# Aptamer-Based Biosensors for the Analytical Determination of Bisphenol A in Foodstuffs
## 4 Detection Techniques Applied in the Analysis of Real Food Samples
### Electrochemical Aptasensors
A very low LOD level resulted in BPA detection by both aptasensors; however, the first case impliesa reduced number of steps during the analyses, which may correlate with cost-effective aptasensor production and limiting data variability in repetitive BPA measurements. [PERSON] et al. [58] used a covalently functionalized acrflavine (ACF) redox probe starting from a GCE that was firstly coated with carbon nanotubes-COOH (CNTs-COOH) and then decorated with golden-platinum nanoparticles (Au-PtNPs) and finally derivatized with ACF. A phosphoramidate bond between ACF-NH\({}_{2}\) and the phosphate group at the 5\({}^{\prime}\)-end of the aptamer led to the final electrode functionalization. The authors demonstrated that each sequential modification of the electrode, GCE\(\rightarrow\)GC/CNTs-COOH\(\rightarrow\)GCE/CNTs/Au-PtNPs\(\rightarrow\)GC/CNTs/Au-PtNPs/ACF, gradually and sensitively increased the cyclic voltammetry (CV) signal up to the coating of the aptamer on the GC/CNTs/Au-PtNPs/ACF electrode, which, conversely, negatively affected the signal. The aptamer-BPA complex formation triggered further decreasing of the CV signal. Despite this strategy implying the use of chemical reactions to conjugate the redox probe to the aptamer, the use of an amide bond can guarantee good stability of the resulting functionalized WE in repetitive detections. [PERSON] et al. [70] used the methylene blue dye as an aptamer intercalating redox probe to avoid aptamer chemical conjugation, a time-consuming and cost-increasing procedure. Indeed, the working electrode was built on a PET film coated with Au and wrapped with carbon tape to achieve an adhesive surface that captured multiwalled CNs in non-covalent way. The obtained electrode surface was then coated with Au and functionalized with the selected aptamer via a disulfide bond. The redox-active dye methylene blue was then intercalated into the aptamer, and the final working electrode was ready to be used. In the absence of BPA, the intercalated dye resulted in a strong electrochemical signal. Once BPA had bonded to its aptamer, the intercalated dye was released, and the electrochemical signal decreased. Both the use of multi-walled CNs and an aptamer-dye complex contributed to a significant signal amplification, allowing for very sensitive BPA detection. However, the use of methylene blue as the intercalating reagent instead of the conjugated reagent, even though simply the chemical functionalization of the WE, also introduces new steps during the analysis, namely the incubation time with the dye to obtain the intercalated methylene blue/aptamer complex, and the subsequent washing of WE to remove the excess of dye molecules, which may contribute to increasing the variability of the results of repetitive measurements. [PERSON] et al. [55] and [PERSON] et al. [64] used enzymes to detect or to amplify, respectively, BPA detection. In the first case, the authors used the system horseradish peroxidase (HRP)/hydroquinone (HQ)/H\({}_{2}\)O\({}_{2}\) to generate the electrochemical signal associated with the formation of the aptamer-BPA complex on the WE. In particular, they functionalized a GCE with gold nanoparticles (AuNPs) carrying a thiolated BPA aptamer. A biotin-modified complementary DNA probe (cDNA) was used to form double-stranded DNA (dsDNA) on a GC/AuNPs/Apt electrode. A subsequent interaction between the biotin and the avidin on streptavidin-modified horseradish peroxidase gold nanoparticles (avidin-HRP-AuNPs) generated a signal of hydroquinone (HQ) oxidation in the presence of H\({}_{2}\)O\({}_{2}\). The oxidized HQ turned back into HQ itself, exchanging electrons with the electrode and causing the amplification of the reduction current. Upon the highly specific interaction between the BPA and its aptamer, cDNA was released from the electrode surface, and BPA was immobilized on the sensing interface. The captured amounts of avidin-HRP-AUNP diminished with the increase in the BPA concentration and produced a series of decreasing catalytic peak currents (Figure 6).