Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy.

There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds-glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)-and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.

Streamlined Fabrication of Hybrid Lipid Bilayer Membranes on Titanium Oxide Surfaces: A Comparison of One- and Two-Tail SAM Molecules.

There is broad interest in fabricating cell-membrane-mimicking, hybrid lipid bilayer (HLB) coatings on titanium oxide surfaces for medical implant and drug delivery applications. However, existing fabrication strategies are complex, and there is an outstanding need to develop a streamlined method that can be performed quickly at room temperature. Towards this goal, herein, we characterized the room-temperature deposition kinetics and adlayer properties of one- and two-tail phosphonic acid-functionalized molecules on titanium oxide surfaces in various solvent systems and identified optimal conditions to prepare self-assembled monolayers (SAMs), upon which HLBs could be formed in select cases. Among the molecular candidates, we identified a two-tail molecule that formed a rigidly attached SAM to enable HLB fabrication via vesicle fusion for membrane-based biosensing applications. By contrast, vesicles adsorbed but did not rupture on SAMs composed of one-tail molecules. Our findings support that two-tail phosphonic acid SAMs offer superior capabilities for rapid HLB coating fabrication at room temperature, and these streamlined capabilities could be useful to prepare durable lipid bilayer coatings on titanium-based materials.

Electricity auto-generating skin patch promotes wound healing process by activation of mechanosensitive ion channels.

Electricity constitutes a natural biophysical component that preserves tissue homeostasis and modulates many biological processes, including the repair of damaged tissues. Wound healing involves intricate cellular events, such as inflammation, angiogenesis, matrix synthesis, and epithelialization whereby multiple cell types sense the environmental cues to rebuild the structure and functions. Here, we report that electricity auto-generating glucose-responsive enzymatic-biofuel-cell (EBC) skin patch stimulates the wound healing process. Rat wounded-skin model and in vitro cell cultures showed that EBC accelerated wound healing by modulating inflammation while stimulating angiogenesis, fibroblast fuctionality and matrix synthesis. Of note, EBC-activated cellular bahaviors were linked to the signalings involved with calcium influx, which predominantly dependent on the mechanosensitive ion channels, primarily Piezo1. Inhibition of Piezo1-receptor impaired the EBC-induced key functions of both fibroblasts and endothelial cells in the wound healing. This study highlights the significant roles of electricity played in wound healing through activated mechanosensitive ion channels and the calcium influx, and suggests the possibility of the electricity auto-generating EBC-based skin patch for use as a wound healing device.

Development of a Glucose Sensor Based on Glucose Dehydrogenase Using Polydopamine-Functionalized Nanotubes.

The electrochemical-based detection of glucose is widely used for diagnostic purposes and is mediated by enzyme-mediated signal transduction mechanisms. For such applications, recent attention has focused on utilizing the oxygen-insensitive glucose dehydrogenase (GDH) enzyme in place of the glucose oxidase (GOx) enzyme, which is sensitive to oxygen levels. Currently used Ru-based redox mediators mainly work with GOx, while Ru(dmo-bpy)2Cl2 has been proposed as a promising mediator that works with GDH. However, there remains an outstanding need to improve Ru(dmo-bpy)2Cl2 attachment to electrode surfaces. Herein, we report the use of polydopamine-functionalized multi-walled carbon nanotubes (PDA-MWCNTs) to effectively attach Ru(dmo-bpy)2Cl2 and GDH onto screen-printed carbon electrodes (SPCEs) without requiring a cross-linker. PDA-MWCNTs were characterized by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, and thermal gravimetric analysis (TGA), while the fabrication and optimization of Ru(dmo-bpy)2Cl2/PDA-MWCNT/SPCEs were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The experimental results demonstrate a wide linear range of glucose-concentration-dependent responses and the multi-potential step (MPS) technique facilitated the selective detection of glucose in the presence of physiologically relevant interfering species, as well as in biological fluids (e.g., serum). The ease of device fabrication and high detection performance demonstrate a viable pathway to develop glucose sensors based on the GDH enzyme and Ru(dmo-bpy)2Cl2 redox mediator and the sensing strategy is potentially extendable to other bioanalytes as well.

