Several classes of copper complexes are known to induce oxidative DNA damage that mediates cell death. These compounds are potentially useful anticancer agents and detailed investigation can reveal the mode of DNA interaction, binding strength, and type of oxidative lesion formed. We recently reported the development of a DNA electrochemical biosensor employed to quantify the DNA cleavage activity of the well-studied [Cu(phen)2]2+ chemical nuclease. However, to validate the broader compatibility of this sensor for use with more diverse—and biologically compatible—copper complexes, and to probe its use from a drug discovery perspective, analysis involving new compound libraries is required. Here, we report on the DNA binding and quantitative cleavage activity of the [Cu(TPMA)(N,N)]2+ class (where TPMA = tris-2-pyridylmethylamine) using a DNA electrochemical biosensor. TPMA is a tripodal copper caging ligand, while N,N represents a bidentate planar phenanthrene ligand capable of enhancing DNA interactions through intercalation. All complexes exhibited electroactivity and interact with DNA through partial (or semi-) intercalation but predominantly through electrostatic attraction. Although TPMA provides excellent solution stability, the bulky ligand enforces a non-planar geometry on the complex, which sterically impedes full interaction. [Cu(TPMA)(phen)]2+ and [Cu(TPMA)(DPQ)]2+ cleaved 39% and 48% of the DNA strands from the biosensor surface, respectively, while complexes [Cu(TPMA)(bipy)]2+ and [Cu(TPMA)(PD)]2+ exhibit comparatively moderate nuclease efficacy (ca. 26%). Comparing the nuclease activities of [Cu(TPMA)(phen)] 2+ and [Cu(phen)2]2+ (ca. 23%) confirms the presence of TPMA significantly enhances chemical nuclease activity. Therefore, the use of this DNA electrochemical biosensor is compatible with copper(II) polypyridyl complexes and reveals TPMA complexes as a promising class of DNA damaging agent with tuneable activity due to coordinated ancillary phenanthrene ligands.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties andin-vitrocytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (whereN,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (K-app) rising from 10(5)to 10(7) m(-1)with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells within-vitroresults surpassing the clinical platinum(II) drug oxaliplatin.

Calcium alginate (CaALG) hydrogel beads and two sets of composite beads, formed from a combination of calcium alginate/propylene glycol alginate/human serum albumin (CaALG/PGA/HSA) and from calcium alginate with the quaternary ammonium salt, (3-(trimethoxysilyl)propyl)-octadecyldimethylammonium chloride (QA), (CaALG/QA), were prepared. Bovine serum albumin (BSA) was condensed with glutaraldehyde (GLA) to form a BSA/GLA hydrogel. The corresponding Ag+-containing gels of all of the above hydrogels were also formed, and slow leaching of the biocidal transition metal ion from the gels bestowed broad spectrum antimicrobial activity. In the absence of added Ag+, CaALG/QA was the only material to deliver marginal to moderate antibacterial and antifungal effects. The Ag+ impregnated hydrogel systems have the potential to maintain the antimicrobial properties of silver, minimising the risk of toxicity, and act as reservoirs to afford ongoing sterility.

According to the American Cancer Society report (2019-2021), the majority (63%) of stage III non-small cell lung cancer (NSCLC) patients are prescribed with chemo and/or radiation therapies, with 5-year relative survival rates of just 19%. Thus, directed drug development, toward personalised cancer treatment, is widely recognised as a necessary strategy in drug discovery research. However, broad generalisations on the modes of action of bioinorganic compounds are not conducive to tailored drug design, hence, fundamental mechanistic research is essential in realising personalised healthcare. In this work, anticancer properties of bis (1,10-phenanthroline) silver (I) acetate monohydrate (Ag-Phen), toward A549 lung cancer cells are presented. Biological assays were carried out to evaluate the effect of Ag-Phen on cell viability, reactive oxygen species generation and mitochondrial membrane potentials. In tandem with the biological assays, electrochemistry was employed to determine the real-time concentrations of intact Ag-Phen and dissociated Ag+ in the extracellular medium using platinum microelectrodes, as a function of cellular exposure time. Observations from the assays conducted include, Ag-Phen induced cytotoxicity (IC50 4.5 mu M at 72 h) and 2-fold ROS generation, and a 50% decrease in mitochondrial membrane potentials with respect to equivalent concentrations of Ag+ and 1,10-phenanthroline. Bio-speciation studies, conducted electrochemically at platinum microelectrodes, revealed almost 50% of the AgPhen had dissociated after 2 h. Significant reductions in concentrations of dissociated Ag+ (from 67.7 mu M to 6.7 mu M), and the Ag-Phen complex (from 50.2 mu M to 11.7 mu M) between 4 and 24 h from the extracellular medium, indicate cellular uptake of both. This novel method facilitates the real-time identification and quantification of electroactive species, both the intact Ag-Phen and Ag+, in the presence of A549 cells.

