Molecular Surface Quantification of Multifunctionalized Gold Nanoparticles Using UV-Visible Absorption Spectroscopy Deconvolution.

Multifunctional gold nanoparticles (AuNPs) are of great interest, owing to their vast potential for use in many areas including sensing, imaging, delivery, and medicine. A key factor in determining the biological activity of multifunctional AuNPs is the quantification of surface conjugated molecules. There has been a lack of accurate methods to determine this for multifunctionalized AuNPs. We address this limitation by using a new method based on the deconvolution and Levenberg-Marquardt algorithm fitting of UV-visible absorption spectrum to calculate the precise concentration and number of cytochrome C (Cyt C) and zinc porphyrin (Zn Porph) bound to each multifunctional AuNP. Dynamic light scattering (DLS) and zeta potential measurements were used to confirm the functionalization of AuNPs with Cyt C and Zn Porph. Transmission electron microscopy (TEM) was used in conjunction with UV-visible absorption spectroscopy and DLS to identify the AuNP size and confirm that no aggregation had taken place after functionalization. Despite the overlapping absorption bands of Cyt C and Zn Porph, this method was able to reveal a precise concentration and number of Cyt C and Zn Porph molecules attached per AuNP. Furthermore, using this method, we were able to identify unconjugated molecules, suggesting the need for further purification of the sample. This guide provides a simple and effective method to quickly quantify molecules bound to AuNPs, giving users valuable information, especially for applications in drug delivery and biosensors.

Hydroxyapatite-filled osteoinductive and piezoelectric nanofibers for bone tissue engineering.

Osteoporotic-related fractures are among the leading causes of chronic disease morbidity in Europe and in the US. While a significant percentage of fractures can be repaired naturally, in delayed-union and non-union fractures surgical intervention is necessary for proper bone regeneration. Given the current lack of optimized clinical techniques to adequately address this issue, bone tissue engineering (BTE) strategies focusing on the development of scaffolds for temporarily replacing damaged bone and supporting its regeneration process have been gaining interest. The piezoelectric properties of bone, which have an important role in tissue homeostasis and regeneration, have been frequently neglected in the design of BTE scaffolds. Therefore, in this study, we developed novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) nanofibers via electrospinning capable of replicating the tissue's fibrous extracellular matrix (ECM) composition and native piezoelectric properties. The developed PVDF-TrFE/HAp nanofibers had biomimetic collagen fibril-like diameters, as well as enhanced piezoelectric and surface properties, which translated into a better capacity to assist the mineralization process and cell proliferation. The biological cues provided by the HAp nanoparticles enhanced the osteogenic differentiation of seeded human mesenchymal stem/stromal cells (MSCs) as observed by the increased ALP activity, cell-secreted calcium deposition and osteogenic gene expression levels observed for the HAp-containing fibers. Overall, our findings describe the potential of combining PVDF-TrFE and HAp for developing electroactive and osteoinductive nanofibers capable of supporting bone tissue regeneration.

Electric Field Induced Biomimetic Transmembrane Electron Transport Using Carbon Nanotube Porins.

Cells modulate their homeostasis through the control of redox reactions via transmembrane electron transport systems. These are largely mediated via oxidoreductase enzymes. Their use in biology has been linked to a host of systems including reprogramming for energy requirements in cancer. Consequently, the ability to modulate membrane redox systems may give rise to opportunities to modulate underlying biology. The current work aims to develop a wireless bipolar electrochemical approach to form on-demand electron transfer across biological membranes. To achieve this goal, it is shown that by using membrane inserted carbon nanotube porins (CNTPs) that can act as bipolar nanoelectrodes, one can control electron flow with externally applied electric fields across membranes. Before this work, bipolar electrochemistry has been thought to require high applied voltages not compatible with biological systems. It is shown that bipolar electrochemical reaction via gold reduction at the nanotubes can be modulated at low cell-friendly voltages, providing an opportunity to use bipolar electrodes to control electron flux across membranes. The authors provide new mechanistic insight into this newly describe phenomena at the nanoscale. The results presented give rise to a new method using CNTPs to modulate cell behavior via wireless control of membrane electron transfer.

Iron-Catalysed Radical Polymerisation by Living Bacteria.

The ability to harness cellular redox processes for abiotic synthesis might allow the preparation of engineered hybrid living systems. Towards this goal we describe a new bacteria-mediated iron-catalysed reversible deactivation radical polymerisation (RDRP), with a range of metal-chelating agents and monomers that can be used under ambient conditions with a bacterial redox initiation step to generate polymers. Cupriavidus metallidurans, Escherichia coli, and Clostridium sporogenes species were chosen for their redox enzyme systems and evaluated for their ability to induce polymer formation. Parameters including cell and catalyst concentration, initiator species, and monomer type were investigated. Water-soluble synthetic polymers were produced in the presence of the bacteria with full preservation of cell viability. This method provides a means by which bacterial redox systems can be exploited to generate "unnatural" polymers in the presence of "host" cells, thus setting up the possibility of making natural-synthetic hybrid structures and conjugates.