Pentacyanoammineferrate-Based Non-Enzymatic Electrochemical Biosensing Platform for Selective Uric Acid Measurement.

The electrochemical-based detection of uric acid (UA) is widely used for diagnostic purposes. However, various interfering species such as ascorbic acid, dopamine, and glucose can affect electrochemical signals, and hence there is an outstanding need to develop improved sensing platforms to detect UA with high selectivity. Herein, we report a pentagonal mediator-based non-enzymatic electrochemical biosensing platform to selectively measure UA in the presence of interfering species. The working electrode was fabricated by electrodepositing polymerized 1-vinylimidazole (PVI), which has an imidazole ligand, onto indium tin oxide (ITO), and then conjugating nickel ions to the PVI-coated ITO electrode. Electrode performance was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements and integrated together with pentacyanoammineferrate, which can bind to the amine groups of UA and function as an electron transferring mediator. The experimental results showed a wide linear range of UA concentration-dependent responses and the multi-potential step (MPS) technique facilitated selective detection of UA in the presence of physiologically relevant interfering species. Altogether, these findings support that pentacyanoammineferrate-based non-enzymatic electrodes are suitable biosensing platforms for the selective measurement of UA, and such approaches could potentially be extended to other bioanalytes as well.

Stopping Membrane-Enveloped Viruses with Nanotechnology Strategies: Toward Antiviral Drug Development and Pandemic Preparedness.

Membrane-enveloped viruses are a leading cause of viral epidemics, and there is an outstanding need to develop broad-spectrum antiviral strategies to treat and prevent enveloped virus infections. In this review, we critically discuss why the lipid membrane surrounding enveloped virus particles is a promising antiviral target and cover the latest progress in nanotechnology research to design and evaluate membrane-targeting virus inhibition strategies. These efforts span diverse topics such as nanomaterials, self-assembly, biosensors, nanomedicine, drug delivery, and medical devices and have excellent potential to support the development of next-generation antiviral drug candidates and technologies. Application examples in the areas of human medicine and agricultural biosecurity are also presented. Looking forward, research in this direction is poised to strengthen capabilities for virus pandemic preparedness and demonstrates how nanotechnology strategies can help to solve global health challenges related to infectious diseases.

Medicinal Activities and Nanomedicine Delivery Strategies for Brucea javanica Oil and Its Molecular Components.

Brucea javanica oil (BJO) is widely used in traditional Chinese medicine to treat various types of cancer and inflammatory diseases. There is significant interest in understanding the medicinal activities of BJO and its molecular components, especially quassinoids, and in exploring how they can be incorporated into nanomedicine delivery strategies for improved application prospects. Herein, we cover the latest progress in developing different classes of drug delivery vehicles, including nanoemulsions, liposomes, nanostructured lipid carriers, and spongosomes, to encapsulate BJO and purified quassinoids. An introduction to the composition and medicinal activities of BJO and its molecular components, including quassinoids and fatty acids, is first provided. Application examples involving each type of drug delivery vehicle are then critically presented. Future opportunities for nanomedicine delivery strategies in the field are also discussed and considered within the context of translational medicine needs and drug development processes.

Motility enhancement of human spermatozoa using electrical stimulation in the nano-Ampere range with enzymatic biofuel cells.

