Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

Modifications of Epitaxial Graphene on SiC for the Electrochemical Detection and Identification of Heavy Metal Salts in Seawater.

The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.

Multilayer Epitaxial Graphene on Silicon Carbide: A Stable Working Electrode for Seawater Samples Spiked with Environmental Contaminants.

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis.

Machine Learning Techniques for Chemical Identification Using Cyclic Square Wave Voltammetry.

Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.

Linear and nonlinear optical characterization of self-assembled, large-area gold nanosphere metasurfaces with sub-nanometer gaps: errata.

We correct a nomenclature error for the plasmon ruler equation used to fit the simulation data in Fig. 2(d) [Opt. Express24, 27360 (2016)].

Paper-Based Electrochemical Detection of Chlorate.

We describe the use of a paper-based probe impregnated with a vanadium-containing polyoxometalate anion, [PMo11VO40]5-, on screen-printed carbon electrodes for the electrochemical determination of chlorate. Cyclic voltammetry (CV) and chronocoulometry were used to characterize the ClO3- response in a pH = 2.5 solution of 100 mM sodium acetate. A linear CV current response was observed between 0.156 and 1.25 mg/mL with a detection limit of 0.083 mg/mL (S/N > 3). This performance was reproducible using [PMo11VO40]5--impregnated filter paper stored under ambient conditions for as long as 8 months prior to use. At high concentration of chlorate, an additional catalytic cathodic peak was seen in the reverse scan of the CVs, which was digitally simulated using a simple model. For chronocoulometry, the charge measured after 5 min gave a linear response from 0.625 to 2.5 mg/mL with a detection limit of 0.31 mg/mL (S/N > 3). In addition, the slope of charge vs. time also gave a linear response. In this case the linear range was from 0.312 to 2.5 mg/mL with a detection limit of 0.15 mg/mL (S/N > 3). Simple assays were conducted using three types of soil, and recovery measurements reported.

A Simple and Inexpensive Electrochemical Assay for the Identification of Nitrogen Containing Explosives in the Field.

We report a simple and inexpensive electrochemical assay using a custom built hand-held potentiostat for the identification of explosives. The assay is based on a wipe test and is specifically designed for use in the field. The prototype instrument designed to run the assay is capable of performing time-resolved electrochemical measurements including cyclic square wave voltammetry using an embedded microcontroller with parts costing roughly $250 USD. We generated an example library of cyclic square wave voltammograms of 12 compounds including 10 nitroaromatics, a nitramine (RDX), and a nitrate ester (nitroglycine), and designed a simple discrimination algorithm based on this library data for identification.

Linear and nonlinear optical characterization of self-assembled, large-area gold nanosphere metasurfaces with sub-nanometer gaps.

We created centimeter-scale area metasurfaces consisting of a quasi-hexagonally close packed monolayer of gold nanospheres capped with alkanethiol ligands on glass substrates using a directed self-assembly approach. We experimentally characterized the morphology and the linear and nonlinear optical properties of metasurfaces. We show these metasurfaces, with interparticle gaps of 0.6 nm, are modeled well using a classical (without charge transfer) description. We find a large dispersion of linear refractive index, ranging from values less than vacuum, 0.87 at 600 nm, to Germanium-like values of 4.1 at 880 nm, determined using spectroscopic ellipsometry. Nonlinear optical characterization was carried out using femtosecond Z-scan and we observe saturation behavior of the nonlinear absorption (NLA) and nonlinear refraction (NLR). We find a negative NLR from these metasurfaces two orders of magnitude larger (n2,sat = -7.94x10-9 cm2/W at Isat,n2 = 0.43 GW/cm2) than previous reports on gold nanostructures at similar femtosecond time scales. We also find the magnitude of the NLA comparable to the largest values reported (ß2,sat = -0.90x105 cm/GW at Isat,ß2 = 0.34 GW/cm2). Precise knowledge of the index of refraction is of crucial importance for emerging dispersion engineering technologies. Furthermore, utilizing this directed self-assembly approach enables the nanometer scale resolution required to develop the unique optical response and simultaneously provides high-throughput for potential device realization.

Plasma-Modified, Epitaxial Fabricated Graphene on SiC for the Electrochemical Detection of TNT.

