3D Conducting Polymeric Membrane and Scaffold Saccharomyces cerevisiae Biofilms to Enhance Energy Conversion in Microbial Fuel Cells.

Microbial fuel cells (MFCs) can spontaneously convert chemical energy into electricity using biocatalytic microorganisms and organic matter as fuel feedstocks. Three-dimensional cross-linked poly(vinyl alcohol)-based membranes were produced by a sol-gel method under homogeneous catalysis and used as the electrolyte to facilitate effective proton conduction. Under dry conditions, these polymeric membranes showed high water uptake (120%) and ionic conductivity (2.815 mS cm-1). In the anode compartment, the scaffold Saccharomyces cerevisiae film biocatalysts were used to improve electron transfer to the cathode, using three major configurations to generate a higher power output. It was found that the graphene anchoring, red light (RL) stimulation, and methylene blue (MB) mediation-enhanced device performance. The electrochemically derived graphene improved the power and current density by 40% because of its high conductivity. The RL stimulation increased the power density by 80% because of a shortened electron flow path to complex III. The MB mediation also yielded a higher current density by 340% because MB can bypass the electron flow from complex II to cytochrome c and transfer electrons directly to complex III. The individual and collective increase in power output was due to more efficient electron flow from the electronic network permeating the biofilm. The generated electrons were transferred either to graphene as an energy-efficient direct transfer mode or to methylene blue as a long-range redox mediator for indirect transfer. Red light stimulation enhanced oxygen utilization efficiency and stimulated electrons in redox proteins enhancing electron flux. These processes generated higher power through the more efficient generation of electrons and faster transport to the external circuit. As society migrates from gasoline consumption to low carbon-based fuels, the MFCs become important in producing electrical energy with low net emissions.

Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth.

Two-photon lithography allows writing of arbitrary nanoarchitectures in photopolymers. This design flexibility opens almost limitless possibilities for biological studies, but the acrylate-based polymers frequently used do not allow for adhesion and growth of some types of cells. Indeed, we found that lithographically defined structures made from OrmoComp do not support E18 murine cortical neurons. We reacted OrmoComp structures with several diamines, thereby rendering the surfaces directly permissive for neuron attachment and growth by presenting a surface coating similar to the traditional cell biology coating achieved with poly-d-lysine (PDL) and laminin. However, in contrast to PDL-laminin coatings that cover the entire surface, the amine-terminated OrmoComp structures are orthogonally modified in deference to the surrounding glass or plastic substrate, adding yet another design element for advanced biological studies.

Nature inspires sensors to do more with less.

The world is filled with widely varying chemical, physical, and biological stimuli. Over millennia, organisms have refined their senses to cope with these diverse stimuli, becoming virtuosos in differentiating closely related antigens, handling extremes in concentration, resetting the spent sensing mechanisms, and processing the multiple data streams being generated. Nature successfully deals with both repeating and new stimuli, demonstrating great adaptability when confronted with the latter. Interestingly, nature accomplishes these feats using a fairly simple toolbox. The sensors community continues to draw inspiration from nature's example: just look at the antibodies used as biosensor capture agents or the neural networks that process multivariate data streams. Indeed, many successful sensors have been built by simply mimicking natural systems. However, some of the most exciting breakthroughs occur when the community moves beyond mimicking nature and learns to use nature's tools in innovative ways.

Graphene veils: A versatile surface chemistry for sensors.

Thin spun-coat films (~4 nm thick) of graphene oxide (GO) constitute a versatile surface chemistry compatible with a broad range of technologically important sensor materials. Countless publications are dedicated to the nuances of surface chemistries that have been developed for sensors, with almost every material having unique characteristics. There would be enormous value in a surface chemistry that could be applied generally with functionalization and passivation already optimized regardless of the sensor material it covered. Such a film would need to be thin, conformal, and allow for multiple routes toward covalent linkages. It is also vital that the film permit the underlying sensor to transduce. Here we show that GO films can be applied over a diverse set of sensor surfaces, can link biomolecules through multiple reaction pathways, and can support cell growth. Application of a graphene veil atop a magnetic sensor array is demonstrated with an immunoassay. We also present biosensing and material characterization data for these graphene veils.

