Light-driven post-translational installation of reactive protein side chains.

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.

Calcifying Coccolithophore: An Evolutionary Advantage Against Extracellular Oxidative Damage.

The evolutionary advantages afforded by phytoplankton calcification remain enigmatic. In this work, fluoroelectrochemical experiments reveal that the presence of a CaCO3 shell of a naturally calcifying coccolithophore, Coccolithus braarudii, offers protection against extracellular oxidants as measured by the time required for the switch-off in their chlorophyll signal, compared to the deshelled equivalents, suggesting the shift toward calcification offers some advantages for survival in the surface of radical-rich seawater.

Quantifying the Extent of Calcification of a Coccolithophore Using a Coulter Counter.

Although, in principle, the Coulter Counter technique yields an absolute measure of particle volume, in practice, calibration is near-universally employed. For regularly shaped and non-biological samples, the use of latex beads for calibration can provide sufficient accuracy. However, this is not the case with particles encased in biogenically formed calcite. To date, there has been no effective route by which a Coulter Counter can be calibrated to enable the calcification of coccolithophores─single cells encrusted with biogenic calcite─to be quantified. Consequently, herein, we seek to answer the following question: to what extent can a Coulter Counter be used to provide accurate information regarding the calcite content of a single-species coccolithophore population? Through the development of a new calibration methodology, based on the measurement and dynamic tracking of the acid-driven calcite dissolution reaction, a route by which the cellular calcite content can be determined is presented. This new method allows, for the first time, a Coulter Counter to be used to yield an absolute measurement of the amount of calcite per cell.

Calcium Carbonate Dissolution from the Laboratory to the Ocean: Kinetics and Mechanism.

The ultimate fate, over the course of millennia, of nearly all of the carbon dioxide formed by humankind is for it to react with calcium carbonate in the world's oceans. Although, this reaction is of global relevance, aspects of the calcite dissolution reaction remain poorly described with apparent contradictions present throughout the expansive literature. In this perspective we aim to evidence how a lack of appreciation of the role of mass-transport may have hampered developments in this area. These insights have important implications for both idealised experiments performed under laboratory conditions and for the measurement and modelling of oceanic calcite sediment dissolution.

Opto-Electrochemical Dissolution Reveals Coccolith Calcium Carbonate Content.

Coccoliths are plates of biogenic calcium carbonate secreted by calcifying marine phytoplankton; annually these phytoplankton are responsible for exporting >1 billion tonnes (1015  g) of calcite to the deep ocean. Rapid and reliable methods for assessing the degree of calcification are technically challenging because the coccoliths are micron sized and contain picograms (pg) of calcite. Here we pioneer an opto-eletrochemical acid titration of individual coccoliths which allows 3D reconstruction of each individual coccolith via in situ optical imaging enabling direct inference of the coccolith mass. Coccolith mass ranging from 2 to 400 pg are reported herein, evidencing both inter- and intra-species variation over four different species. We foresee this scientific breakthrough, which is independent of knowledge regarding the species and calibration-free, will allow continuous monitoring and reporting of the degree of coccolith calcification in the changing marine environment.

Visualising electrochemical reaction layers: mediated vs. direct oxidation.

Electrochemical treatments are widely used for 'clean up' in which toxic metals and organic compounds are removed using direct or mediated electrolysis. Herein we report novel studies offering proof of concept that spectrofluorometric electrochemistry can provide important mechanistic detail into these processes. A thin layer opto-electrochemical cell, with a carbon fibre (radius 3.5 µm) working electrode, is used to visualise the optical responses of the oxidative destruction of a fluorophore either directly, on an electrode, or via the indirect reaction of the analyte with an electrochemically formed species which 'mediates' the destruction. The optical responses of these two reaction mechanisms are first predicted by numerical simulation followed by experimental validation of each using two fluorescent probes, a redox inactive (in the electrochemical window) 1,3,6,8-pyrenetetrasulfonic acid and the redox-active derivative 8-hydroxypyrene-1,3,6-trisulfonic acid. In the vicinity of a carbon electrode held at different oxidative potentials, the contrast between indirect electro-destruction, chlorination, and direct oxidation is very obvious. Excellent agreement is seen between the numerically predicted fluorescence intensity profiles and experiment.

