Ronald F. Renzi

Automated Digital Microfluidic Sample Preparation for Next-Generation DNA Sequencing

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80% ± 4.8%.

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.

A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

A laser-based technique to continuously monitor metal aerosol emissions

Abstract We are developing an instrument to continuously monitor metal aerosol emissions in applications including industrial process vents (e.g., exhaust stacks from electroplating baths), waste treatment processes (incinerators), and boilers and industrial furnaces (coal-fired power plants). The monitoring technique is based on laser spark spectroscopy (LASS; also known as laser-induced breakdown spectroscopy - LIBS), in which a pulsed laser is used to rapidly heat a particle and produce a plasma (or laser ‘spark’). The light emission from the spark is spectrally resolved and analyzed to identify the elemental constituents of the particle and quantify the abundance of the measured species. One feature of LASS is that it can measure atomic species embedded in either solid particles or fine liquid droplets, which account for a large percentage of metal emissions from applications of interest. In the initial work described here, we have focused on the application of the LASS technique for measuring chromium emissions from electroplating baths. This paper describes the approach used for measuring the total chromium concentration in laboratory simulations of electroplating aerosols. Chromium concentrations less than 1 mg/scm can be measured. This work forms the basis for future applications to incineration and fossil power plants.

Microfluidic routing of aqueous and organic flows at high pressures: fabrication and characterization of integrated polymer microvalve elements

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.

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.

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.

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.

Size and Concentration Measurements of Particles Produced in Commercial Chromium Plating Processes

Abstract Optical measurements of particle size and concentration were made at the chromium plating tank and exhaust system at a commercial hexavalent chromium plating facility. Particles were examined at three locations in the exhaust system: 1) directly at the hexavalent chromium plating bath surface, 2) at the exit of a cyclone separator located in the exhaust system approximately three to four meters downstream of the bath, and 3) in the exhaust stack, downstream of the induced draft fan and all abatement devices. Particle diameters at the bath surface ranged from 0.3 to 25 μm. Downstream of the cyclone exit and mesh pad filters, particle top sizes were approximately 5 and 0.7 mm, respectively. On a mass basis, the collection efficiency of all abatement devices was 99.997%. Assuming that droplets in the flow consist primarily of water and chromium, correcting the total particle mass flow against water content gives a chromium emission rate of 64,000 μg/hr, which compares favorably with a value of 77,00...

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

Field‐deployable detection technologies in the nations 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 Sandias μChemLab™ 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.