A new class of DNA intercalating metallodrug is reported. These agents contain a polypyridyl caging ligand called DPA which is bound to a copper ion ligated to a phenanthrene (Phen) DNA intercalator. The lead agent is Cu-DPA-DPPZ and it can recognize the minor groove of G–C rich DNA sequences and displays promising activity against several pancreatic cancer cell lines. The graphic shows the Cu-DPA-DPPZ complex intercalating the minor groove of a G–C DNA sequence taken from the protein data bank (2ANA). The modeling was completed using PyMOL together with the X-ray crystal structure of the complex. More information can be found in the Full Paper by A. Kellett et al. on page 971.

The technique chosen to immobilise DNA onto electrodes can determine the density and stability of the resultant immobilised layer. DNA-modified electrodes were prepared using four common DNA immobilisation methods and characterised using ferrocyanide. The negatively charged DNA strands should repel ferrocyanide anions. The DNA layers created using adsorption at glassy carbon electrodes were unstable, while those created through chemisorption of thiol-modified DNA onto gold electrodes were repeatable and stable, and returned, on average, 94 % repulsion of the probe. The presented results show how the immobilisation protocol, and DNA type, affects the stability, repeatability, and integrity of resultant DNA layers.

Isoxazolo-pyrene tethered calix[4]arenes selectively detect copper(II) ions without interference from related perchlorate ions. The fluorescence emission of the probes, synthesised by nitrile oxide alkyne cycloaddition, and characterised by spectroscopic and crystallographic data, is rapidly reduced by Cu(II) ions. Detection limits are in the micromolar or sub-micromolar range (0.3-3.6 μM) based on a 1 : 1 sensor:analyte interaction. Voltammetric behaviour and 1 H NMR data provide new insights into the sensing mechanism which is dependent on the calixarene substitution pattern. When the calixarene lower rim is fully substituted, Cu(II) detection occurs through a traditional chelation mechanism. In contrast, for calixarenes 1,3-disubstituted on the lower rim, detection takes place through a chemodosimetric redox reaction. The isolation of a calix[4]diquinone from the reaction with excess Cu(ClO4 )2 provides confirmation that the sensor-analyte interaction culminates in irreversible sensor oxidation.

The electrochemical determination of low concentrations of silver, and the short-lived highly reactive oxygen and nitrogen species (ROS/RNS), requires reliable, reproducible measurements with sensitive analytical methods. In this work, gold, platinum and platinum black micro and nanoelectrodes were fabricated. Characterisation revealed flat disk-shaped working areas of 20 - 25 µm (micro) and 1 - 10 nm (nano) in diameter, derived from steady-state limiting currents and validated using FE-SEM.[1] Reported studies on silver detection have been carried out under a myriad of conditions but, to date, electrochemical determination of silver in biological buffers and media has not been successful.[2] Ultra-low concentrations of silver (1 nM – 80 nM) were determined, by anodic stripping voltammetry (ASV) with Ag/AgBr as a reference electrode, using micro- and nanoelectrodes of platinum and gold in chloride-free phosphate buffer (PB, pH 7.4). Laser pulled electrodes exhibit highly reproducible stripping voltammetry data for the determination of silver and returned linear calibration curves. A low detection limit of 1.3 pM in this medium provides the ability to explore silver and silver-based bioinorganic drug uptake by tumour and microbial cells. [3] This study also includes the fabrication of Pt/Pt-black nanoelectrodes for the detection of ROS/RNS in chloride-free PB (pH 7.4). These data should help with the identification and quantitation of cellular ROS/RNS release. In particular, monitoring oxidative stress, as a cellular response on exposure to bioinorganic drugs, should help to elucidate the interaction pathway of bioinorganic anti-cancer drugs. We envisage that electrochemical speciation studies will help to elucidate the mechanism of action and cellular uptake of bioinorganic silver-based anti-microbial and anti-cancer drugs in cell sustaining media for in vitro and, potentially, in vivo single cell analysis. [1] Percival, S. J. et al., RSC Adv. 2014, 4 (21), 10491. [2] Radulescu, M. C. et al., Sensors 2010, 10 (12), 11340. [3] Prabhakar Sidambaram. et al., J. Electrochem. Soc. 2019, 166, 6, B532.

