Electrochemically stimulating developments in bioelectronic medicine

Cellular homeostasis is in part controlled by biological generated electrical activity. By interfacing biology with electronic devices this electrical activity can be modulated to actuate cellular behaviour. There are current limitations in merging electronics with biology sufficiently well to target and sense specific electrically active components of cells. By addressing this limitation, researchers give rise to new capabilities for facilitating the twoway transduction signalling mechanisms between the electronic and cellular components. This is required to allow significant advancement of bioelectronic technology which offers new ways of treating and diagnosing diseases. Most of the progress that has been achieved to date in developing bioelectronic therapeutics stimulate neural communication, which ultimately orchestrates organ function back to a healthy state. Some devices used in therapeutics include cochlear and retinal implants and vagus nerve stimulators. However, all cells can be effected by electrical inputs which gives rise to the opportunity to broaden the use of bioelectronic medicine for treating disease. Electronic actuation of non-excitable cells has been shown to lead to ‘programmed’ cell behaviour via application of electronic input which alter key biological processes. A neglected form of cellular electrical communication which has not yet been considered when developing bioelectronics therapeutics is faradaic currents. These are generated during redox reactions. A precedent of electrochemical technology being used to modulate these reactions thereby controlling cell behaviour has already been set. In this mini review we highlight the current state of the art of electronic routes to modulating cell behaviour and identify new ways in which electrochemistry could be used to contribute to the new field of bioelectronic medicine

An electrochemical system for the study of trans-plasma membrane electron transport in whole eukaryotic cells

The development of new assays to study trans-plasma membrane electron transport (tPMET) in eukaryotic systems is paramount for a number reasons, which include the further understanding of the underlying biology which can then potentially be applied to innovate technological advancements in biosensing, microbial fuel, and pharmaceutical fields. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes, or the detection of ferrocyanide using colorimetric methods. Developing a method to simultaneously analyze the redox state of a reporter molecule would give advantages in probing the underlying biology. Herein we present an electrochemical based method that allows for the quantification of both ferricyanide and ferrocyanide redox states to a highly sensitive degree. We have applied this system to a novel application of assessing oncogenic cell-driven iron reduction, and have shown that it can effectively quantitate and identify differences in iron reduction capability of three lung epithelial cell lines. Importantly, the development of the technology has led to new biological hypothesis which now need addressing

Switching specific biomolecular interactions on surfaces under complex biological conditions

Herein, electrically switchable mixed self-assembled monolayers based on oligopeptides have been developed and investigated for their suitability in achieving control over biomolecular interactions in the presence of complex biological conditions. Our model system, a biotinylated oligopeptide tethered to gold within a background of tri(ethylene glycol) undecanethiol, is ubiquitous in both switching specific protein interactions in highly fouling media while still offering the non-specific protein-resistance to the surface. Furthermore, the work demonstrated that the performance of the switching on the electro-switchable oligopeptide is sensitive to the characteristics of the media, and in particular, its protein concentration and buffer composition, and thus such aspects should be considered and addressed to assure maximum switching performance. This study lays the foundation for developing more realistic dynamic extracellular matrix models and is certainly applicable in a wide variety of biological and medical applications

Electrochemical analysis of gold nanoparticles multifunctionalised withCytochrome c and a zinc Porphyrin

Cytochrome c (Cyt c), known for its functional redox capabilities, plays a pivotal role in biologicalprocesses such as the electron transport chain and apoptosis. However, understanding how differentconjugation strategies impact its structural and redox characteristics is limited. To fill this gap, weinvestigated the effects of conjugating Cyt c and a zinc(II) porphyrin (Zn Porph) to gold nanoparticles(AuNPs). We used circular dichroism (CD) spectroscopy to detect structural conformational changesin Cyt c upon conjugation and time-of-flight secondary ion mass spectrometry (TOF-SIMS) toidentify protein orientation. Cyt c was predicted to have different orientations depending on the sizeof AuNPs and methods used to conjugate the protein, it was hypothesised that the orientation of Cythttps://doi.org/10.26434/chemrxiv-2023-rsrwv ORCID: https://orcid.org/0000-0002-4872-8928 Content not peer-reviewed by ChemRxiv. License: CC BY 4.02c may influence the redox properties of the protein. The electrochemical properties of Cyt c wereassessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We used DPVbasedto determine the heterogeneous rate constant (k0). The results show a lower k0 for conjugatedCyt c than free Cyt c, likely due to structural changes in the protein. The spatial orientation of Cyt chad minimal influence on k0, while ligand density and AuNP size had an effect. The k0 value of ZnPorph did not decrease on conjugation. Despite these changes, Cyt c and Zn Porph maintained theirelectrochemical capabilities after conjugation.</p

An electrochemical system for the study of trans-plasma membrane electron transport in whole eukaryotic cells

The development of new assays to study trans-plasma membrane electron transport (tPMET) in eukaryotic systems is paramount for a number reasons, which include the further understanding of the underlying biology which can then potentially be applied to innovate technological advancements in biosensing, microbial fuel, and pharmaceutical fields. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes, or the detection of ferrocyanide using colorimetric methods. Developing a method to simultaneously analyze the redox state of a reporter molecule would give advantages in probing the underlying biology. Herein we present an electrochemical based method that allows for the quantification of both ferricyanide and ferrocyanide redox states to a highly sensitive degree. We have applied this system to a novel application of assessing oncogenic cell-driven iron reduction, and have shown that it can effectively quantitate and identify differences in iron reduction capability of three lung epithelial cell lines. Importantly, the development of the technology has led to new biological hypothesis which now need addressing