Mammalian-Cell-Driven Polymerisation of Pyrrole.

A model cancer cell line was used to initiate polymerisation of pyrrole to form the conducting material polypyrrole. The polymerisation was shown to occur through the action of cytosolic exudates rather than that of the membrane redox sites that normally control the oxidation state of iron as ferricyanide or ferrocyanide. The data demonstrate for the first time that mammalian cells can be used to initiate synthesis of conducting polymers and suggest a possible route to detection of cell damage and/or transcellular processes through in situ and amplifiable signal generation.

Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport in Whole Eukaryotic Cells.

The study of trans-plasma membrane electron transport (tPMET) in oncogenic systems is paramount to the further understanding of cancer biology. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes in conjunction with cell surface oxygen consumption, or the detection of an electron acceptor using colorimetric methods. However, when using an iron redox based system to probe tPMET, there is yet to be a method that allows for the simultaneous quantification of iron redox states while providing an exceptional level of sensitivity. 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.

Tailoring the Electrochemical Properties of Carbon Nanotube Modified Indium Tin Oxide via in Situ Grafting of Aryl Diazonium.

Our ability to tailor the electronic properties of surfaces by nanomodification is paramount for various applications, including development of sensing, fuel cell, and solar technologies. Moreover, in order to improve the rational design of conducting surfaces, an improved understanding of structure/function relationships of nanomodifications and effect they have on the underlying electronic properties is required. Herein, we report on the tuning and optimization of the electrochemical properties of indium tin oxide (ITO) functionalized with single-walled carbon nanotubes (SWCNTs). This was achieved by controlling in situ grafting of aryl amine diazonium films on the nanoscale which were used to covalently tether SWCNTs. The structure/function relationship of these nanomodifications on the electronic properties of ITO was elucidated via time-of-flight secondary ion mass spectrometry and electrochemical and physical characterization techniques which has led to new mechanistic insights into the in situ grafting of diazonium. We discovered that the connecting bond is a nitro group which is covalently linked to a carbon on the aryl amine. The increased understanding of the surface chemistry gained through these studies enabled us to fabricate surfaces with optimized electron transfer kinetics. The knowledge gained from these studies allows for the rational design and tuning of the electronic properties of ITO-based conducting surfaces important for development of various electronic applications.

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.

Wireless electrical-molecular quantum signalling for cancer cell apoptosis.

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments.

Engineering nanowires in bacteria to elucidate electron transport structural-functional relationships.

Bacterial pilin nanowires are protein complexes, suggested to possess electroactive capabilities forming part of the cells' bioenergetic programming. Their role is thought to be linked to facilitating electron transfer between cells and the external environment to permit metabolism and cell-to-cell communication. There is a significant debate, with varying hypotheses as to the nature of the proteins currently lying between type-IV pilin-based nanowires and polymerised cytochrome-based filaments. Importantly, to date, there is a very limited structure-function analysis of these structures within whole bacteria. In this work, we engineered Cupriavidus necator H16, a model autotrophic organism to express differing aromatic modifications of type-IV pilus proteins to establish structure-function relationships on conductivity and the effects this has on pili structure. This was achieved via a combination of high-resolution PeakForce tunnelling atomic force microscopy (PeakForce TUNA™) technology, alongside conventional electrochemical approaches enabling the elucidation of conductive nanowires emanating from whole bacterial cells. This work is the first example of functional type-IV pili protein nanowires produced under aerobic conditions using a Cupriavidus necator chassis. This work has far-reaching consequences in understanding the basis of bio-electrical communication between cells and with their external environment.

Biocompatible metallosurfactant-based nanocolloid-loaded Rose Bengal with excellent singlet oxygen-induced phototoxicity efficiency against cancer cells.

