World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood.

Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 µL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 µL) and further purification in microscale volumes (5-15 µL) on the DMF device itself. Processing by the system was >2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications.

Quality control of next-generation sequencing library through an integrative digital microfluidic platform.

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/µL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

Integrated fiber optic incoherent broadband cavity enhanced absorption spectroscopy detector for near-IR absorption measurements of nanoliter samples.

An integrated fiber-optic sensor is described that uses incoherent broadband cavity enhanced absorption spectroscopy for sensitive detection of aqueous samples in nanoliter volumes. Absorption was measured in a 100 µm gap between the ends of two short segments of multimode graded-index fiber that were integrated into a capillary using a precision machined V-grooved fixture that allowed for passive fiber alignment. The other ends of the fibers were coated with dielectric mirrors to form a 9.5 cm optical resonator. Light from a fiber-coupled superluminescent diode was directly coupled into one end of the cavity, and transmission was measured using a fiber-coupled silicon photodiode. Dilute aqueous solutions of near infrared dye were used to determine the minimum detectable absorption change of 2.4×10(-4) under experimental conditions in which pressure fluctuations limited performance. We also determined that the absolute minimum detectable absorption change would be 1.6×10(-5) for conditions of constant pressure in which absorption measurement is limited by electronic and optical noise. Tolerance requirements for alignment are also presented.

An ultra-high temperature flow-through capillary device for bacterial spore lysis.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.

Development of an integrated microfluidic instrument for unattended water-monitoring applications.

Field-deployable detection technologies in the nation's water supplies have become a high priority in recent years. The unattended water sensor is presented which employs microfluidic chip-based gel electrophoresis for monitoring proteinaceous analytes in a small integrated sensor platform. The instrument collects samples directly from a domestic water flow. The sample is then processed in an automated microfluidic module using in-house designed fittings, microfluidic pumps and valves prior to analysis via Sandia's microChemLab module, which couples chip-based electrophoresis separations with sensitive LIF detection. The system is controlled using LabVIEW software to analyze water samples about every 12 min. The sample preparation, detection and data analysis has all been fully automated. Pressure transducers and a positive control verify correct operation of the system, remotely. A two-color LIF detector with internal standards allows corrections to migration time to account for ambient temperature changes. The initial unattended water sensor prototype is configured to detect protein biotoxins such as ricin as a first step toward a total bioanalysis capability based on protein profiling. The system has undergone significant testing at two water utilities. The design and optimization of the sample preparation train is presented with results from both laboratory and field testing.

Isotropically etched radial micropore for cell concentration, immobilization, and picodroplet generation.

To enable several on-chip cell handling operations in a fused-silica substrate, small shallow micropores are radially embedded in larger deeper microchannels using an adaptation of single-level isotropic wet etching. By varying the distance between features on the photolithographic mask (mask distance), we can precisely control the overlap between two etch fronts and create a zero-thickness semi-elliptical micropore (e.g. 20 microm wide, 6 microm deep). Geometrical models derived from a hemispherical etch front show that micropore width and depth can be expressed as a function of mask distance and etch depth. These models are experimentally validated at different etch depths (25.03 and 29.78 microm) and for different configurations (point-to-point and point-to-edge). Good reproducibility confirms the validity of this approach to fabricate micropores with a desired size. To illustrate the wide range of cell handling operations enabled by micropores, we present three on-chip functionalities: continuous-flow particle concentration, immobilization of single cells, and picoliter droplet generation. (1) Using pressure differentials, particles are concentrated by removing the carrier fluid successively through a series of 44 shunts terminated by 31 microm wide, 5 microm deep micropores. Theoretical values for the concentration factor determined by a flow circuit model in conjunction with finite volume modeling are experimentally validated. (2) Flowing macrophages are individually trapped in 20 microm wide, 6 microm deep micropores by hydrodynamic confinement. The translocation of transcription factor NF-kappaB into the nucleus upon lipopolysaccharide stimulation is imaged by fluorescence microscopy. (3) Picoliter-sized droplets are generated at a 20 microm wide, 7 microm deep micropore T-junction in an oil stream for the encapsulation of individual E. coli bacteria cells.

Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.

The ability to monitor cell signaling events is crucial to the understanding of immune defense against invading pathogens. Conventional analytical techniques such as flow cytometry, microscopy, and Western blot are powerful tools for signaling studies. Nevertheless, each approach is currently stand-alone and limited by multiple time-consuming and labor-intensive steps. In addition, these techniques do not provide correlated signaling information on total intracellular protein abundance and subcellular protein localization. We report on a novel phosphoFlow Chip (pFC) that relies on monolithic microfluidic technology to rapidly conduct signaling studies. The pFC platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. pFC allows host-pathogen phosphoprofiling in 30 min with an order of magnitude reduction in the consumption of reagents. For pFC validation, we monitor the mitogen-activated protein kinases ERK1/2 and p38 in response to Escherichia coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7). pFC permits ERK1/2 phosphorylation monitoring starting at 5 s after LPS stimulation, with phosphorylation observed at 5 min. In addition, ERK1/2 phosphorylation is correlated with subsequent recruitment into the nucleus, as observed from fluorescence microscopy performed on cells upstream of flow cytometric analysis. The fully integrated cell handling has the added advantage of reduced cell aggregation and cell loss, with no detectable cell activation. The pFC approach is a step toward unified, automated infrastructure for high-throughput systems biology.

An integrated microfluidic platform for sensitive and rapid detection of biological toxins.

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of approximately 10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody-analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is <10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.

Magnetic bead based immunoassay for autonomous detection of toxins.

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

An embedded microretroreflector-based microfluidic immunoassay platform.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.