Dieter Frense

Biofilms are ubiquitous at interfaces of natural and technical origin. Depending on type and application, biofilm formation is desired or has to be prevented. Therefore, reliable detection of initial biofilm growth is essential in many areas. One method of biofilm monitoring is the electrochemical impedance spectroscopy. Among other factors, this method is heavily dependent on the electrode geometry. In order to achieve a high measurement sensitivity, the electrode size must be chosen according to the biofilm that is to be measured. This paper presents an approach for simulating and modeling the optimal electrode geometry for a specific biofilm. First, a geometric model of a biofilm with up to 6000 individual bacteria is generated. The simulated impedances are used to calculate which electrode geometry maximizes sensitivity depending on the biofilm height. In the chosen example of an E. coli biofilm in a nutrient solution, the optimum size of an interdigital electrode (bar gap equals width) was 2.5 µm for a biofilm height of up to 2 µm. The used algorithms and models can be simply adapted for other biofilms. In this way, the most sensitive electrode geometry for a specific biofilm measurement can be determined with minimal effort.

Biosensors offer exciting opportunities for various clinical applications and constitute a rapidly growing research branch due to new generations of bioreceptors, transducers, and biomaterials with versatile characteristics, such as conductive polymers. Polythiophene is a prominent example of conducting polymers and convinces with excellent conductivity and stability—and is yet barely used for the construction of biosensors. We want to address the fundamental lack of straightforward fabrication procedures for bioreceptor immobilization platforms based on polythiophene. We investigate the literature-known monomer 3-thiopheneacetic acid and present our newly developed method for electrochemical coupling of the linker p-aminobenzoic acid to deposited polythiophene films. Aminated bioreceptors can subsequently be immobilized via EDC/NHS click chemistry. Films were electropolymerized and modified by chronopotentiometry, characterized by electrochemical impedance spectroscopy, surface-enhanced Raman spectroscopy, as well as energy-dispersive X-ray spectroscopy. Both of the presented methods allow for the fabrication of functionalized polythiophene thin films of high conductivity and good reproducibility while convincing with their ease in synthesis.

Due to their unique properties, polythiophene and other conductive polymers have become the subject of intensive research and are promising substrate materials for innovative and trendsetting applications. To this day, boron trifluoride diethyl etherate (BFEE) is the preferred solvent for the electropolymerization of thiophene, although it does not allow for reproducible film qualities due to its decomposition under ambient conditions. We therefore want to equip the reader with a starter kit for the electropolymerization of high-quality polythiophene films from stable solvents and a simple yet efficient method to remove the deposited films from the electrodes for their reuse. By drying the working solution prior to its utilization, and by adding a Lewis acid catalyst, films that display enhanced electron transfer and a smooth surface topography can be obtained, which can both be beneficial for the analytic performance of a subsequently built biosensor.

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.

An aptamer-based biosensor was developed for the detection of doxorubicin using electrochemical impedance spectroscopy. Doxorubicin and its 14-dehydroxylated version daunorubicin are anthracyclines often used in cancer treatment. Due to their mutagenic and cardiotoxic effects, detection in groundwater is desirable. We developed a biosensor using the daunorubicin-binding aptamer as biological recognition element. The aptamer was successfully co-immobilized with mercaptohexanol on gold and a density of 1.3*10¹³ ± 2.4*10¹² aptamer molecules per cm² was achieved. The binding of doxorubicin to the immobilized aptamer was detected by electrochemical impedance spectroscopy. The principle is based on the inhibition of electron transfer between electrode and ferro-/ferricyanide in solution caused by the binding of doxorubicin to the immobilized aptamer. A linear relationship between the charge transfer resistance (R_ct) and the doxorubicin concentration was obtained over the range of 31 nM to 125 nM doxorubicin, with an apparent binding constant of 64 nM and a detection limit of 28 nM. With the advantages of high sensitivity, selectivity, and simple sensor construction, this method shows a high potential of impedimetric aptasensors in environmental monitoring.

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.