Using square wave voltammetry, we show an increase in the electrochemical detection of trinitrotoluene (TNT) with a working electrode constructed from plasma modified graphene on a SiC surface vs. unmodified graphene. The graphene surface was chemically modified using electron beam generated plasmas produced in oxygen or nitrogen containing backgrounds to introduce oxygen or nitrogen moieties. The use of this chemical modification route enabled enhancement of the electrochemical signal for TNT, with the oxygen treatment showing a more pronounced detection than the nitrogen treatment. For graphene modified with oxygen, the electrochemical response to TNT can be fit to a two-site Langmuir isotherm suggesting different sites on the graphene surface with different affinities for TNT. We estimate a limit of detection for TNT equal to 20 ppb based on the analytical standard S/N ratio of 3. In addition, this approach to sensor fabrication is inherently a high-throughput, high-volume process amenable to industrial applications. High quality epitaxial graphene is easily grown over large area SiC substrates, while plasma processing is a rapid approach to large area substrate processing. This combination facilitates low cost, mass production of sensors.

Integrating Paper Chromatography with Electrochemical Detection for the Trace Analysis of TNT in Soil.

We report on the development of an electrochemical probe for the trace analysis of 2,4,6-trinitrotoluene (TNT) in soil samples. The probe is a combination of graphite electrodes, filter paper, with ethylene glycol and choline chloride as the solvent/electrolyte. Square wave chromatovoltammograms show the probes have a sensitivity for TNT of 0.75 nA/ng and a limit of detection of 100 ng. In addition, by taking advantage of the inherent paper chromatography step, TNT can be separated in both time and cathodic peak potential from 4-amino-dinitrotolene co-spotted on the probe or in soil samples with the presence of methyl parathion as a possible interferent.

Synthesis of a 2,2'-bipyridyl functionalized oligovinylene-phenylene using Heck and Horner-Wadsworth-Emmons reactions and X-ray crystal structure of E-(4-(4-bromostyryl)phenyl)(methyl)sulfane.

The synthesis of a new 2,2'-bipyridyl functionalized oligovinylenephenylene (OVP-5) containing a methyl protected thiol using Heck coupling and the Horner-Wadsworth-Emmons reaction and is described. A key step involving a diisopropylcarbodiimide promoted dehydration of a stable ß-hydroxyphosphonate intermediate was identified. The structure of precursor E-(4-(4-bromostyryl)phenyl)(methyl)sulfane was determined using X-ray crystallography.

Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing.

The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O(2) at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine's inherent redox chemistry is presented.

Observation of two discrete conductivity states in quinone-oligo(phenylene vinylene).

The single-molecule conductivity of quinone-oligo(phenylene vinylene) (Q-OPV) attached to a gold substrate was studied using electrochemical scanning tunnelling microscopy. The results show that the molecule has two discrete conductivity states: a low-conductivity state, when it is oxidized, and a high-conductivity state, when reduced. The electron transport through the molecule in both states occurs via coherent tunnelling. The molecular conductivity in either oxidation state is independent from the electrochemical gate potential; however, the gate potential can be used to switch the oxidation state of the molecule. Numerical calculations suggest that the highest occupied molecular orbital (HOMO) of Q-OPV controls tunnelling through the molecule and that the independence of conductivity from the electrochemical gate in either oxidation state originates from strong penetration of HOMO into the substrate. In addition, the greater delocalization of HOMO in the reduced state than in the oxidized state explains the greater conductivity of Q-OPV in the former than in the latter.

Fluorescence-based sensing of 2,4,6-trinitrotoluene (TNT) using a multi-channeled poly(methyl methacrylate) (PMMA) microimmunosensor.

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1-10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT.

Multiplex charge-transfer interactions between quantum dots and peptide-bridged ruthenium complexes.

Simultaneous detection of multiple independent fluorescent signals or signal multiplexing has the potential to significantly improve bioassay throughput and to allow visualization of concurrent cellular events. Applications based on signal multiplexing, however, remain hard to achieve in practice due to difficulties in both implementing hardware and the photophysical liabilities associated with available organic dye and protein fluorophores. Here, we used charge-transfer interactions between luminescent semiconductor quantum dots (QDs) and proximal redox complexes to demonstrate controlled quenching of QD photoemission in a multiplexed format. In particular, we show that, because of the ability of the Ru complex to effectively interact with CdSe-ZnS QDs emitting over a broad window of the optical spectrum, higher orders of multiplexed quenching can be achieved in a relatively facile manner. Polyhistidine-appended peptides were site-specifically labeled with a redox-active ruthenium (Ru) phenanthroline complex and self-assembled onto QDs, resulting in controlled quenching of the QD emission. Different QD colors either alone or coupled to Ru-phen-peptide were then mixed together and optically interrogated. Composite spectra collected from mixtures ranging from four up to eight distinct QD colors were deconvoluted, and the individual QD photoluminescence (PL) loss due to charge transfer was quantified. The current multiplexing modality provides a simpler format for exploiting the narrow, size-tunable QD emissions than that offered by resonance energy transfer; for the latter, higher orders of multiplexing are limited by spectral overlap requirements.

