Electrochemical characterization of the prepared aptasensor
Fig.2 showed the EIS characterization for the aptasensor fabrication. The charge transfer resistance (Rct) increased from 251.3 ohm of the bare AuE (a) to 1219 ohm of the cDNA/AuE (b), indicating that the cDNA was immobilized to the electrode surface. For the Apt/cDNA/AuE (c), the Rct was increased to 1381 ohm, indicating that Apt hybridized successfully with cDNA on the electrode surface. After the Apt/cDNA/AuE was incubated by 1 μg·mL-1 FB1 and Exo-I respectively, the Rct (d) was decreased to 836 ohm. This was because that Apt was specifically combined with FB1 and released from the electrode, and cDNA was digested by Exo-I due to the expose of its 3’ end, resulting in the less negative charge on the electrode surface.
The detection of FB1 on Apt/cDNA/AuE sensor
Fig.3 showed the DPV results of MB on the Exo-I/Apt/cDNA/AuE (a), FB1/Apt/cDNA/AuE (b) and Exo-I/FB1/Apt/cDNA/AuE (c) in Tris-HCl buffer. In the absence of FB1, the Exo-I/Apt/cDNA/AuE showed an initial peak current of 7.29 μA (a). With the addition of 1 μg·mL-1 FB1, the peak current of FB1/Apt/cDNA/AuE (b) decreased to 4.02 μA. This is because that in the presence of FB1, the formation of Apt-FB1 composite made Apt release from double-stranded DNA on the electrode surface, resulting in that the amounts of MB intercalated into the double-stranded DNA were decreased. After the addition of Exo-I, the DPV value of Exo-I/FB1/Apt/cDNA/AuE (c) further decreased to 2.41 μA, indicating that Exo-I could digest the single-stranded cDNA on the electrode surface and achieve the signal amplification.
Optimization of the aptasensor
Fig.4 showed the effect of FB1 incubation time (A), Exo-I amount (B) and Exo-I incubation time (C) on the electrochemical signal. As shown in Fig.4 A, it can be seen that ΔI increased with the increasing of FB1 incubation time and reached the maximum of 5.3 μA at 10 min. Therefore, 10 min was selected as the optimal FB1 incubation time. As can be seen from Fig.4 B, ΔI increased with increasing of Exo-I amount and reached the maximum at 5 U, then decreased when the amount was further increased. This may due to that the limit of active surface area on the fabricated electrode led to the inefficiency of redundant Exo-I. So, 5 U of Exo-I was used for the subsequent experiments. As shown in Fig.4 C, the ΔI increased quickly with increasing the incubation time in the first 30 min, then changed slightly when the incubation time was more than 30 min. Therefore, 30 min was used as the optimal Exo-I incubation time.
Analytical performance of the designed aptasensor
Fig.5 showed the calibration plot of the fabricated aptasensor for FB1 detection. With the concentration range of 1×10-3~1000 ng·mL-1, a linear relationship between ∆I and Lg [CFB1] was observed, and the linear regression equation was ∆I=0.71036 Lg[CFB1]+3.18714 (R2=0.998). The limit of detection (LOD) was calculated to be 0.15 pg·mL-1 at a signal-to-ratio of 3. Compared to the previous reports, the designed aptasensor obtained a wider linear range and lower LOD, and the result was shown in Table 1.
Specificity, reproducibility, repeatability and stability
The specificity of the aptasensor to ochratoxin A (OTA), zearalenone (ZEA) and aflatoxin B1 (AFB1) was studied, and the results were shown in Fig.6. Only when the prepared aptasensor was incubated in FB1, the peak current decreased significantly, indicating that the designed aptasensor had good specificity and could meet the experimental requirements.
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℃. 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
The accuracy of the fabricated aptasensor was evaluated by studying the recovery of FB1 in beer samples and corn samples, and the results were shown in Table 2. Beer samples were filtrated through a 0.45 µm membrane, and used for subsequent tests by spiking different concentrations of FB1. Non-contaminated corn samples were finely milled to obtain corn powder, and 0.5 g of the corn powder was extracted with methanol-water (60:40, v/v. 5 mL) using an orbital shaker for 30 min. After centrifugation for 15 min, the extract was used for analysis by spiking different concentrations of FB1. By addition of 100 ng·mL−1, 1 ng·mL−1 and 1×10-2 ng·mL−1 of FB1, for the beer samples, the average recoveries were 88.5%, 96.1% and 98.6% respectively. For the corn samples, the average recoveries were 91.4%, 87.3% and 106.8% respectively. These results indicated that the fabricated aptasensor can be applied in FB1 detection of the food samples.