Anthony J. Makarewicz

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Environmental Monitoring for Biological Threat Agents Using the Autonomous Pathogen Detection System with Multiplexed Polymerase Chain Reaction

We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instruments response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.

Performance Characteristics of the Aerosol Collectors of the Autonomous Pathogen Detection System (APDS)

This research analyzes the physical performance characteristics of the aerosol collectors of the autonomous pathogen detection system (APDS) that was recently developed by the Lawrence Livermore National Laboratory. The APDS is capable of continuous and fully autonomous monitoring for multiple airborne threat organisms and can be used as part of a monitoring network for urban areas and major public gatherings. The system has already been successfully tested with airborne Bacillus anthracis and Yersinia pestis biowarfare agents. The APDS aerosol collection system consists of a PM-style cap to remove large particles and a low-pressure drop virtual impactor preconcentrator positioned in front of a wetted-wall cyclone. The aerosol collectors operate at flow rates as high as 3750 l/min and collect airborne particles into 4 ml of liquid for subsequent detection. In our tests we determined the overall collection efficiency of the system by measuring the difference between inlet and outlet particle concentrations. The tests were performed with polydisperse oleic acid and monodisperse polystyrene latex (PSL) particles (0.6–3.1 µ m), and for three values of the major air flow rates in the virtual impactor (1760, 2530, and 3300 l/min), two values of the product, or cyclone, flow rates (375 and 450 l/min), and two different volumes of collection liquid (4 and 6 ml). We found that the cutoff size (d50 ) of the entire collection system varied from 1.5 to 2.0 µ m when collecting PSL particles, with 3.1 µ m PSL particles being collected with efficiency of approximately 85%. When collecting oleic acid particles the d50 of the entire system varied from 1.1 to 1.6 µ m. The concentration rates of the aerosol collection system were found to increase with increasing overall collection flow rate and approached one million per minute at the highest tested flowrates. Such high concentrating rates and high air sample volumes make the APDS collection system highly suitable for detecting low concentrations of airborne pathogens.

Hydrodynamic modeling of tissue ablation with a free-electron laser

The Vanderbilt University free-electron laser (FEL) provides a continuously tunable ((lambda) equals 2 - 10 micrometer) source of pulsed IR radiation with a pulse structure unlike those of conventional lasers (a macropulse of 5 microseconds consisting of a train of 1 ps micropulses at a frequency of 3 GHz). A numerical hydrodynamic code at Lawrence Livermore National Laboratory, known as LASTIS3D, was used to model the ablation of tissue using the FEL. This study investigates the role of the FEL pulse structure by comparing the results from simulations using a time-averaged energy deposition and a pulsetrain energy deposition.

Apds, a Network-Ready, Broad Spectrum, Environmental Pathogen Detection System

The Autonomous Pathogen Detection System (APDS) 1 is a stand-alone pathogen detection system capable of rapid, continuous, low cost environmental monitoring of multiple airborne biological threat agents. Its basic design comprises aerosol sampling, in-line sample preparation, multiplex detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. Its primary application is to warn civilians and emergency preparedness personnel of a terrorist attack, the same system could also have a role in protecting military personnel from biological warfare attacks. APDS instruments can be used at high profile events such as the Olympics for short-term, intensive monitoring or more permanent installation in major public buildings or transportation nodes. All of these units can be networked to a single command center so that a small group of technical experts could maintain and respond to alarms at any of the sensors. The APDS has several key advantages over competing technologies: (1) the ability to measure up to 100 different agents and controls in a single sample, (2) the flexibility and ease with which new bead-based assays can be developed and integrated into the system, (3) the presence of an orthogonal, real-time detection module for highly sensitive and selective nucleic acid amplification and detection, (4) the ability to use the same basic system components for multiple deployment architectures, and (5) the relatively low cost per assay (<

Modeling optically generated focused stress waves

2 per 10-plex or

Droplet-based assay system

0.20 per assay) and minimal consumables.

Autonomous detection of aerosolized bacillus anthracis and Yersinia pestis

Stress waves generated at the end of optical fibers through thermoelastic expansion have been used for several purposes, including the destruction of blood clots, the destruction of kidney and gallstones, and the induction of cell permeability changes for drug delivery. We have undertaken the modeling of stress wave propagation in an effort to optimize the stress wave effects in these therapies. In particular, we have studied techniques to focus the stress wave in order to increase the pressure magnitude for a given pulse energy and to alter the compressive/tensile pressure ratio. This study includes the effects of acoustic wavelength and focusing fiber tip shape.