Photodynamic therapy (PDT) is facing challenges such as poor solubility, precise delivery, self-aggregation, and photobleaching of photosensitizers with cancer cells due to their less tendency to accumulate in tumor tissues. To address these challenges, we have explored a Rose Bengal (RB)-loaded metallocatanionic vesicles (MCVs) nanosystem for the phototoxicity of cancer cells. Different sets of MCVs were prepared by two different cationic single-chain metallosurfactants, i.e., hexadecylpyridinium trichlorocuprate (CuCPC I) and hexadecylpyridinium trichloroferrate (FeCPC I) in combination with anionic double-chain sodium bis(2-ethylhexyl)sulfosuccinate (AOT) surfactant in phosphate buffer saline of pH 7.4. The RB-loaded CuCPC I:AOT and FeCPC I:AOT vesicles enhanced the maximum singlet oxygen (1O2) generation by 1-fold and 3-fold, respectively, compared to pure RB. Upon irradiation with a 532 nm laser for 10 min, these RB-loaded CuCPC I:AOT and FeCPC I:AOT MCVs significantly decreased the metabolic activity of U-251 cells by 70% and 85% at MCVs concentration of 0.75 µM, respectively. Furthermore, RB-loaded MCVs showed the highest intracellular 1O2-mediated membrane damage and cell-killing effect as confirmed by singlet oxygen sensor green and differential nuclear staining assay, which is attributed to the cellular uptake profile of different RB-loaded MCVs fractions. Caspase 3/7 assay confirmed the apoptotic pathway of cell death by activating caspase. Therefore, the photoactivation of RB-loaded MCVs led to a significant reduction in the viability of U-251 cells (maximum 85%), which resulted in cell death. Our study demonstrated the advantage of using these dual-charge and biocompatible metallocatanionic vesicles as a promising delivery system of photodynamic therapy that can enhance 1O2 generation from PS and can be further utilized in photomedicine.

Electrochemical Immunosensor for Ultra-Low Detection of Human Papillomavirus Biomarker for Cervical Cancer.

Human papillomavirus (HPV) is the causative agent for cervical cancer. Of the various types of HPV, the high-risk HPV-16 type is the most important antigenic high-risk HPV. In this work, the antigenic HPV-16 L1 peptide was immobilized on a glassy carbon electrode and used to detect several concentrations of the anti-HPV-16 L1 antibody, and vice versa. Two electrode platforms were used: onion-like carbon (OLC) and its polyacrylonitrile (OLC-PAN) composites. Both platforms gave a wide linear concentration range (1.95 fg/mL to 6.25 ng/mL), excellent sensitivity (>5.2 µA/log ([HPV-16 L1, fg/mL]), and extra-ordinarily low limit of detection (LoD) of 1.83 fg/mL (32.7 aM) and 0.61 fg/mL (10.9 aM) for OLC-PAN and OLC-based immunosensors, respectively. OLC-PAN modified with the HPV-16 L1 protein showed low LoD for the HPV-16 L1 antibody (2.54 fg/mL, i.e., 45.36 aM), proving its potential use for screening purposes. The specificity of detection was proven with the anti-ovalbumin antibody (anti-OVA) and native ovalbumin protein (OVA). An immobilized antigenic HPV-16 L1 peptide showed insignificant interaction with anti-OVA in contrast with the excellent interaction with anti-HPV-16 L1 antibody, thus proving high specificity. The application of the immunosensor as a potential point-of-care (PoC) diagnostic device was investigated with screen-printed carbon electrodes, which detected ultra-low (ca. 0.7 fg/mL ≈ 12.5 aM) and high (ca. 12 µg/mL ≈ 0.21 µM) concentrations. This study represents the lowest LoD reported for HPV-16 L1. It opens the door for further investigation with other electrode platforms and realization of PoC diagnostic devices for screening and testing of HPV biomarkers for cervical cancer.

Electric field responsive nanotransducers for glioblastoma.

BACKGROUND: Electric field therapies such as Tumor Treating Fields (TTFields) have emerged as a bioelectronic treatment for isocitrate dehydrogenase wild-type and IDH mutant grade 4 astrocytoma Glioblastoma (GBM). TTFields rely on alternating current (AC) electric fields (EF) leading to the disruption of dipole alignment and induced dielectrophoresis (DEP) during cytokinesis. Although TTFields have a favourable side effect profile, particularly compared to cytotoxic chemotherapy, survival benefits remain limited (~ 4.9 months) after an extensive treatment regime (20 hours/day for 18 months). The cost of the technology also limits its clinical adoption worldwide. Therefore, the discovery of new technology that can enhance both the therapeutic efficiency and efficacy of these TTFields will be of great benefit to cancer treatment and decrease healthcare costs worldwide. METHODS: In this work, we report the role of electrically conductive gold (GNPs), dielectric silica oxide (SiO2), and semiconductor zinc oxide (ZnO) nanoparticles (NPs) as transducers for enhancing EF mediated anticancer effects on patient derived GBM cells. Physicochemical properties of these NPs were analyzed using spectroscopic, electron microscopy, and light-scattering techniques. RESULTS: In vitro TTFields studies indicated an enhanced reduction in the metabolic activity of patient-derived Glioma INvasive marginal (GIN 28) and Glioma contrast enhanced core (GCE 28) GBM As per our journal style, article titles should not include capitalised letters unless these are proper nouns/acronyms. We have therefore used the article title "Electric field responsive nanotransducers for glioblastoma" as opposed to "Electric Field Responsive Nanotransducers for Glioblastoma" as given in the submission system. Please check if this is correct.cells in groups treated with NPs vs. control groups, irrespective of NPs dielectric properties. Our results indicate the inorganic NPs used in this work enhance the intracellular EF effects that could be due to the virtue of bipolar dielectrophoretic and electrophoretic effects. CONCLUSIONS: This work presents preliminary evidence which could help to improve future EF applications for bioelectronic medicine. Furthermore, the merits of spherical morphology, excellent colloidal stability, and low toxicity, make these NPs ideal for future studies for elucidating the detailed mechanism and efficacy upon their delivery in GBM preclinical models.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Metallocatanionic vesicle-mediated enhanced singlet oxygen generation and photodynamic therapy of cancer cells.