A new methodology for quantitative LSPR biosensing and imaging.

A new quantitative analysis methodology for localized surface plasmon resonance (LSPR) biosensing which determines surface-receptor fractional occupancy, as well as an LSPR imaging technique for the spatiotemporal mapping of binding events, is presented. Electron beam nanolithography was used to fabricate 20 × 20 arrays of gold nanostructures atop glass coverslips. A single biotinylated array was used to measure the association kinetics of neutravidin to the surface by spectroscopically determining the fractional occupancy as a function of time. By regenerating the same array, a reliable comparison of the kinetics could be made between control samples and neutravidin concentrations ranging from 1 µM to 50 nM. CCD-based imagery of the array, taken simultaneously with the spectroscopic measurements, reveals the binding of neutravidin to the surface as manifested by enhanced scattering over the majority of the resonance peak. The temporal resolution of the LSPR imaging technique was 200 ms and the spatial resolution was 8 µm(2).

High-quality uniform dry transfer of graphene to polymers.

In this paper we demonstrate high-quality, uniform dry transfer of graphene grown by chemical vapor deposition on copper foil to polystyrene. The dry transfer exploits an azide linker molecule to establish a covalent bond to graphene and to generate greater graphene-polymer adhesion compared to that of the graphene-metal foil. Thus, this transfer approach provides a novel alternative route for graphene transfer, which allows for the metal foils to be reused.

Fluidic force discrimination assays: a new technology for tetrodotoxin detection.

Tetrodotoxin (TTX) is a low molecular weight (approximately 319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of approximately 15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.

Evolution of a magnetic-based biomolecular detection system.

Amongst the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection has emerged as a promising approach. Magnetic labels can be detected by a wide range of non-invasive methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Over a decade ago, the U. S. Naval Research Laboratory pioneered the use of giant magnetoresistive (GMR) sensors to detect biomolecules labeled with paramagnetic microbeads. Since then, our various investigations and engineering efforts have resulted in significant improvements in both the magnetoelectronic instrumentation and the assays associated with these magnetic labels. This paper and subsequent presentation provides a synopsis of the development of our technology which has evolved into a highly sensitive detection method.

Reusable, compression-sealed fluid cells for surface mounting to planar substrates.

We have developed a universal structure and mechanism for the repeatable, rapid-attachment of a fluid cell to a planar substrate. The fluid cell and all fluidic connections are completely contained in a plastic body such that attachment requires neither adhesives nor modification of the substrate. The geometry of the fluid cell is defined by the active area of the planar substrate (e.g. a sensor array). All required components have been quickly prototyped using Computer Numerical Control (CNC) machining. It is also straight-forward to create an array of fluid cells to attach to a single substrate (e.g. a standard microscope slide). All components are easy to assemble and can be cleaned and reused, making this flexible approach applicable for a wide range of lab-on-a-chip applications.

Formation of primary amines on silicon nitride surfaces: a direct, plasma-based pathway to functionalization.

Silicon nitride is the most commonly used passivation layer in biosensor applications where electronic components must be interfaced with ionic solutions. Unfortunately, the predominant method for functionalizing silicon nitride surfaces, silane chemistry, suffers from a lack of reproducibility. As an alternative, we have developed a silane-free pathway that allows for the direct functionalization of silicon nitride through the creation of primary amines formed by exposure to a radio frequency glow discharge plasma fed with humidified air. The aminated surfaces can then be further functionalized by a variety of methods; here we demonstrate using glutaraldehyde as a bifunctional linker to attach a robust NeutrAvidin (NA) protein layer. Optimal amine formation, based on plasma exposure time, was determined by labeling treated surfaces with an amine-specific fluorinated probe and characterizing the coverage using X-ray photoelectron spectroscopy (XPS). XPS and radiolabeling studies also reveal that plasma-modified surfaces, as compared with silane-modified surfaces, result in similar NA surface coverage, but notably better reproducibility.

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays.

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.