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.

Development of a Colorimetric Sensor for Autonomous, Networked, Real-Time Application.

This review describes an ongoing effort intended to develop wireless sensor networks for real-time monitoring of airborne targets across a broad area. The goal is to apply the spectrophotometric characteristics of porphyrins and metalloporphyrins in a colorimetric array for detection and discrimination of changes in the chemical composition of environmental air samples. The work includes hardware, software, and firmware design as well as development of algorithms for identification of event occurrence and discrimination of targets. Here, we describe the prototype devices and algorithms related to this effort as well as work directed at selection of indicator arrays for use with the system. Finally, we review the field trials completed with the prototype devices and discuss the outlook for further development.

Field Demonstration of a Distributed Microsensor Network for Chemical Detection.

We have developed the ABEAM-15, a custom-built multiplexed reflectance device for the detection of vapor phase and aerosolized chemical plumes. The instrument incorporates fifteen individual sensing elements, has wireless communications, offers support for a battery pack, and is capable of both live and fully autonomous operation. Two housing options have been fabricated: a compact open housing for indoor use and a larger weather-sealed housing for outdoor use. Previously developed six-plex analysis algorithms are extended to 15-plex format and implemented on a laptop computer. We report the results of recent outdoor field trials with this instrument in Denver, CO in a stadium security scenario. Through software, the wireless modules on each instrument were configured to form a six-instrument, star-point topology, distributed microsensor network with live reporting and real-time data analysis. The network was tested with aerosols of methyl salicylate.

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.

Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens.

The fast-growing nonmodel marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order to further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences, and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that, while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this nonmodel chassis.

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.

Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine-Mediated Photodynamic Therapy.

Current challenges in photodynamic therapy (PDT) include both the targeted delivery of the photosensitizer (PS) to the desired cellular location and the maintenance of PS efficacy. Zinc phthalocyanine (ZnPc), a macrocyclic porphyrin and a potent PS for PDT, undergoes photoexcitation to generate reactive singlet oxygen that kills cells efficiently, particularly when delivered to the plasma membrane. Like other commonly employed PS, ZnPc is highly hydrophobic and prone to self-aggregation in aqueous biological media. Further, it lacks innate subcellular targeting specificity. Cumulatively, these attributes pose significant challenges for delivery via traditional systemic drug delivery modalities. Here, we report the development and characterization of a liquid crystal nanoparticle (LCNP)-based formulation for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer. ZnPc was coloaded with the organic fluorophore, perylene (PY), in the hydrophobic polymeric matrix of the LCNP core. PY facilitated the fluorescence-based tracking of the LCNP carrier while also serving as a Förster resonance energy transfer (FRET) donor to the ZnPc acceptor. This configuration availed efficient singlet oxygen generation via enhanced excitation of ZnPc from multiple surrounding PY energy donors. When excited in a FRET configuration, cuvette-based assays revealed that singlet oxygen generation from the ZnPc was ∼1.8-fold greater and kinetically 12 times faster compared to when the ZnPc was excited directly. The specific tethering of the LCNPs to the plasma membrane of HEK 293 T/17 and HeLa cells was achieved by surface functionalization of the NPs with PEGylated cholesterol. In HeLa cells, LCNPs coloaded with PY and ZnPc, when photoexcited in a FRET configuration, mediated 70% greater cell killing compared to LCNPs containing ZnPc alone (direct excitation of ZnPc). This was attributed to a significant increase of the oxidative stress in the cells during the PDT. Overall, this work details the ability of the LCNP platform to facilitate (1) the specific tethering of the PY-ZnPc FRET pair to the plasma membrane and (2) the FRET-mediated, augmented singlet oxygen generation for enhanced PDT relative to the direct excitation of ZnPc alone.

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.

Reflectance-based detection for long term environmental monitoring.

Here, the potential of colorimetric sensors utilizing porphyrin indicators for long term environmental monitoring is demonstrated. Prototype devices based on commercial color sensing chips (six per device) were combined with in-house developed algorithms for data analysis. The devices are intended to provide real-time sensing of threats. An initial outdoor data set was collected using prototype devices with occasional spiked exposure to targets. This data was supported by similar data collected in a controlled indoor environment. Weaknesses in the noted performance of the devices during these experiments were addressed through altering device parameters, algorithm parameters, and array element composition. Additional outdoor data sets totaling 1,616 h and indoor data sets totaling 728 h were collected in support of assessing these changes to the system configuration. The optimized system provided receiver operating characteristics (ROC) of specificity 0.97 and sensitivity 1.0.

Development of a Detection Algorithm for Use with Reflectance-Based, Real-Time Chemical Sensing.