Sperm motility is a crucial factor for normal fertilisation that is partly supported by mitochondrial activity. Enzymatic biofuel cells (EBFCs) generate electric currents by an electron grade from anodic to cathodic electrodes in a culture media. We demonstrate that electrical stimulation by EBFC at the nano-Ampere range enhances sperm motility that can potentially allow the development of a new therapeutic tool for male infertility, including poor motility. EBFC was set up with three different electrical currents (112 nA/cm2 and 250 nA/cm2) at two different times (1 h, 2 h). Each sample was evaluated for its motility by computer-assisted sperm analyses and sperm viability testing. In the expanded study, we used the optimal electrical current of the EBFC system to treat asthenozoospermia and sperm with 0% motility. Results showed that optimal electrical stimulation schemes with EBFCs enhanced sperm motility by 30-40% compared with controls. Activated spermatozoa led to tyrosine phosphorylation in the tail area of the sperm following the electrical stimulation in the nano-Ampere range. However, the electrically stimulated group did not exhibit increased acrosomal reaction rates compared with the control group. In cases related to asthenozoospermia, 40% of motility was recovered following the electrical stimulation at the nano-Ampere range. However, motility is not recovered in sperm with 0% motility. In conclusion, we found that sperm motility was enhanced by exposure to electrical currents in the nano-Ampere range induced by optimal EBFCs. Electrical stimulation enhanced the motility of the sperm though tyrosine phosphorylation in spermatozoa. Therefore, our results show that electrical currents in the nano-Ampere range can be potentially applied to male infertility therapy as enhancers of sperm motility in assisted reproductive technology.

Biologically interfaced nanoplasmonic sensors.

Understanding biointerfacial processes is crucial in various fields across fundamental and applied biology, but performing quantitative studies via conventional characterization techniques remains challenging due to instrumentation as well as analytical complexities and limitations. In order to accelerate translational research and address current challenges in healthcare and medicine, there is an outstanding need to develop surface-sensitive technologies with advanced measurement capabilities. Along this line, nanoplasmonic sensing has emerged as a powerful tool to quantitatively study biointerfacial processes owing to its high spatial resolution at the nanoscale. Consequently, the development of robust biological interfacing strategies becomes imperative to maximize its characterization potential. This review will highlight and discuss the critical role of biological interfacing within the context of constructing nanoplasmonic sensing platforms for biointerfacial science applications. Apart from paving the way for the development of highly surface-sensitive characterization tools that will spur fundamental biological interaction studies and improve the overall understanding of biological processes, the basic principles behind biointerfacing strategies presented in this review are also applicable to other fields that involve an interface between an inorganic material and a biological system.

Author Correction: Performance of a glucose-reactive enzyme-based biofuel cell system for biomedical applications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Performance of a glucose-reactive enzyme-based biofuel cell system for biomedical applications.

A glucose-reactive enzyme-based biofuel cell system (EBFC) was recently introduced in the scientific community for biomedical applications, such as implantable artificial organs and biosensors for drug delivery. Upon direct contact with tissues or organs, an implanted EBFC can exert effects that damage or stimulate intact tissue due to its byproducts or generated electrical cues, which have not been investigated in detail. Here, we perform a fundamental cell culture study using a glucose dehydrogenase (GDH) as an anode enzyme and bilirubin oxidase (BOD) as a cathode enzyme. The fabricated EBFC had power densities of 15.26 to 38.33 nW/cm2 depending on the enzyme concentration in media supplemented with 25 mM glucose. Despite the low power density, the GDH-based EBFC showed increases in cell viability (~150%) and cell migration (~90%) with a relatively low inflammatory response. However, glucose oxidase (GOD), which has been used as an EBFC anode enzyme, revealed extreme cytotoxicity (~10%) due to the lethal concentration of H2O2 byproducts (~1500 µM). Therefore, with its cytocompatibility and cell-stimulating effects, the GDH-based EBFC is considered a promising implantable tool for generating electricity for biomedical applications. Finally, the GDH-based EBFC can be used for introducing electricity during cell culture and the fabrication of organs on a chip and a power source for implantable devices such as biosensors, biopatches, and artificial organs.

Ultrasonic synthesis and characterization of poly(acrylamide)-co-poly(vinylimidazole)@MWCNTs composite for use as an electrochemical material.

Applying a nanocomposite to increase the conductivity of an electrode can facilitate electrochemical analysis. In this regard, multi-walled carbon nanotubes (MWCNTs) evenly dispersed in hydrophilic solution can play an important role in electrochemical bio-sensing due to their unique properties, such as their high electrical conductivity and ability to conjugate with hydrophilic enzymes. Herein, we report the simple ultrasonic synthesis of a highly dispersible, enzyme-binding nanocomposite, poly(acrylamide)-co-poly(vinyl imidazole) (7:1 mol ratio)-MWCNTs (PAA-PVI@MWCNTs). This material, having a zeta potential of 36.6 ±â€¯0.53 mV, was applied as a film to an electrode surface and stably bound with glucose oxidase to transfer an electron between the enzyme and electrode in the presence of glucose. The PAA-PVI@MWCNTs composite, which was readily dispersed in deionized water, can be used as a biocompatible material for applications such as bio-sensing, point-of-care testing (POCT), and other health care functions.

