Shawn P. Mulvaney

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.

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.

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.

Fluidic Force Discrimination Assays: A New Technology for Tetrodotoxin Detection

Tetrodotoxin (TTX) is a low molecular weight (~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 ~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.

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.

Biosensors: Magnets tackle kinetic questions

Interactions between biomolecules can be probed with the help of technology that was developed for reading data stored on magnetic disk drives.

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.

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 natures 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 natures tools in innovative ways.

Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

POINT-OF-NEED DIAGNOSTICS: BIOSURVEILLANCE WITH A DEVICE2CLOUD CAPABILITY IN SIERRA LEONE

Background Infectious diseases contribute to a high burden of diseases globally. Surveillance using low-cost technology, combined with cutting edge platforms offers a path for understanding the disease ecology of locations of interest in resource-poor countries. Our goal was to pilot a bio surveillance system comprising of rapid lateral flow immunoassays, rapid PCR and a cloud database. Methods The study was carried out in Bo, Sierra Leone at the Mercy Hospital. We recruited 1570 subjects over a period of two years. Inclusion criteria for the study were being febrile, being at least five years of age, living within the city of Bo or its neighbouring villages and agreeing to participate in the study. The assays used included a DPP multiplex lateral flow assay for dengue, Burkholderia pseudomallei, Yersinia pestis, malaria Pf/Pan, a Film Array PCR platform with multiplex Biothreat and SASFI panels that together detect over 30 pathogen targets. We used a Deki Reader to upload lateral flow images to the cloud database. The Deki reader quantitates test results, such that scores at ≥1.75 are considered positive. A special computer program was designed to upload pdf images of PCR results to the cloud database. The cloud database was designed for automated quality assessment and remote monitoring. Results Preliminary results show that out of 1570 samples processed by DPP, 30(1.9%) were positive for Burkholderia, 41(2.6%) were positive for Dengue NS1 antigen, 22(1.4%) were positive for Yesinia pestis fraction 1 antigen, and 340(21.7%) were positive for malaria. When a cross-section of results obtained by eye was compared with results automatically detected by the D2C platform, there was 95.2% concordance between results obtained by eye and those obtained automatically by the Deki reader to the cloud database. Conclusions Active disease surveillance and the ability to remotely monitor activities in peripheral health units are critical needs in many poor countries. Our results provide additional perspectives on the twin problem of surveillance and remote quality assessment.