Optimising amperometric pH sensing in blood samples: an iridium oxide electrode for blood pH sensing.

Amperometric pH sensing in blood samples has been studied using iridium oxide electrodes. The iridium oxide electrodes are made by electrodeposition of iridium oxide onto an iridium micro-disc electrode from an alkaline solution of iridium(iii) oxide. The response of the electrode is studied in aqueous solutions and authentic samples of sheep's blood employing both cyclic voltammetry and square wave voltammetry. Uncertainties of pH measurement in blood samples via cyclic voltammetry (±0.07 pH units) were improved by a factor of two using square wave voltammetry (±0.03 pH units). Limitations of amperometric pH sensing in blood samples are considered as caused by the uncertainty of the required reference measurements (via a conventional glass electrode) and also the use of matrix-free and low ionic strength buffers to calibrate a standard glass electrode for the measurement of blood pH.

Voltammetric demonstration of thermally induced natural convection in aqueous solution.

In electrochemical systems imperfect thermostating inevitably leads to the presence of bulk convective flows. As recognised by Nernst [Z. Phys. Chem., 1904, 52] damping of these bulk convective flows next to a solid surface, or at the electrode, leads to diffusional mass transport predominating locally. This work questions the exclusivity of diffusional transport and provides hitherto unexplored physical insights into how thermally induced flows in bulk solution can, on both macro- and microelectrodes, influence a voltammetric measurement. Imperfect thermostating results in flows in the bulk solution which are predicted and here expeimentally shown to be of the order of 100 µm s-1. Here we show that even in the absence of natural convective flows induced by the electrochemical reaction itself, this thermally induced bulk convection can significantly affect the voltammetric response. First, evaporative losses from an open electrochemical cell can be sufficient to produce convective flows that can alter the electrochemical response. Second, electrodes with various sizes and geometries have been investigated and experimental results evidence that the sensitivity of an electrode to these flows in bulk solution is to a large extent controlled by the size of the surrounding non-conductive supporting substrate used to insulate parts of the electrode.

Porosity controls the catalytic activity of platinum nanoparticles.

Dendritic/mesoporous nanoparticle structures arise naturally and result from aggregation based growth mechanisms. The resulting porous particles exhibit high total surface areas (internal and external) but determining the magnitude of the interface remains challenging. Furthermore, assessing the chemical accessibility of the catalytic interface presents an additional difficulty. Taking three structurally related but different sized platinum nanoparticle samples (30-70 nm), we demonstrate how the catalytic rate of two archetypal surface limited reactions scale not with the square of the particle radius but with a power law of 2.6-2.9. This power law directly reflects the mesoporosity of the nanoparticles; the internal surface of the nanoparticles is both chemically accessible and contributes to the catalytic activity. For the 70 nm particles, up to 60% of the catalytic surface is contained in the internal structure of the particle.

Correction: Chemical analysis in saliva and the search for salivary biomarkers - a tutorial review.

Correction for 'Chemical analysis in saliva and the search for salivary biomarkers - a tutorial review' by Kamonwad Ngamchuea, et al., Analyst, 2018, 143, 81-99.

Simultaneous activity and surface area measurements on single mesoporous nanoparticle aggregates.

The underpotential deposition of hydrogen and the hydrogen evolution reaction is studied at individual mesoporous nanoparticles. This work shows how the electroactive surface area and catalytic activity of these individual particles can be simultaneously measured.

A thermostated cell for electrochemistry: minimising natural convection and investigating the role of evaporation and radiation.

