A point-of-care diagnostic for differentiating Ebola from endemic febrile diseases.

Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.

Surface-enhanced Raman scattering (SERS) cytometry.

Significant advances have been made in the preparation and applications of surface-enhanced Raman scattering (SERS)-active materials for biomolecular analysis. Bright signals, photostability, and narrow spectral features of SERS-active materials offer attractive advantages for cytometric analyses. However, SERS cytometry is still in an early stage of development, and advances in both instrumentation and reagents will be necessary to realize its full potential. In this chapter, we discuss the challenges of expanding the numbers of fluorescent labels that can be measured in cytometry, and introduce SERS tags with extremely narrow spectral peaks as an approach to make more efficient use of the optical spectrum and increase the number of parameters in cytometry.

Leveraging nanoscale plasmonic modes to achieve reproducible enhancement of light.

The strongly enhanced and localized optical fields that occur within the gaps between metallic nanostructures can be leveraged for a wide range of functionality in nanophotonic and optical metamaterial applications. Here, we introduce a means of precise control over these nanoscale gaps through the application of a molecular spacer layer that is self-assembled onto a gold film, upon which gold nanoparticles (NPs) are deposited electrostatically. Simulations using a three-dimensional finite element model and measurements from single NPs confirm that the gaps formed by this process, between the NP and the gold film, are highly reproducible transducers of surface-enhanced resonant Raman scattering. With a spacer layer of roughly 1.6 nm, all NPs exhibit a strong Raman signal that decays rapidly as the spacer layer is increased.

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

High throughput single nanoparticle spectroscopy.

Progress in the development and application of nanoengineered systems is limited by the availability of quantitative measurement techniques. For the engineering of nanoparticle (NP)-based systems, single NP characterization is essential, but existing methods are slow and low throughput. We demonstrate a flow spectroscopy technique capable of analyzing hundreds of nanoparticles per second and use this technique for the high throughput analysis of nanoparticle surface-enhanced resonant Raman scattering (SERRS) tags. By measuring Rayleigh and Raman scattering from thousands of individual tags, tag preparations can be characterized based on their brightness and uniformity. The rapid analysis of individual nanoparticles using high spectral resolution flow spectroscopy will be useful in many areas of nanoengineering.

Reconfigurable core-satellite nanoassemblies as molecularly-driven plasmonic switches.

Molecular control of plasmon coupling is investigated in sub-100 nm assemblies composed of 13 nm gold "satellite" particles tethered by reconfigurable DNA nanostructures to a 50 nm gold "core" particle. Reconfiguration of the DNA nanostructures from a compact to an extended state results in blue shifting of the assembly plasmon resonance, indicating reduced interparticle coupling and lengthening of the core-satellite tether. Scattering spectra of the core-satellite assemblies before and after reconfiguration are compared with spectra calculated using a structural model that incorporates the core/satellite ratio determined by TEM imaging and estimates of tether length based upon prior measurements of interparticle separation in DNA linked nanoparticle networks. A strong correspondence between measured and simulated difference spectra validates the structural models that link the observed plasmon modulation with DNA nanostructure reconfiguration.