Cy R. Tamanaha

The BARC biosensor applied to the detection of biological warfare agents.

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.

A DNA array sensor utilizing magnetic microbeads and magnetoelectronic detection

We describe a multi-analyte biosensor that uses magnetic microbeads as labels to detect DNA hybridization on a micro-fabricated chip. The beads are detected by giant magnetoresistance (GMR) magnetoelectronic sensors embedded in the chip. The prototype device is a tabletop unit containing electronics, a chip carrier with a microfluidic flow cell, and a compact electromagnet and is capable of simultaneous detection of eight different analytes.

Magnetic labeling, detection, and system integration

Among the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection is emerging as a promising new approach. Magnetic labels can be non-invasively detected by a wide range of methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Here we provide an overview of the various approaches developed for magnetic labeling and detection as applied to biosensing. We illustrate the challenges to integrating one such approach into a complete sensing system with a more detailed discussion of the compact Bead Array Sensor System developed at the U.S. Naval Research Laboratory, the first system to use magnetic labels and microchip-based detection.

Real-Time DNA Detection Using Reduced Graphene Oxide Field Effect Transistors

www.MaterialsViews.com C O M M Real-Time DNA Detection Using Reduced Graphene Oxide Field Effect Transistors U N IC A By Rory Stine , Jeremy T. Robinson , Paul E. Sheehan , and Cy R. Tamanaha * IO N Rapidly detecting and identifying biomolecules in solution is a pressing need in areas as diverse as medical diagnostics, food safety, and national defense. While traditional laboratory techniques using secondary labels such as fl uorophores or enzymes offer the greatest sensitivity, they cannot monitor in real time probe/target interactions and increase signifi cantly the cost and complexity of screening for biological agents in the fi eld. As such, numerous studies have been undertaken to develop labelfree sensors that directly detect the binding of a target. Multiple transduction approaches have been examined. Surface plasmon resonance (SPR) has long been considered the gold-standard for label-free biological detection, [ 1 ] and has achieved sensitivities in the low nanomolar range [ 2 , 3 ] for direct, non-amplifi ed DNA detection. Recently, sensors based on silicon nanowire [ 4 , 5 ] and carbon nanotube [ 6 ] fi eld effect transistors (FETs) have shown higher sensitivities. These transistors, which are gated by the adsorption of charged biomolecules, have shown DNA detection limits from low n M [ 7 , 8 ] to high p M [ 9 , 10 ] in solutions at or near physiological salt concentrations. Detection limits as low as f M [ 11 , 12 ] have also been shown but only for salt-free solutions that decrease the Debye screening. [ 13 ]

Chemically isolated graphene nanoribbons reversibly formed in fluorographene using polymer nanowire masks

We demonstrated the fabrication of graphene nanoribbons (GNRs) as narrow as 35 nm created using scanning probe lithography to deposit a polymer mask(1-3) and then fluorinating the sample to isolate the masked graphene from the surrounding wide band gap fluorographene. The polymer protected the GNR from atmospheric adsorbates while the adjacent fluorographene stably p-doped the GNRs which had electron mobilities of ∼2700 cm2/(V·s). Chemical isolation of the GNR enabled resetting the device to nearly pristine graphene.

Aminated graphene for DNA attachment produced via plasma functionalization

We demonstrate the use of a unique plasma source to controllably functionalize graphene with nitrogen and primary amines, thereby tuning the chemical, structural, and electrical properties. Critically, even highly aminated graphene remains electronically conductive, making it an ideal transduction material for biosensing. Proof-of-concept testing of aminated graphene as a bio-attachment platform in a biologically active field-effect transistor used for DNA detection is demonstrated.

A simple pen-spotting method for arraying biomolecules on solid substrates.

We describe a simple, relatively inexpensive method for depositing biomolecules on a solid substrate using Rapidograph drafting pens. The pens can be used without modification to accurately deposit spots between approximately 100 and 600 microm in diameter. When mounted on a suitable microtranslation stage, the pens can be used to easily deposit tens of spots aligned with underlying substrate features such as microfabricated sensors. The pens are particularly convenient because pre-mixed solutions can be stored in the pens for multiple uses. We demonstrate the use of this approach to deposit DNA probes on a microsensor array.

Direct detection of genomic DNA with fluidic force discrimination assays.

Herein, we describe the direct detection of genomic DNA using fluidic force discrimination (FFD) assays. Starting with extracted bacterial DNA, samples are fragmented by restriction enzymes or sonication, then thermocycled in the presence of blocking and labeling sequences, and finally detected with microbead-based FFD assays. Both strain and species identification of extracted Bacillus DNA have been demonstrated in <30 min, without amplification (e.g., PCR). Femtomolar assays can be achieved with this rapid and simple procedure.

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.

Trace explosives sensor testbed (TESTbed)

A novel vapor delivery testbed, referred to as the Trace Explosives Sensor Testbed, or TESTbed, is demonstrated that is amenable to both high- and low-volatility explosives vapors including nitromethane, nitroglycerine, ethylene glycol dinitrate, triacetone triperoxide, 2,4,6-trinitrotoluene, pentaerythritol tetranitrate, and hexahydro-1,3,5-trinitro-1,3,5-triazine. The TESTbed incorporates a six-port dual-line manifold system allowing for rapid actuation between a dedicated clean air source and a trace explosives vapor source. Explosives and explosives-related vapors can be sourced through a number of means including gas cylinders, permeation tube ovens, dynamic headspace chambers, and a Pneumatically Modulated Liquid Delivery System coupled to a perfluoroalkoxy total-consumption microflow nebulizer. Key features of the TESTbed include continuous and pulseless control of trace vapor concentrations with wide dynamic range of concentration generation, six sampling ports with reproducible vapor profile outputs, limited low-volatility explosives adsorption to the manifold surface, temperature and humidity control of the vapor stream, and a graphical user interface for system operation and testing protocol implementation.