FB1, the formation of Apt-FB1 made aptamer release from the electrode surface. Then, the effect of Exo-I on G-rich cDNA of the electrode surface resulted in the less access of MB, which further decreased the electrochemical signal and amplified ΔI. The change of MB electrochemical signal can be applied for FB1 detection.
In virtue of the favorable combination of MB with double-stranded DNA and G-rich cDNA, and the advantages of Exo-I including easy design, simple operation, high amplification efficiency and excellent selectivity, the proposed signal amplification strategies can save the tedious preparation process and is beneficial to the experimental stability.
Materials and chemicals
The used oligonucleotides were provided by Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China), and their sequences were as follows: cDNA: 5′-SH-GAG GGG TGG GCG GGA GGG AGA TTG CAC GGA CTA TCT AAT TGA ATA AGC-3′. Apt: 5′-ATA CCA GCT TAT TCA ATT AAT CGC ATT ACC TTA TAC CAG CTT ATT CAA TTA CGT CTG CAC ATA CCA GCT TAT TCA AGT AGA TAG TAA GTG CAA TCT-3′. FB1 and Exo-I were purchased from Acros and TaKaRa, respectively. 0.05 M of pH 7.4 Tris–HCl buffer (containing 0.05 M Tris, 0.2 M NaCl and 0.001 M EDTA) was used.
The CHI 660E Electrochemical Workstation was used for the electrochemical experiments (Shanghai Chenhua Instrument Corporation, China). The gold electrode (AuE) was used as working electrode. Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were used for the electrochemical measure.
Fabrication and mechanism of the aptasensor
When FB1 was absent, cDNA and Apt could not be degraded by Exo-I because that the 3′ end of both cDNA and Apt were protected by the formation of double-stranded DNA. MB could intercalate into G-rich cDNA and double-stranded DNA, and produce a strong current signal. When FB1 was present, the complex of Apt and FB1 was formed and released from the surface of the electrode, leading to the expose of 3′ end of single-stranded cDNA on the electrode surface. When Exo-I was added onto the electrode surface, the single-stranded cDNA was degraded in the 3′-5′ direction. The decrease of double-stranded DNA and G-rich cDNA resulted in the less access of MB to the electrode surface and the decrease of the electrochemical signal. The change of MB electrochemical signal can be applied for FB1 detection.
Results and discussion
Electrochemical characterization of the prepared aptasensor
The detection of FB1 on Apt/cDNA/AuE sensor
Optimization of the aptasensor
Analytical performance of the designed aptasensor
Comparison with other reported methods for FB1 detection
Linearity, (ng mL−1)
LOD, (ng mL−1)
Chemiluminescence and enzyme-linked immunosorbent
ECL-ELISA based on anti-FB1 IgG and HRP
0.21 × 10−3
Fluorescence resonance energy transfer
1 × 10−3 ~ 100
0.35 × 10−3
2 × 10−3
Electrochemical magneto immunosensor
1 × 10−2~50
3.4 × 10−3
1 × 10−3~1000
1 × 10−3
1 × 10−3~1000
0.15 × 10−3
Specificity, reproducibility, repeatability and stability
Under the optimized conditions, the reproducibility and the repeatability of the fabricated aptasensor was respectively evaluated with inter-assay and intra-assay. Under the same experimental conditions, five fabricated aptasensors were tested by monitoring the peak current of MB with 1 μg mL−1 FB1 on the FB1/Apt/cDNA/AuE, and a relative standard deviation (RSD) of 5.72% was calculated, implying that the fabricated sensor had satisfactory reproducibility. The one aptasensor was investigated by monitoring the peak current of MB in the presence of 1 μg mL−1 FB1 for five replicate determinations under the same conditions, and RSD of 5.38% was calculated, implying that the fabricated aptasensor had acceptable repeatability.
For the study on stability of the fabricated aptasensor, the peak current of MB on the three Exo-I/Apt/cDNA/AuE was detected, and the average peak current is 7.21 μA. Then the fabricated aptasensors were stored at 4 °C. After a 35-day storage period, the average peak current of MB on the Exo-I/Apt/cDNA/AuE was 6.14 μA, and the aptasensor retained 85.2% of its initial current response, indicating the acceptable stability.
Analysis of FB1 in food samples
Recovery of FB1 in food samples
Added (ng mL−1)
Average found (ng mL−1)
Average recovery (%)
RSD (%), n = 3
1 × 10−2
0.986 × 10−2
1 × 10−2
1.068 × 10−2
In summary, on the basis of DNA and Exo-I as signal amplification strategy, a novel and facile signal-off aptasensor was developed for FB1 detection. Utilizing the favorable combination of MB with double-stranded DNA and G-rich cDNA, the specific DNA was designed to enrich abundant MB for initial signal amplification. On the other hand, with the advantages of easy design, simple operation, high amplification efficiency and excellent selectivity, Exo-I was used to design a novel signal-off aptasensor for amplifying the ΔI. These two signal amplification strategies can avoid the complicated nanomaterial preparation and instability. As a result, this proposed aptasensor showed the favorable performance with simple preparation, good selectivity, reproducibility, repeatability, stability as well as a wider linear range with lower LOD, providing a promising potential for application in food safety detection.
MW, FZ, SF and HJ conceived and designed the experiments; FZ and SF performed the experiments; MW and HJ analyzed the data; MW, FZ, and SF wrote and modified the paper. All authors read and approved the final manuscript.
This study was supported by the Natural Science Foundation of Henan Province (182300410188) in the design of the study, and collection, analysis, and interpretation of data; supported by the Fundamental Research Funds for the Henan Provincial Colleges and Universities in Henan University of Technology (2016RCJH04) in collection, analysis, and interpretation of data; supported by Key Scientific and Technological Project of Henan Province (192102310255) in writing the manuscript.
The authors declare that they have no competing interests.
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