In clinics, photodynamic therapy (PDT) is established as a non-invasive therapeutic modality for certain types of cancers and skin disease. However, due to poor water solubility, photobleaching, and the dark toxicity of photosensitizers (PSs), further developments are required to improve the efficiency of PDT. Herein, we report the role of metallocatanionic vesicles (MCVs) in enhancing the phototoxicity of methylene blue (MB) against cancer cells. These MCVs were prepared via a facile and quick solution-solution mixing method using a cationic single-chain metallosurfactant (FeCPC I) in combination with anionic sodium oleate (Na Ol). For singlet oxygen (1O2) generation and PDT studies, two fractions of FeCPC I : Na Ol, i.e., 30 : 70 (V37) and 70 : 30 (V73), were chosen based on their long-term stability in aqueous media. A cationic PS MB was loaded into these vesicles. The MB-loaded MCV 30 : 70 and 70 : 30 fractions enhanced the 1O2 generation by 0.10- and 0.40-fold, respectively, compared with MB alone. Upon illumination using a 650 nm laser, these MB-loaded V73 and V37 MCVs significantly decreased the metabolic activity of MCF-7 cells by ≤50% at a concentration of 0.75 µM. Furthermore, the SOSG assay revealed that the synthesized MCVs enhanced the intracellular 1O2 compared with MB alone. The MB-loaded V73 MCVs showed the highest 1O2-mediated membrane damage and cell-killing effect, as confirmed using the differential nuclear staining assay (DNS), which is attributed to the cellular uptake profile of the different MCV fractions. Altogether, this work shows the advantage of using these biocompatible and dual-charge MCVs as promising delivery vehicles that can enhance the 1O2 generation from the PS. This work suggests the future application of these Fe-MCVs in magnetically guided PDT.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

Printing biohybrid materials for bioelectronic cardio-3D-cellular constructs.

Conductive hydrogels are emerging as promising materials for bioelectronic applications as they minimize the mismatch between biological and electronic systems. We propose a strategy to bioprint biohybrid conductive bioinks based on decellularized extracellular matrix (dECM) and multiwalled carbon nanotubes. These inks contained conductive features and morphology of the dECM fibers. Electrical stimulation (ES) was applied to bioprinted structures containing human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). It was observed that in the absence of external ES, the conductive properties of the materials can improve the contractile behavior of the hPSC-CMs, and this effect is enhanced under the application of external ES. Genetic markers indicated a trend toward a more mature state of the cells with upregulated calcium handling proteins and downregulation of calcium channels involved in the generation of pacemaking currents. These results demonstrate the potential of our strategy to manufacture conductive hydrogels in complex geometries for actuating purposes.

Reproducible fabrication of robust, renewable vertically aligned multiwalled carbon nanotube/epoxy composite electrodes.

We describe the reproducible fabrication of robust, vertically aligned multiwalled carbon nanotube (VACNT)/epoxy composite electrodes. The electrodes are characterized by cyclic voltammetry, impedance spectroscopy, and scanning electron and atomic force microscopies. Low background currents are obtained at the electrodes, and common redox probe molecules and NADH show excellent voltammetric behavior. When electrode performance deteriorates due to fouling, the electrode surfaces can be reproducibly renewed by mechanical polishing followed by O(2) plasma treatment. The electrochemical performance of the electrodes is maintained after more than 100 cycles of use and renewal.

Toward nanobioelectronic medicine: Unlocking new applications using nanotechnology.

Bioelectronic medicine aims to interface electronic technology with biological components and design more effective therapeutic and diagnostic tools. Advances in nanotechnology have moved the field forward improving the seamless interaction between biological and electronic components. In the lab many of these nanobioelectronic devices have the potential to improve current treatment approaches, including those for cancer, cardiovascular disorders, and disease underpinned by malfunctions in neuronal electrical communication. While promising, many of these devices and technologies require further development before they can be successfully applied in a clinical setting. Here, we highlight recent work which is close to achieving this goal, including discussion of nanoparticles, carbon nanotubes, and nanowires for medical applications. We also look forward toward the next decade to determine how current developments in nanotechnology could shape the growing field of bioelectronic medicine. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing.