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.
Table 1 Comparison with other reported methods for FB1 detection
Method
|
Amplification strategy
|
Linearity
(ng·mL−1)
|
LOD
(ng·mL−1)
|
Ref.
|
Chemiluminescence &enzyme-linked immunosorbent
|
ECL-ELISA based on anti-FB1 IgG and HRP
|
0.14~0.9
|
0.09
|
[17]
|
Chemiluminescence
|
Charge-coupled device
|
2.5~500
|
2.5
|
[18]
|
Fluorescence
|
Anti apt/Apt-NH2/TiO2-PSi
|
0.001~10
|
0.21×10-3
|
[5]
|
Fluorescence resonance energy transfer
|
AuNPs-MB-UCNPs
|
0.01~100
|
0.01
|
[19]
|
Electrochemiluminescence
|
MIP/Ru@SiO2/CS/AuNPs/GCE
|
1×10-3~100
|
0.35×10-3
|
[6]
|
Electrochemical immunosensor
|
AP-anti-antibody/anti-FB1/FB1-BSA-SWNTs/CS/GCE
|
0.01~1000
|
2×10-3
|
[20]
|
Electrochemical immunosensor
|
Ab-AuNPs-PPy/ErGO-SPE
|
200~4500
|
4.2
|
[21]
|
Electrochemical magneto immunosensor
|
FB1-HRP/Ab-FB1/MB&protein G/CSPE
|
0.73~11.2
|
0.33
|
[22]
|
Electrochemical aptasensor
|
Apt-AuNPs-SPCE
|
1×10-2~50
|
3.4×10-3
|
[7]
|
Electrochemical aptasensor
|
GS-TH/S2/S1/Au/GCE
|
1×10-3~1000
|
1×10-3
|
[23]
|
Electrochemical aptasensor
|
Exo-I/Apt/cDNA/AuE
|
1×10-3~1000
|
0.15×10-3
|
This work
|
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. With the FB1 concentration of 1 μg·mL−1, the relative standard deviation (RSD) was 5.72% for five different electrodes, and 5.38 for one electrode with five repetitive measurements, confirming the satisfactory reproducibility and repeatability of the designed aptasensor. The fabricated aptasensor was stored at 4℃. After a 35-day storage period, 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 the results were shown in Table 2. By addition of 100 ng·mL−1, 1 ng·mL−1 and 1×10-2 ng·mL−1 of FB1, the average recoveries were 88.5%, 96.1% and 98.6% respectively, indicating that the fabricated aptasensor can be applied in FB1 detection of the food samples.
Table 2 Recovery of FB1 in beer samples
Sample
|
Added (ng·mL−1)
|
Found (ng·mL−1)
|
Average Recovery
(%)
|
RSD (%)
n=3
|
1
|
100
|
88.5
|
88.5
|
1.75
|
86.8
|
90.3
|
2
|
1
|
0.94
|
96.1
|
2.25
|
0.98
|
0.96
|
3
|
1×10-2
|
0.94×10-2
|
98.6
|
5.65
|
1.05×10-2
|
0.96×10-2
|