An optimised thermostated electrochemical cell is designed and implemented. This is informed by experimental and computational studies characterizing the extent to which the thermostating of an electrochemical cell via a heated bath can be realised, both with the cell closed and open to the environment. The heat transfer in the system is simulated and probed experimentally; special emphasis is put on heat loss due to radiation and evaporation. Experiments and simulations demonstrate that these two mechanisms of heat transfer lead to a steady temperature in the cell that differs from that of the thermostat by ∼0.1 K. Simulations indicate that spatial inhomogeneities in the stationary temperature drive natural convective flows with a significant velocity. These new physical insights inform the optimization of a new electrochemical cell and its application in measurements of the impact frequency of silver nanoparticles as a function of temperature.

A quantitative methodology for the study of particle-electrode impacts.

Herein we provide a generic framework for use in the acquisition and analysis of the electrochemical responses of individual nanoparticles, summarising aspects that must be considered to avoid mis-interpretation of data. Specifically, we threefold highlight the importance of the nanoparticle shape, the effect of the nanoparticle diffusion coefficient on the probability of it being observed and the influence of the used measurement bandwidth. Using the oxidation of silver nanoparticles as a model system, it is evidenced that when all of the above have been accounted for, the experimental data is consistent with being associated with the complete oxidation of the nanoparticles (50 nm diameter). The duration of many single nanoparticle events are found to be ca. milliseconds in duration over a range of experiments. Consequently, the insight that the use of lower frequency filtered data yields a more accurate description of the charge passed during a nano-event is likely widely applicable to this class of experiment; thus we report a generic methodology. Conversely, information regarding the dynamics of the nano redox event is obscured when using such lower frequency measurements; hence, both data sets are complementary and are required to provide full insight into the behaviour of the reactions at the nanoscale.

Singlet Oxygen and the Origin of Oxygen Functionalities on the Surface of Carbon Electrodes.

The generation of oxygen-containing functionalities on pristine carbon surfaces is investigated and shown to be light sensitive, specifically to infra-red radiation. A mechanistic route involving singlet oxygen, 1 O2 , is proposed and evidenced.

Dynamics of Silver Nanoparticles in Aqueous Solution in the Presence of Metal Ions.

Using a combined UV-vis, DLS, and electrochemical approach, this work experimentally studies the physical origin of the observed colorimetric sensitivity of aqueous silver nanoparticles toward divalent metal ions. In the presence of Pb2+, AgNPs are slow to reversibly form agglomerates (the time scale of the reverse deagglomeration process is of the order of hours). This agglomeration is shown to be induced by complex formation between Pb2+ and citrate groups localized on the AgNPs, reducing surface charges (zeta-potential) and hence electrostatic repulsion between the AgNPs. Other divalent metal ions including Ca2+, Cd2+, Zn2+, Ni2+, Co2+, and Sn2+ are also studied, and the resulting sizes of the AgNPs clusters and the extents of the UV-vis spectrum red-shift in λmax have a strong positive correlation with the metal-ligand (citrate) complex formation constant (Kf). This work thus serves as a guide for the selection of capping agents on the basis of Kf and demonstrates the correlation between sizes and spectrophotometric as well as electrochemical responses of the AgNPs clusters. Importantly, we give further physical insights into the size-dependent properties of AgNPs and emphasize the difference between theoretical and experimental values of extinction coefficients, where the latter is affected by the angle-dependent scattering intensities and the measurement technique used.

Rapid Method for the Quantification of Reduced and Oxidized Glutathione in Human Plasma and Saliva.

A new method is developed to determine the concentrations of reduced (GSH) and oxidized glutathione (GSSG), and it enables the calculation of the GSH:GSSG ratios in human plasma and saliva samples. The assay is based on the masking of GSH in a GSH and GSSG mixture via a 1,4-addition reaction with p-benzoquinone (BQ), followed by enzymatic kinetic measurement. The enzyme, glutathione reductase, is highly specific to glutathione. Excess BQ can thus be easily removed by the addition of non-GSH thiols. The assay takes less than 2 min, is suitable for a short-time-scale study, and minimizes the in vitro underestimation of the GSH:GSSG ratio arising from the degradation of GSH and formation of GSSG. We further show in this paper that the stability of the total glutathione content (GSH + GSSG) and GSH in saliva is significantly greater than in plasma, encouraging the development of noninvasive saliva sensing.

