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.

Multi-Enzyme Assembly on T4 Phage Scaffold.

Over the past two decades, various scaffolds have been designed and synthesized to organize enzyme cascades spatially for enhanced enzyme activity based on the concepts of substrate channeling and enhanced stability. The most bio-compatible synthetic scaffolds known for enzyme immobilization are protein and DNA nanostructures. Herein, we examined the utility of the T4 phage capsid to serve as a naturally occurring protein scaffold for the immobilization of a three-enzyme cascade: Amylase, Maltase, and Glucokinase. Covalent constructs between each of the enzymes and the outer capsid protein Hoc were prepared through SpyTag-SpyCatcher pairing and assembled onto phage capsids in vitro with an estimated average of 90 copies per capsid. The capsid-immobilized Maltase has a fourfold higher initial rate relative to Maltase free in solution. Kinetic analysis also revealed that the immobilized three-enzyme cascade has an 18-fold higher converted number of NAD+ to NADH relative to the mixtures in solution. Our results demonstrate that the T4 phage capsid can act as a naturally occurring scaffold with substantial potential to enhance enzyme activity by spatially organizing enzymes on the capsid Hoc.

Microbial Nanocellulose Printed Circuit Boards for Medical Sensing.

We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing-potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.

Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles.

The sequence fitness of a llama single-domain antibody with an unusually high thermal stability is explored by a combined computational and experimental study. Starting with the X-ray crystallographic structure, RosettaBackrub simulations were applied to model sequence-structure tolerance profiles and identify key substitution sites. From the model calculations, an experimental site-directed mutagenesis was used to produce a panel of mutants, and their melting temperatures were determined by thermal denaturation. The results reveal a sequence fitness of an excess stability of approximately 12 °C, a value taken from a decrease in the melting temperature of an electrostatic charge-reversal substitution in the CRD3 without a deleterious effect on the binding affinity to the antigen. The tolerance for the disruption of antigen recognition without loss in the thermal stability was demonstrated by the introduction of a proline in place of a tyrosine in the CDR2, producing a mutant that eliminated binding. To further assist the sequence design and the selection of engineered single-domain antibodies, an assessment of different computational strategies is provided of their accuracy in the detection of substitution "hot spots" in the sequence tolerance landscape.

A Fully-Flexible Solution-Processed Autonomous Glucose Indicator.

We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM-45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries.

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.

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm.

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together ß-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.

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.

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9) M to 7.0×10(-10) M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5) pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

Selection and evaluation of single domain antibodies toward MS2 phage and coat protein.

MS2 phage (MS2 Ø) is a coli phage, non-pathogenic to eukaryotic cells, which has been used as a simulant for viral biothreats, such as those causing smallpox and hemorrhagic fever. MS2 Ø consists of an icosahedral capsid, 28nm in diameter, and a single stranded RNA genome; the viral capsid is composed of 180 copies of coat protein (CP). In this study, we isolated anti-MS2 Ø single domain antibodies (sdAbs) for the sensitive detection of the MS2 Ø. To achieve this, a first immune sdAb library was prepared from llamas immunized with purified coat protein and a second from animals immunized with MS2 Ø. By panning the two libraries against CP, MS2 Ø, or alternating between the two targets, anti-MS2 Ø and anti-CP sdAbs were selected, sequenced, and characterized for their binding affinity. Both direct binding assays and capture sandwich assays were performed on the MAGPIX platform. One of the best anti-MS2 Ø sdAb, Lib2CP12H, could detect MS2 Ø concentrations as low as 1.45ng/mL (∼5.0E+6pfu/mL), providing equivalent detection to conventional antibodies. This sdAb is thermally stable with a melting temperature around 60°C and recovered 80% of its secondary structure after heat denaturation.

Statistical analysis of plating variable effects on the electrical conductivity of electroless copper patterns on paper.

We describe a process for selective metallization of paper substrates bearing inkjet printed patterns of a commercial Pd/Sn colloidal catalyst ink plated using a commercial electroless Cu bath. The electrical conductivity of the Cu films is analyzed as a function of feature geometry (line dimensions (L) and spacing (S)), type of paper (P), age of the Pd/Sn patterns (A), plating time (T), and plating temperature (H) using a two-level factorial design. Conductivity is influenced predominantly by the P, T, and H factors, with lesser contributions attributed to pair-wise interactions among several of the variables studied. Increases in T and/or H enhance conductivity of the Cu films, whereas increases in P, corresponding to the use of rougher, more porous, paper substrates, yield Cu films exhibiting decreased conductivity. Our analysis leads to a model that predicts Cu film conductivity well over the ranges of variables examined, provides guidelines for identification of optimum conditions for plating highly conductive Cu films, and identifies areas for further process improvement.

Selective electroless metallization of patterned polymeric films for lithography applications.

The fabrication of electrical interconnects to provide power for and communication with computers as their component complementary metal oxide semiconductor (CMOS) devices continue to shrink in size presents significant materials and processing compatibility challenges. We describe here our efforts to address these challenges using top-surface imaging and hybrid photoresist/self-assembled monolayer patterning approaches, in conjunction with selective electroless metal deposition, to develop processes capable of fabricating appropriate submicron and nanoscale metal features useful as electrical interconnects, as well as plasma-etch-resistant masks and metal diffusion barriers. Our efforts focus on the development of cost-effective methods compatible with a manufacturing environment that satisfy materials and process constraints associated with CMOS device production. We demonstrate the fabrication of approximately 50-nm-width features in metal with high fidelity and sufficient control of edge acuity to satisfy current industry design rules using our processes and discuss the challenges and opportunities for fabrication of analogous sub-10-nm metal features.

Identification of explosives with two-dimensional ultraviolet resonance Raman spectroscopy.

The first two-dimensional (2D) resonance Raman spectra of TNT, RDX, HMX, and PETN are measured with an instrument that sequentially and rapidly switches between laser wavelengths, illuminating these explosives with forty wavelengths between 210 nm and 280 nm. Two-dimensional spectra reflect variations in resonance Raman scatter with illumination wavelength, adding information not available from single or few one-dimensional spectra, thereby increasing the number of variables available for use in identification, which is especially useful in environments with contaminants and interferents. We have recently shown that 2D resonance Raman spectra can identify bacteria. Thus, a single device that identifies the presence of explosives, bacteria, and other chemicals in complex backgrounds may be feasible.

Identification of bacteria from two-dimensional resonant-Raman spectra.

We present the first measurements of two-dimensional resonant-Raman spectra and demonstrate the applicability of the method to the identification of bacteria, including differentiation of genetically similar species. A new device that sequentially illuminates bacteria with different ultraviolet wavelengths and measures a spectrum at each was developed for this purpose. We anticipate that information within such two-dimensional spectra will allow identification of bacteria and chemicals in environments containing multiple organisms and chemicals, leading, for example, to instruments that rapidly identify bacteria in hospital and food plant settings, for screening large populations, and for biochemical-threat warning systems.