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.

Multi-focal multiphoton lithography.

Multiphoton lithography (MPL) provides unparalleled capabilities for creating high-resolution, three-dimensional (3D) materials from a broad spectrum of building blocks and with few limitations on geometry, qualities that have been key to the design of chemically, mechanically, and biologically functional microforms. Unfortunately, the reliance of MPL on laser scanning limits the speed at which fabrication can be performed, making it impractical in many instances to produce large-scale, high-resolution objects such as complex micromachines, 3D microfluidics, etc. Previously, others have demonstrated the possibility of using multiple laser foci to simultaneously perform MPL at numerous sites in parallel, but use of a stage-scanning system to specify fabrication coordinates resulted in the production of identical features at each focal position. As a more general solution to the bottleneck problem, we demonstrate here the feasibility for performing multi-focal MPL using a dynamic mask to differentially modulate foci, an approach that enables each fabrication site to create independent (uncorrelated) features within a larger, integrated microform. In this proof-of-concept study, two simultaneously scanned foci produced the expected two-fold decrease in fabrication time, and this approach could be readily extended to many scanning foci by using a more powerful laser. Finally, we show that use of multiple foci in MPL can be exploited to assign heterogeneous properties (such as differential swelling) to micromaterials at distinct positions within a fabrication zone.

Dynamic remodeling of subcellular chemical gradients using a multi-directional flow device.

Elucidation of the mechanisms by which external chemical cues regulate polarized cellular behaviors requires tools that can rapidly recast chemical landscapes with subcellular resolution. Here, we describe an approach for creating steep microscopic gradients of cellular effectors at any desired position in culture that can be reoriented rapidly to evaluate dynamic responses. In this approach, micrometre pores are ablated in a membrane that supports cell adherence, allowing dosing reagent from an underlying reservoir to enter the cell-culture flow chamber as sharp streams that are directed at subcellular targets by using a system of paired sources and drains to specify flow direction. This tool substantially extends capabilities for chemical interaction with cultured cells, enabling investigations of chemotaxis via precise placement and reorientation of peptide gradients formed at the boundaries of dosing streams. These studies demonstrate that neutrophil precursor cells can repolarize and redirect their migration paths using morphological responses that depend on the subcellular localization of chemoattractant gradients.

Microreplication and design of biological architectures using dynamic-mask multiphoton lithography.

A strategy for rapidly printing three-dimensional (3D) microscopic replicas using multiphoton lithography directed by a dynamic electronic mask is reported. Morphological descriptions of 3D structures are encoded as stacks of 2D slices created from tomographic and computer-designed instruction sets. In this manner, digital images serve as input for a sequence of reflective photomasks on a digital micromirror device to direct replication of a structure. By scanning a laser focus across the face of the intrinsically aligned masks, tomographic and computed data can be translated into protein-based 3D reproductions with submicrometer feature sizes within 1 min. This straightforward and highly versatile approach may provide improved routes for the development of 3D cellular scaffolds, rapid prototyping of microanalytical devices, and production of custom tissue replacements.

Parallel chemical dosing of subcellular targets.

To characterize the role of spatially heterogeneous signaling in cellular function, methods are required for differentially exposing distinct regions of individual cells to externally applied reagents. Although a range of standard approaches exists for generating localized chemical gradients in culture, including puffer pipet spritzing and photolytic release of caged effectors, each is limited in key respects. Here, we report development of a cell-dosing strategy that addresses these limitations, providing the means to create steep gradients of any aqueous-miscible compound at essentially unlimited numbers of sites in parallel. In this approach, cells are cultured on a micrometer-thick polymer membrane that serves as a barrier between two stacked laminar-flow channels: one containing the cell culture and the other serving as a reagent flow cell. By focusing a pulsed laser beam onto one or more selected membrane positions, micrometer-diameter pores can be ablated upstream of desired cellular targets. Nascent pores thus serve as ports of entry into the culture environment for reagent streams capable of modifying subcellular features at positions potentially hundreds of micrometers from ablation sites. Importantly, individual reagent streams also can be rapidly eliminated by photo-cross-linking a protein plug over a selected pore. This versatile strategy for dynamically reshaping the chemical microenvironments in which cells reside should be useful in a variety of cell biology applications, ranging from neurotrophic modulation of neurite pathfinding to stimulation of cellular networks.

Direct-write fabrication of functional protein matrixes using a low-cost Q-switched laser.

We report the use of an inexpensive, small, and "turn-key" Q-switched 532-nm Nd:YAG laser as a source for nonlinear, direct-write protein microfabrication. In this approach, microJoule pulses (pulse widths, approximately 600 ps) are focused using high numerical aperture optics to submicrometer focal spots, creating instantaneous intensities great enough to promote multiphoton excitation of a photosensitizer and subsequent intermolecular cross-linking of protein molecules. By scanning the femtoliter focal volume through reagent solution, extended protein-based structures can be fabricated with precise, three-dimensional topographies. As with earlier studies using a femtosecond titanium:sapphire laser costing more than 100K, physically robust and chemically responsive microstructures can be fashioned rapidly with feature sizes smaller than 0.5 microm, and cross-linking can be achieved using both biologically benign sensitizers (e.g., flavins) and by using the proteins themselves to sensitize cross-linking. We demonstrate in situ fabrication to corral neurite outgrowth and show the ability to functionalize avidin structures with biotinylated reagents, an approach that enables chemical sensing to be performed in specified microenvironments. Characterization of this inexpensive, low-power source will greatly broaden access to direct-write protein microfabrication.

Catalytic three-dimensional protein architectures.

We demonstrate a strategy for microfabricating catalytically active, three-dimensional matrixes composed of cross-linked protein in cellular and microfluidic environments. In this approach, a pulsed femtosecond laser is used to excite photosensitizers via multiphoton absorption within three-dimensionally defined volumes, a process that promotes cross-linking of protein residue side chains in the vicinity of the laser focal point. In this manner, it is possible to fabricate protein microparticles with dimensions on the order of the multiphoton focal volume (less than 1 microm(3)) or, by scanning the position of a laser focal point relative to a specimen, to generate surface-adherent matrixes or cables that extend through solution for hundreds of micrometers. We show that protein matrixes can be functionalized either through direct cross-linking of enzymes, by decoration of avidin matrixes with biotinylated enzymes, or by cross-linking biotinylated proteins that then are linked to biotinylated enzymes via an avidin couple. Several formats are explored, including microparticles that can be translocated to desired sites of action (including cytosolic positions), protein pads that generate product gradients within cell cultures, and on-column nanoreactors for microfluidic systems. These biomaterial fabrication technologies offer opportunities for studying a variety of cell functions, ranging from single-cell biochemistry and development to perturbation and analysis of small populations of cultured cells.