Heterogeneity in a cell population is a universal phenomenon in all biological systems including whole tissues, and cell cultures.[1] Intra-tumour heterogeneity has been widely reported for decades from morphological perspectives, whereas phenotypic and genotypic heterogeneity has also been detected.[2] DNA platination is widely assumed as the central mode of action for most platinum anti-cancer drugs. This generalisation on the mode of action for conventional chemotherapy may actually impede effective drug development. Clinical approval for most novel anti-cancer drugs has been denied due to uncertainty in optimal usage, and the modes of action unclear.[3] For a better understanding of variations, from cell to cell, in response to metal-based drugs, single cell analysis is the key. Single cell experiments should provide critical information about the pathway and disease state, which will guide personalised medicine and therapeutic strategies. Electrochemistry is the foremost approach for studying the uptake of metal-based drugs in single cells. This strategy involves positioning a micro or nanoelectrodes inside or near the surface of a single cell for the electrochemical monitoring of individual cellular events.[4] In this work, the aim is to exploit single cell electrochemical analysis, to study the uptake of silver-based bioinorganic drugs and to elucidate the mode of action. Single cell electrochemistry experiments were carried out using the platinum and platinum-black nanoelectrodes on human lung carcinoma (A549) cell lines. Speciation studies of the silver-phenanthroline bioinorganic drugs was examined using stripping voltammetry for metal detection, and the redox behaviour analysis by cyclic voltammetry for in vitro and single cell analysis. [1] Andrew G. Ewing. et al., Anal. Chem. 2019, 91, 1, 588-621 [2] Glenn Deng. et al., Front Cell Dev Biol. 2016, 4: 116. [3] Walter Berger. et al., ESMO Open. 2017, 2(3): e000239. [4] Dechen Jiang. et al., ACS Sens. 2018, 3 (2), 242–250

The electrochemical determination of ultra-low concentrations of silver requires reliable, reproducible measurements using sensitive analytical techniques. To date, the electrochemical determination of silver in biological buffers and pH neutral media has not been successful in terms of reproducibility. In this work, we report on the determination of ultra-low concentrations of silver in chloride-free phosphate buffer solution (PB, pH 7.4). Detection was conducted at gold and platinum micro and nanoelectrodes using anodic stripping voltammetry (ASV). The micro and nanoelectrodes were fabricated using a Sutter P-2000 laser puller, with physical and electrochemical characterization revealing flat disk-shaped working surfaces of 10-15 mu m (microelectrode) and 10-100 nm (nanoelectrodes) in radius. These dimensions were calculated from steady-state limiting currents and confirmed using FE-SEM. The laser pulled electrodes exhibit excellent electrochemical activity when characterized using ferrocene, without the addition of supporting electrolyte, and reproducible stripping voltammetric profiles for the determination of silver, with a LoD of 1.3 pM(1.8%) were obtained in 0.1 M chloride-free phosphate at platinum nanoelectrode. Determination of ultra-low concentrations of silver in chloride-free PB provides the scope to explore the mechanism of action of bioinorganic silver-based anti-bacterial, anti-fungal and anti-cancer drugs in cell media for in vitro and, potentially, in vivo analysis. (c) 2019 The Electrochemical Society.

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([CuII(phen)2]2+), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [CuII(phen)2]2+, a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [CuII(phen)2]2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [CuI(phen)2]+, with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [CuII(phen)2]2+.

The present work describes a novel approach for the determination of dopamine (DA) using carbon paste electrodes modified with sulfated β-cyclodextrin (S-β-CDCPE). The electrodes show high affinity towards DA electrochemical oxidation. DA returned a concentration detection range 5 × 10−7 M to 5 × 10−4 M and a detection limit of 1.33 × 10−7 M. DA, ascorbic acid (AA), and serotonin (5-HT) in a solution mixture can be simultaneously oxidized at significantly different potentials in the presence of S-β-CDCPE, while the unmodified CPE returned an overlapping response. In particular, S-β-CD-modified carbon paste microelectrode exhibits clear peak potential separations of 0.161, 0.101, and 0.258 V (vs. Ag/AgCl) for AA/DA, DA/5-HT, and AA/5-HT, respectively, in artificial cerebrospinal fluid (aCSF).