Surface reactivity of the quinone/hydroquinone redox center tethered to gold: comparison of delocalized and saturated bridges.

We found that when a quinone headgroup, present in a mixed self-assembled monolayer on gold, reacts with a nucleophile, dissolved in the bulk phase, the reaction rate widely depends on the chemical nature of the tether, being 7 times faster for quinones attached via a delocalized bridge as compared to a saturated alkane chain. Cyclic voltammetry (CV) of the quinone/hydroquinone redox couple was used to monitor the nucleophilic addition, while simulated CVs compared to experimental runs permitted the determination of rate constants. Analysis of CV data also suggests that the delocalized oligo(phenylene ethynylene) bridge facilitates the addition of two mercaptoethanol molecules as compared to the alkane bridge, where only one molecule is being added. The use of delocalized bridges for tethering quinones to electrodes is of great potential in electrochemically controlled "tuning" of surfaces needed in biosensor applications.

Nanoporous organosilicas as preconcentration materials for the electrochemical detection of trinitrotoluene.

We describe the use of nanoporous organosilicas for rapid preconcentration and extraction of trinitrotoluene (TNT) for electrochemical analysis and demonstrate the effect of template-directed molecular imprinting on TNT adsorption. The relative effects of the benzene (BENZ)- and diethylbenzene (DEB)-bridged organic-inorganic polymers, having narrow or broad pore size distributions, respectively, on electrochemical response and desorption behavior were examined. Sample volumes of 0.5-10 mL containing 5-1000 ppb TNT in a phosphate-buffered saline buffer were preconcentrated in-line before the detector using a microcolumn containing 10 mg of imprinted BENZ or DEB. Square-wave voltammetry was used to detect the first reduction peak of TNT in an electrochemical flow cell using a carbon working electrode and a Ag/AgCl reference electrode. Imprinted BENZ released TNT faster than imprinted DEB with considerably less peak tailing and displayed enhanced sensitivity and an improvement in the limit of detection (LOD) owing to more rapid elution of TNT from that material with increasing signal amplitude. For imprinted BENZ, the slope of signal versus concentration scaled linearly with increasing preconcentration volume, and for preconcentrating 10 mL of sample, the LOD for TNT was estimated to be 5 ppb. Template-directed molecularly imprinted DEB (TDMI-DEB) was 7-fold more efficient in adsorption of TNT from aqueous contaminated soil extract than nonimprinted DEB.

Non-photochemical catalytic hydrolysis of methyl parathion using core-shell Ag@TiO2 nanoparticles.

Highly water-dispersible core-shell Ag@TiO2 nanoparticles were prepared and shown to be catalytically active for the rapid degradation of the organothiophosphate pesticide methyl parathion (MeP). Formation of the hydrolysis product, p-nitrophenolate was monitored at pH 7.5 and 8.0, using UV-Vis spectroscopy. 31P NMR spectroscopy confirmed that hydrolysis is the predominant pathway for substrate breakdown under non-photocatalytic conditions. We have demonstrated that the unique combination of TiO2 with silver nanoparticles is required for catalytic hydrolysis with good recyclability. This work represents the first example of MeP degradation using TiO2 doped with AgNPs under mild and ambient conditions. Analysis of catalytic data and a proposed dark mechanism for MeP hydrolysis using core-shell Ag@TiO2 nanoparticles are described.

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates.

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/µM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms.

Effect of protein orientation on electron transfer between photosynthetic reaction centers and carbon electrodes.

A new type of monolayer of photosynthetic reaction centers (RC) with the primary donor facing the carbon electrode has been constructed using a new bifunctional linker and genetically engineered protein. Comparison of protein in two different orientations with linkers binding to the opposite sides of the protein demonstrates the possibility of utilizing the constructed surfaces as photoelectronic devices. The results show improvement of the electron transfer efficiency when RC is bound with the primary donor (P) facing the electrode (P-side). In either protein orientation, electron transfer within the protein is unidirectional and when applying a voltage RC operates as a photorectifier. Electron transfer between the protein and carbon electrodes in the constructed devices is most likely occurring by tunneling.