Here, we describe our efforts focused on development of an algorithm for identification of detection events in a real-time sensing application relying on reporting of color values using commercially available color sensing chips. The effort focuses on the identification of event occurrence, rather than target identification, and utilizes approaches suitable to onboard device incorporation to facilitate portable and autonomous use. The described algorithm first excludes electronic noise generated by the sensor system and determines response thresholds. This automatic adjustment provides the potential for use with device variations as well as accommodating differing indicator behaviors. Multiple signal channels (RGB) as well as multiple indicator array elements are combined for reporting of an event with a minimum of false responses. While the method reported was developed for use with paper-supported porphyrin and metalloporphyrin indicators, it should be equally applicable to other colorimetric indicators. Depending on device configurations, receiver operating characteristic (ROC) sensitivities of 1 could be obtained with specificities of 0.87 (threshold 160 ppb, ethanol).

Thermally activated long range electron transport in living biofilms.

Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.

Electric Field Modulation of Semiconductor Quantum Dot Photoluminescence: Insights Into the Design of Robust Voltage-Sensitive Cellular Imaging Probes.

The intrinsic properties of quantum dots (QDs) and the growing ability to interface them controllably with living cells has far-reaching potential applications in probing cellular processes such as membrane action potential. We demonstrate that an electric field typical of those found in neuronal membranes results in suppression of the QD photoluminescence (PL) and, for the first time, that QD PL is able to track the action potential profile of a firing neuron with millisecond time resolution. This effect is shown to be connected with electric-field-driven QD ionization and consequent QD PL quenching, in contradiction with conventional wisdom that suppression of the QD PL is attributable to the quantum confined Stark effect.

Load-Induced Hydrodynamic Lubrication of Porous Films.

We present an exploratory study of the tribological properties and mechanisms of porous polymer surfaces under applied loads in aqueous media. We show how it is possible to change the lubrication regime from boundary lubrication to hydrodynamic lubrication even at relatively low shearing velocities by the addition of vertical pores to a compliant polymer. It is hypothesized that the compressed, pressurized liquid in the pores produces a repulsive hydrodynamic force as it extrudes from the pores. The presence of the fluid between two shearing surfaces results in low coefficients of friction (µ ≈ 0.31). The coefficient of friction is reduced further by using a boundary lubricant. The tribological properties are studied for a range of applied loads and shear velocities to demonstrate the potential applications of such materials in total joint replacement devices.

Sweet substrate: a polysaccharide nanocomposite for conformal electronic decals.

A conformal electronic decal based on a polysaccharide circuit board (PCB) is fabricated and characterized. The PCBs are laminates composed of bioderived sugars - nanocellulose and pullulan. The PCB and decal transfer are a bioactive material system for supporting electronic devices capable of conforming to bio-logical surfaces.

Reconfigurable acquisition system with integrated optics for a portable flow cytometer.

Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.

Catch and release: integrated system for multiplexed detection of bacteria.

An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10(2)-10(6) cells/mL of target bacteria. Reversal of magnets' rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.

Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven.

Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential), forming a biofilm extending many cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors, thought to involve extracellular matrix c-type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 µm away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode surface. The magnitude of the redox gradient seems to correlate with current, which is consistent with electron transport from cells in the biofilm to the anode, where electrons effectively diffuse from areas of high to low concentration, hopping between redox cofactors. Comparison with gate measurements, when one electrode is used as an electron source and the other electrode is used as an electron drain, suggests that there are multiple types of redox cofactors in Geobacter biofilms spanning a range in oxidation potential that can engage in electron transport. The majority of these redox cofactors, however, seem to have oxidation potentials too negative to be involved in electron transport when acetate is the electron source.

Design and fabrication of gecko-inspired adhesives.

Recently, there has been significant interest in developing dry adhesives mimicking the gecko adhesive system, which offers several advantages compared to conventional pressure-sensitive adhesives. Specifically, gecko adhesive pads have anisotropic adhesion properties; the adhesive pads (spatulae) stick strongly when sheared in one direction but are non-adherent when sheared in the opposite direction. This anisotropy property is attributed to the complex topography of the array of fine tilted and curved columnar structures (setae) that bear the spatulae. In this study, we present an easy, scalable method, relying on conventional and unconventional techniques, to incorporate tilt in the fabrication of synthetic polymer-based dry adhesives mimicking the gecko adhesive system, which provides anisotropic adhesion properties. We measured the anisotropic adhesion and friction properties of samples with various tilt angles to test the validity of a nanoscale tape-peeling model of spatular function. Consistent with the peel zone model, samples with lower tilt angles yielded larger adhesion forces. The tribological properties of the synthetic arrays were highly anisotropic, reminiscent of the frictional adhesion behavior of gecko setal arrays. When a 60° tilt sample was actuated in the gripping direction, a static adhesion strength of ~1.4 N/cm(2) and a static friction strength of ~5.4 N/cm(2) were obtained. In contrast, when the dry adhesive was actuated in the releasing direction, we measured an initial repulsive normal force and negligible friction.