The complexes [Ag2(OOC-(CH2)n-COO)] (n=1-10) (1-10) were synthesised and reacted with 1,10-phenanthroline (phen) to yield derivatives formulating as [Ag2(phen)x(OOC-(CH2)y-COO)]·zH2O (x=2 or 3; y=1-10; z=1-4) (11-20) which are highly water-soluble and photo-stable in aqueous solution. The phen derivatives 11-20 exhibit chemotherapeutic potential against Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and against cisplatin-sensitive breast (MCF-7) and resistant ovarian (SKOV-3) cancer cell lines. Cyclic voltammetric analysis and DNA binding and intercalation studies indicate that the mechanism of action of 11-20 is significantly different to that of their silver(I) dicarboxylate precursors and they do not induce DNA damage or ROS generation in mammalian cells. The representative complexes 9 and 19 (containing the undecanedioate ligand) were both found to significantly reduce superoxide and hydrogen peroxide induced oxidative stress in the yeast S. cerevisiae.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Here we report the synthesis and isolation of a series of bis-chelate Cu(2+) phenanthroline-phenazine cationic complexes of [Cu(DPQ)(Phen)](2+), [Cu(DPPZ)(Phen)](2+), and [Cu(DPPN)(Phen)](2+) (where Phen = 1,10-phenanthroline, DPQ = dipyridoquinoxaline, DPPZ = dipyridophenazine, and DPPN = benzo[i]dipyridophenazine). These compounds have enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2](2+) (bis-Phen), with calf thymus DNA binding constants of DPQ and DPPZ agents (∼10(7) M(bp)(-1)) being the highest currently known for Cu(2+) phenanthrene compounds. Complex DNA binding follows DPQ ≈ DPPZ > DPPN > bis-Phen, with fluorescence quenching and thermal melting experiments on poly[d(A-T)2] and poly[d(G-C)2] supporting intercalation at both the minor and major groove. Phenazine complexes, however, show enhanced targeting and oxidative cleavage on cytosine-phosphate-guanine-rich DNA and have comparable in vitro cytotoxicity toward the cisplatin-resistant ovarian cancer line, SKOV3, as the clinical oxidative DNA-damaging drug doxorubicin (Adriamycin). In this study we also describe how a novel "on-chip" method devised for the Bioanalyser 2100 was employed to quantify double-stranded DNA damage, with high precision, by the complex series on pUC19 DNA (49% A-T, 51% G-C). Both DPQ and bis-Phen complexes are highly efficient oxidizers of pUC19, with DPQ being the most active of the overall series. It is apparent, therefore, that oxidative chemical nuclease activity on homogeneous canonical DNA is not entirely dependent on dynamic nucleotide binding affinity or intercalation, and this observation is corroborated through catalytic interactions with the superoxide anion radical and Fenton breakdown of hydrogen peroxide.

The synthetic chemical nuclease, [Cu(1,10-phenanthroline)2](2+), has stimulated research within metallonuclease development and in the area of cytotoxic metallodrug design. Our analysis reveals, however, that this agent is "promiscuous" as it binds both dsDNA and protein biomolecules, without specificity, and induces general toxicity to a diversity of cell lineages. Here, we describe the synthesis and characterization of small-molecule metallonucleases containing the redox-active cation, [Cu(RCOO)(1,10-phen)2](+), where 1,10-phen = 1,10-phenanthroline and R = -H, -CH3, -C2H5, -CH(CH3)2, and -C(CH3)3. The presence of coordinated carboxylate groups in the complex cation functions to enhance dsDNA recognition, reduce serum albumin binding, and offer control of toxicity toward human cancer cells, Gram positive and negative bacteria, and fungal pathogens. The induction of genomic dsDNA breaks (DSBs) were identified in ovarian adenocarcinoma cells using immunodetection of γ-H2AX. Formate, acetate, and pivalate functionalized complexes induced DSBs in a higher percentage of cells compared with [Cu(1,10-phen)2](2+), which supports the importance of inner-sphere modification toward enhancing targeted biological application.

1,10-Phenanthroline-5,6-dione and l-tyrosine methyl ester react to form phenanthroline-oxazine (PDT) from which [Cu(PDT)(2)](ClO(4))(2) and [Ag(PDT)(2)]ClO(4)·2MeOH are obtained. Binding to calf-thymus DNA by Ag(I) and Cu(II) PDT complexes exceed bis-1,10-phenanthroline analogues and the minor groove binding drugs, pentamidine and netropsin. Furthermore, unlike the artificial metallonuclease, [Cu(phen)(2)](2+), the [Cu(PDT)(2)](2+) complex does not cleave DNA in the presence of added reductant indicating unique interaction with DNA.

Poly (3,4-ethylene dioxythiophene)/sulphated β-cyclodextrin (PEDOT/S-β-CD) films, deposited onto gold working electrodes, were investigated for ascorbic acid (AA) and catecholamine detection using cyclic voltammetry, hydrodynamic voltammetry and amperometry. The thin PEDOT/S-β-CD films were fabricated via three electropolymerisation cyclic voltammetry cycles, on gold working macroelectrodes. A limit of detection (LoD) of 1.3×10−7M was determined at PEDOT/S-β-CD modified gold electrodes, via cyclic voltammetry, for dopamine (DA) in the presence of 1×10−3M of AA in human urine samples.

The di-copper(II) cation, [Cu(2)(μ-terephthalate)(1,10-phen)(4)](2+), is a powerful, non-sequence-specific, minor-groove oxidizer of duplex DNA which, unlike copper(II) bis-1,10-phenanthroline chloride, operates independently of exogenous reagents. The agent displays excellent in vitro cytoxicity towards cisplatin-resistant ovarian cancer cells, producing intracellular reactive oxygen species upon nano-molar exposure.