Supported Microwires for Electroanalysis: Sensitive Amperometric Detection of Reduced Glutathione.

A carbon microfiber (7 µm diameter) is employed herein as an electroanalytical sensor. The fabricated sensor is cheap, is disposable, and requires only 150 µL of samples. The carbon fiber is surface-mounted onto an inert surface to overcome the problems of the fragility of the microwire and the possible interference of convective force due to the nonrigid nature of the wires, as well as to improve the reproducibility in length and the amperometric responses. As the cylindrical electrode is supported on a surface, the diffusion of redox-active species to the electrode is partially blocked by the substrate. A theoretical model is developed to account for this hindered diffusion. The mass-transport regime is altered from "linear" at very short time, where the amperometric responses of the supported microwire closely resemble that of an isolated free-standing cylinder (current ∝ electrode area), to "convergent" at long time where its response now tends toward that of a hemicylinder of equal radius. The model is validated using chronoamperometry and cyclic voltammetry of an ideal outer-sphere redox probe, reversible ferrocene methanol oxidation. The fabricated microwire electrode is further applied to the system of irreversible 2-nitro-5-thiobenzoate oxidation used in the detection of reduced glutathione (GSH). The microwire electrode shows significantly higher ratio of Faradaic to non-Faradaic currents as compared to microdisk, macrodisk or carbon nanotube modified electrodes. Using the fabricated microwire, GSH can be detected with the sensitivity of 0.7 nA µM-1 and the limit of detection of 0.5 µM (3 sB/m).

Reaction Layer Imaging Using Fluorescence Electrochemical Microscopy.

The chemical confinement of a pH sensitive fluorophore to a thin-reaction layer adjacent to an electrode surface is explored as a potentially innovative route to improving the spatial resolution of fluorescence electrochemical microscopy. A thin layer opto-electrochemical cell is designed, facilitating the visualization of a carbon fiber (diameter 7.0 µm) electrochemical interface. Proton consumption is driven at the interface by the reduction of benzoquinone to hydroquinone and the resulting interfacial pH change is revealed using the fluorophore 8-hydoxypyrene-1,3,6-trisulfonic acid. It is demonstrated that the proton depletion zone may be constrained and controlled by the addition of a finite acid concentration to the system. Simulation of the resulting fluorescence intensity profiles is achieved on the basis of a finite difference model, with excellent agreement between the theoretical and experimental results.

Potassium (De-)insertion Processes in Prussian Blue Particles: Ensemble versus Single Nanoparticle Behaviour.

Potassium (de-)insertion from Prussian blue (PB) is investigated at the single and multi-particle scale. The electrochemical behaviour is found to differ between the two measurement types. At the single particle level, oxidation of the PB nanoparticles with concomitant K+ deinsertion occurs more readily than the associated reduction, relating to K+ insertion. In contrast, the cyclic voltammetry of PB in a composite electrode containing conductive additives and polymeric binder suggests the opposite behaviour. Implications for assessing battery materials are discussed.

Aqueous Voltammetry in the Near Absence of Electrolyte.

In order to minimize the incidence of the CO2 hydrolysis and conduct aqueous electrochemistry in the virtual absence of electrolyte, a novel methodology is developed to achieve the near minimum conductivity (≈60 nS cm-1 ) for an aqueous solution through in situ deionization with ion exchange resin beads. Aqueous electrochemistry studying the oxidations of platinum, ferrocenemethanol, and hydrogen (H2 ) were conducted in the near complete absence of trace ionic species at a platinum microelectrode (d=10 µm). Both surface and solution phase electrochemical reactions were clearly observed, indicating that under these conditions there is a sufficiently compressed double layer for an interfacial electron transfer to be driven and the iR effects are significantly smaller than theoretically expected.