Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin.

We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-µL required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.

Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant.

Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.

Rapid, Portable, Multiplexed Detection of Bacterial Pathogens Directly from Clinical Sample Matrices.

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.

MiRNA detection at single-cell resolution using microfluidic LNA flow-FISH.

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. We also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10 µl to >100 µl are concentrated within a sub-50 nl region at the PAM filter edge in the microchannel, thus concentrating them over 1000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0×2.0×0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 µl of lysed blood sample can be analyzed within 60 min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases.

Single cell microRNA analysis using microfluidic flow cytometry.

MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent in situ hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ∼10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.

The O/OREOS mission: first science data from the Space Environment Survivability of Living Organisms (SESLO) payload.

We report the first telemetered spaceflight science results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment, executed by one of the two 10 cm cube-format payloads aboard the 5.5 kg Organism/Organic Exposure to Orbital Stresses (O/OREOS) free-flying nanosatellite. The O/OREOS spacecraft was launched successfully to a 72° inclination, 650 km Earth orbit on 19 November 2010. This satellite provides access to the radiation environment of space in relatively weak regions of Earth's protective magnetosphere as it passes close to the north and south magnetic poles; the total dose rate is about 15 times that in the orbit of the International Space Station. The SESLO experiment measures the long-term survival, germination, and growth responses, including metabolic activity, of Bacillus subtilis spores exposed to the microgravity, ionizing radiation, and heavy-ion bombardment of its high-inclination orbit. Six microwells containing wild-type (168) and six more containing radiation-sensitive mutant (WN1087) strains of dried B. subtilis spores were rehydrated with nutrient medium after 14 days in space to allow the spores to germinate and grow. Similarly, the same distribution of organisms in a different set of microwells was rehydrated with nutrient medium after 97 days in space. The nutrient medium included the redox dye Alamar blue, which changes color in response to cellular metabolic activity. Three-color transmitted intensity measurements of all microwells were telemetered to Earth within days of each of the 48 h growth experiments. We report here on the evaluation and interpretation of these spaceflight data in comparison to delayed-synchronous laboratory ground control experiments.