The development of reliable biosensing platforms plays a key role in the detection of proteins in clinically and environmentally derived samples for diagnostics, as well as for process monitoring in biotechnological productions. For this purpose, the biosensor has to be stable and reproducible, and highly sensitive to detect potentially extremely low concentrations and prevent the nonspecific binding of interfering compounds. In this review, we present an overview of recently published (2017–2019) immobilization techniques for aptamers on gold electrodes for the electrochemical detection of proteins. These include the direct immobilization of thiolated aptamers and the utilization of short linkers, streptavidin/biotin interaction, as well as DNA nanostructures and reduced graphene oxide as immobilization platforms. Applied strategies for signal amplification and the prevention of biofouling are additionally discussed, as they play a crucial role in the design of biosensors. While a wide variety of amplification strategies are already available, future investigations should aim to establish suitable antifouling strategies that are compatible with electrochemical measurements. The focus of our review lies on the detailed discussion of the underlying principles and the presentation of utilized chemical protocols in order to provide the reader with promising ideas and profound knowledge of the subject, as well as an update on recent discoveries and achievements.

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 10¹³ aptamer molecules per cm². As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL⁻¹ and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Prion diseases are caused by the aggregation of the native α-helical prion protein PrP^C into its pathological β-sheet-rich isoform PrP^Sc. In current models of PrP^Sc, helix1 is assumed to be preferentially converted into β-sheet during aggregation of PrP^C. This was supported by the NMR structure of PrP^C since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP_23-159 including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP_23-144 corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP_23-159 aggregated significantly faster compared with the truncated fragment humPrP_23-144, clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP_23-159, suggesting that helix1 is not converted to β-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in β-sheet content of humPrP_23-159 fibrils compared with humPrP_23-144. In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into β-sheet during aggregation of PrP^C to PrP^Sc.