A Simple Interfacial Platform for Homogeneous Electrochemical Immunoassays Using a Poly(Vinylimidazole)-Modified Electrode.

In this study, a homogeneous method featuring simple, one-step detection was developed to analyze hippuric acid (HA), a major metabolite of toluene. High sensitivity was achieved with the facile immobilization of poly(vinylimidazole) (PVI) on an indium tin oxide (ITO) electrode. Using a previously developed approach, pentacyanoferrate was coordinated with pyridyl-N ligands, and the redox-active Fe(II/III) centers were bound to Ni(II) ions on the electrode via electrostatic cyanide bridges. The detection was accomplished by the competitive binding of free HA and pentacyanoferrate-(4-aminomethylpyridine-hippuric acid) (Fe-HA, the electron transfer mediator) to the HA antibody on the Ni(II) ions-modified PVI-ITO (Ni-PVI-ITO) electrode. The electrical and physicochemical characterization of the electrode was carried out by cyclic voltammetry, differential pulse voltammetry, field emission scanning electron microscopy, and X-ray photoelectron spectroscopy. At low mediator concentrations, the electrical signals were proportional to the HA concentration between 0.1 µg/mL and 1.0 mg/mL. The same method may be extended to other small organic molecules.

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Disposable Non-Enzymatic Glucose Sensors Using Screen-Printed Nickel/Carbon Composites on Indium Tin Oxide Electrodes.

Disposable screen-printed nickel/carbon composites on indium tin oxide (ITO) electrodes (DSPNCE) were developed for the detection of glucose without enzymes. The DSPNCE were prepared by screen-printing the ITO substrate with a 50 wt% nickel/carbon composite, followed by curing at 400 °C for 30 min. The redox couple of Ni(OH)2/NiOOH was deposited on the surface of the electrodes via cyclic voltammetry (CV), scanning from 0-1.5 V for 30 cycles in 0.1 M NaOH solution. The DSPNCE were characterized by field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and electrochemical methods. The resulting electrical currents, measured by CV and chronoamperometry at 0.65 V vs. Ag/AgCl, showed a good linear response with glucose concentrations from 1.0-10 mM. Also, the prepared electrodes showed no interference from common physiologic interferents such as uric acid (UA) or ascorbic acid (AA). Therefore, this approach allowed the development of a simple, disposable glucose biosensor.

Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells.

Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration.

Homogeneous electrochemical detection of hippuric acid in urine based on the osmium-antigen conjugate.

A homogeneous electrochemical immunoassay is based on the interaction of osmium-antigen conjugate with its antibody. The novelty presented herein is the direct conjugation of the osmium complex to a small antigen and the application of the quantitative analysis of the antigen and its antibody as the electrical signal for homogeneous immunoassay. The small antigen chosen is hippuric acid (HA), a major urinary metabolite in toluene-exposed humans. As a redox mediator, [Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine-HA)Cl](+/2+) (Os-HA antigen) has been synthesized and characterized on screen-printed carbon electrodes. The synthesized Os-HA antigen shows reversible redox peaks at E(½)=0.056 V versus Ag/AgCl. The homogeneous competitive immunoassay relies on the interaction between Os-HA antigen conjugate and free antigen to its antibody, which can generate electrical signals linearly proportional to the free antigen monitored by cyclic voltammetry and differential pulse voltammetry in the range of 10 µg mL(-1) to 5.12 mg mL(-1). The cutoff concentration of HA in urine samples is 2.0 mg mL(-1), so the method can be used to develop a HA immunosensor. Moreover, the proposed homogeneous electrochemical immunoassay method can be applied to detect low concentrations of small antigens found in the healthcare area.