Reversible, High-Affinity Surface Capturing of Proteins Directed by Supramolecular Assembly

The ability to design surfaces with reversible, high-affinity protein binding sites represents a significant step forward in the advancement of analytical methods for diverse biochemical and biomedical applications. Herein, we report a dynamic supramolecular strategy to directly assemble proteins on surfaces based on multivalent host–guest interactions. The host–guest interactions are achieved by one-step nanofabrication of a well-oriented β-cyclodextrin host-derived self-assembled monolayer on gold (β-CD-SAM) that forms specific inclusion complexes with hydrophobic amino acids located on the surface of the protein. Cytochrome c, insulin, α-chymotrypsin, and RNase A are used as model guest proteins. Surface plasmon resonance and static time-of-flight secondary ion mass spectrometry studies demonstrate that all four proteins interact with the β-CD-SAM in a specific manner via the hydrophobic amino acids on the surface of the protein. The β-CD-SAMs bind the proteins with high nanomolar to single-digit micromolar dissociation constants (KD). Importantly, while the proteins can be captured with high affinity, their release from the surface can be achieved under very mild conditions. Our results expose the great advantages of using a supramolecular approach for controlling protein immobilization, in which the strategy described herein provides unprecedented opportunities to create advanced bioanalytic and biosensor technologies

Real-time electrocatalytic sensing of cellular respiration

In the present work we develop a real-time electrochemical mediator assay to enable the assessment of cell numbers and chemical toxicity. This allowed us to monitor metabolism down to a single cell in a low cost easy to use rapid assay which is not possible with current technology. The developed assay was based on the determination of oxygen. This was made possible via the use of electrochemical mediator ferrocene carboxylic acid (FcA). The FcA showed distinctive catalytic properties in interacting with reactive oxygen species generated from oxygen when compared to ferrocene methanol (FcMeOH). A deeper insight into the chemistry controlling this behaviour is provided. The behaviour is then taken advantage of to develop a cellular aerobic respiration assay. We describe the properties of the FcA system to detect, in real-time, the oxygen consumption of Escherichia coli DH5-α (E. coli). We demonstrated that the FcA-based oxygen assay is highly sensitive, and using a population of cells, oxygen consumption rates could be calculated down to a single cell level. More importantly, the results can be accomplished in minutes, considerably outperforming current commercially available biooxygen demand assays. The developed assay is expected to have a significant impact in diverse fields and industries, ranging from environmental toxicology through to pharmaceutical and agrochemical industries

Top-Down Characterisation of an Antimicrobial Sanitiser, Leading from Quenchers of Efficacy to Mode of Action

We developed a top-down strategy to characterise an antimicrobial, oxidising sanitiser, which has diverse proposed applications including surface-sanitisation of fresh foods, and with benefits for water resilience. The strategy involved finding quenchers of antimicrobial activity then antimicrobial mode of action, by identifying key chemical reaction partners starting from complex matrices, narrowing down reactivity to specific organic molecules within cells. The sanitiser electrolysed-water (EW) retained partial fungicidal activity against the food-spoilage fungus Aspergillus niger at high levels of added soils (30–750 mg mL-1), commonly associated with harvested produce. Soil with high organic load (approx. 98 mg g-1) gave stronger EW inactivation. Marked inactivation by a complex organics mix (YEPD medium) was linked to its protein-rich components. Addition of pure proteins or amino acids (≤1 mg mL-1) fully suppressed EW activity. Mechanism was interrogated further with the yeast model, corroborating marked suppression of EW action by the amino acid methionine. Pre-culture with methionine increased resistance to EW, sodium hypochlorite, or chlorine-free ozonated water. Overexpression of methionine sulfoxide reductases (which reduce oxidised methionine) protected against EW. Fluoroprobe-based analyses indicated that methionine and cysteine inactivate free chlorine species in EW. Intracellular methionine oxidation can disturb cellular FeS-clusters and we showed that EW treatment impairs FeS-enzyme activity. The study establishes the value of a top-down approach for multi-level characterisation of sanitiser efficacy and action. The results reveal proteins and amino acids as key quenchers of EW activity and, among the amino acids, the importance of methionine oxidation and FeS-cluster damage for antimicrobial mode-of-action

Impedimetric Characterization of Bipolar Nanoelectrodes with Cancer Cells

Merging of electronics with biology, defined as bioelectronics, at the nanoscale holds considerable promise for sensing and modulating cellular behavior. Advancing our understanding of nanobioelectronics will facilitate development and enable applications in biosensing, tissue engineering, and bioelectronic medicine. However, studies investigating the electrical effects when merging wireless conductive nanoelectrodes with biology are lacking. Consequently, a tool is required to develop a greater understanding of merging conductive nanoparticles with cells. Herein, this challenge is addressed by developing an impedimetric method to evaluate bipolar electrode (BPE) systems that could report on electrical input. A theoretical framework is provided, using impedance to determine if conductive nanoparticles can be polarized and used to drive current. It is then demonstrated that 125 nm of gold nanoparticle (AuNP) bipolar electrodes (BPEs) could be sensed in the presence of cells when incorporated intracellularly at 500 μg/mL using water and phosphate-buffered saline (PBS) as electrolytes. These results highlight how nanoscale BPEs act within biological systems. This research will impact the rational design of using BPE systems in cells for both sensing and actuating applications