br Afterwards when the probe DNA and the
Afterwards, when the probe DNA and the aptamer were covalently
In the EIS technique, the linear part at low frequencies associates
bound onto the Bio AuNP/CD/GCE surface, the electron-transfer re-
with the diﬀusion-limited process, and the semicircle portion at high
sistance enhanced. This demonstrated that the probe DNA and the ap-
frequencies relates to the FF-MAS transfer-limited process. Fig. 6 de-
tamer sequence had been immobilized on the surface of the Bio AuNP/
monstrates the Nyquist plots of EIS associated with the construction of
CD/GCE. Furthermore, due to the hybridization of the probe DNA, the target DNA and the aptamer sequence with the target antigen, the electron-transfer resistance increased the most. As it can be seen in Fig. 6, the EIS analyses also confirmed the CV and DPV results .
Through electrochemical methods, chronoamperometry made it possible to register quick and reproducible signals. Indeed, the registry was possible after the addition of the CD, Bio AuNP, aptamer sequence, antigen, probe DNA and target oligonucleotide. According to the data, the chronoamperometry results confirm the CV, DPV and EIS graphs (Fig. S6).
The incubation time of the DNA probe and the aptamer sequence was experimentally optimized. According to the results, 1 h and 2 h were the optimum values found for the DNA probe and the aptamer sequence respectively (Fig. S7). Generally, they are considered as short durations. Therefore, 2 h and 3 h were chosen as optimum hybridiza-tion times for the target DNA and the target antigen respectively (Fig. S8), which proved to be really good durations.
3.4. Repeatability and stability of the biosensor
To check the repeatability of the biosensor, three parallel im-mobilizations of the probe DNA and the aptamer were done on the modified GCE. Then, hybridization was carried out with the target DNA and the CEA antigen. Three DNA biosensors, which were made in-dividually, showed a totally acceptable relative standard deviation of 0.014 and 0.012 for the DNA sensor and the aptasensor respectively (Fig. S9).
Generally, the stability of DNA sensors and aptasensors plays an important role in gaining an appropriate degree of sensitivity. To test the stability of the aptasensor and the DNA sensor in this study, cyclic voltammetry was repeated for three times, but no considerable change was observed in the Ipeak values. Besides, when the modified electrode was stored at 4 °C for about a week, the results remained almost un-changed.
3.5. Evaluation of the selectivity of the DNA biosensor
To evaluate the selectivity of the aptasensor and the DNA sensor, an APTA/Bio AuNP/CD/GCE and an ssDNA/Bio AuNP/CD/GCE were used to appraise the hybridization process. In this regard, it has been found that there was a decrement in the current signal. This means that hy-bridization took place between the ssDNA and the complementary DNA as well as between the aptamer and the target antigen [22,23].
In our study, however, there was no obvious decrease in the peak current after hybridization with the mismatch target (bovine serum albumin) and the non-complementary DNA sequence. This demon-strates that there occurs no hybridization and that the slight reduction of the peak current might be due to the non-specific adsorption (Fig. S10).
3.6. Sensitivity of the electrochemical DNA biosensor
Biosensor sensitivity was tested by applying the modified probe DNA electrode and an aptamer sequence hybridized with various con-centrations of the complementary DNA strand and the target antigen respectively. As it can be seen in Fig. 7, the DPV peak current of ca-techol on the mentioned biosensors decreased, along with the en-hancement in the concentration of either the complementary DNA strand or the target antigen in each biosensor.