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.

Multiplex primer prediction software for divergent targets.

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ( approximately 3700 18-mers or approximately 2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus.

On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets.

The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused-silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment and will be useful in viral discovery and gene-profiling applications.

Development of an automated DNA purification module using a micro-fabricated pillar chip.

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 x 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 microm, respectively, which provides a relatively large surface area (ca. 3 cm(2)) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

High-throughput quantitative polymerase chain reaction in picoliter droplets.

Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/microL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.

On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets.

The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 106 smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet motion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of approximately 18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

Fabricating optical fiber imaging sensors using ink jet printing technology: a pH sensor proof-of-concept.

We demonstrate the feasibility of using Drop-on-Demand microjet printing technology for fabricating imaging sensors by reproducibly printing an array of photo-polymerizable sensing elements, containing a pH sensitive indicator, on the surface of an optical fiber image guide. The reproducibility of the microjet printing process is excellent for microdot (i.e. micrometer-sized polymer) sensor diameter (92.2+/-2.2 microm), height (35.0+/-1.0 microm), and roundness (0.00072+/-0.00023). pH sensors were evaluated in terms of pH sensing ability (< or =2% sensor variation), response time, and hysteresis using a custom fluorescence imaging system. In addition, the microjet technique has distinct advantages over other fabrication methods, which are discussed in detail.

Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation.

The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.

Temporal dependence of the enhancement of material removal in femtosecond-nanosecond dual-pulse laser-induced breakdown spectroscopy.

Despite the large neutral atomic and ionic emission enhancements that have been noted in collinear and orthogonal dual-pulse laser-induced breakdown spectroscopy, the source or sources of these significant signal and signal-to-noise ratio improvements have yet to be explained. In the research reported herein, the combination of a femtosecond preablative air spark and a nanosecond ablative pulse yields eightfold and tenfold material removal improvement for brass and aluminum, respectively, but neutral atomic emission is enhanced by only a factor of 3-4. Additionally, temporal correlation between enhancement of material removal and of atomic emission is quite poor, suggesting that the atomic-emission enhancements noted in the femtosecond-nanosecond pulse configuration result in large part from some source other than simple improvement in material removal.

Spatial and temporal dependence of interspark interactions in femtosecond-nanosecond dual-pulse laser-induced breakdown spectroscopy.

A femtosecond air spark has recently been combined with a nanosecond ablative pulse in order to map the spatial and temporal interactions of the two plasmas in femtosecond-nanosecond orthogonal preablation spark dual-pulse laser-induced breakdown spectroscopy (LIBS). Good spatial and temporal correlation was found for reduced atomic emission from atmospheric species (nitrogen and oxygen) and increased atomic emission from ablated species (copper and aluminum) in the femtosecond-nanosecond plasma, suggesting a potential role for atmospheric pressure or nitrogen/oxygen concentration reduction following air spark formation in generating atomic emission enhancements in dual-pulse LIBS.

A non-lensed fiber-optic resonance-enhanced multiphoton ionization probe.

We have developed a miniature fiber-optic probe with no focusing optics for in situ analysis of volatile organic compounds (VOCs). The probe uses an optical fiber to transmit a laser pulse to a vapor sample causing it to ionize adjacent to the fiber tip through a resonance-enhanced multiphoton ionization (REMPI) process. The distal end of the optical fiber is contained co-axially within 2-mm-inner-diameter stainless steel tubing that serves as an electrode. The electrode is biased at a high positive potential to collect electrons. The current generated is shown to be proportional over about two orders of magnitude to the concentration of the species ionized. Visible wavelength REMPI spectroscopy is used to determine probe sensitivities of 20 ppb (benzene) and 43 ppb (toluene). Designing the probe without focusing optics specifies an achromatic ionization region constant in size and position as the laser wavelength is scanned, which simplifies data collection and reduction. Focusing achromatic systems are discussed and the potential signal improvement is estimated.

Effects of sample temperature in femtosecond single-pulse laser-induced breakdown spectroscopy.

As much as tenfold atomic emission enhancements have been observed in experiments combining nanosecond (ns) and femtosecond (fs) laser pulses in an orthogonal dual-pulse configuration for laser-induced breakdown spectroscopy (ns-fs orthogonal dual-pulse LIBS). In the examination of one of several potential sources of these atomic emission enhancements (sample heating by a ns air spark), minor reductions in atomic emission and as much as 15-fold improvements in mass removal have been observed for fs single-pulse LIBS of heated brass and aluminum samples. These results suggest that, although material removal with a high-powered, ultrashort fs pulse is temperature dependent, sample heating by the ns air spark is not the source of the atomic emission enhancements observed in ns-fs orthogonal dual-pulse LIBS.

Serum protein profile alterations in hemodialysis patients.

BACKGROUND: Serum protein profiling patterns can reflect the pathological state of a patient and therefore may be useful for clinical diagnostics. Here, we present results from a pilot study of proteomic expression patterns in hemodialysis patients designed to evaluate the range of serum proteomic alterations in this population. METHODS: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to analyze serum obtained from patients on periodic hemodialysis treatment and healthy controls. Serum samples from patients and controls were first fractionated into six eluants on a strong anion exchange column, followed by application to four array chemistries representing cation exchange, anion exchange, metal affinity and hydrophobic surfaces. A total of 144 SELDI-TOF-MS spectra were obtained from each serum sample. RESULTS: The overall profiles of the patient and control samples were consistent and reproducible. However, 30 well-defined protein differences were observed; 15 proteins were elevated and 15 were decreased in patients compared to controls. Serum from 1 patient exhibited novel protein peaks suggesting possible additional changes due to a secondary disease process. CONCLUSION: SELDI-TOF-MS demonstrated consistent serum protein profile differences between patients and controls. Similarity in protein profiles among dialysis patients suggests that patient physiological responses to end-stage renal disease and/or dialysis therapy have a major effect on serum protein profiles.

Dual-pulse laser-induced breakdown spectroscopy with combinations of femtosecond and nanosecond laser pulses.

Nanosecond and femtosecond laser pulses were combined in an orthogonal preablation spark dual-pulse laser-induced breakdown spectroscopy (LIBS) configuration. Even without full optimization of interpulse alignment, ablation focus, large signal, signal-to-noise ratio, and signal-to-background ratio enhancements were observed for both copper and aluminum targets. Despite the preliminary nature of this study, these results have significant implications in the attempt to explain the sources of dual-pulse LIBS enhancements.

A reusable flow-through polymerase chain reaction instrument for the continuous monitoring of infectious biological agents.

Continuous monitoring of the environment for infectious diseases and related biowarfare agents requires the implementation of practical cost-effective methodologies that are highly sensitive and specific. One compatible method employed in clinical diagnostics is real-time polymerase chain reaction (PCR) analysis. The utility of this technique for environmental monitoring is limited, however, by the utilization of single-use consumables in commercial PCR instruments. This greatly increases mechanical complexity, because sophisticated robotic mechanisms must replenish the disposable elements. An alternative strategy develops an autonomous monitoring system consisting of reusable modules that readily interface with fluidic circuitry in a flow-through scheme. The reduced complexity should increase reliability while decreasing operating costs. In this report, we describe a reusable, flow-through PCR module that functions as one component in such a system. This module was rigorously evaluated with Bacillus anthracis genomic DNA and demonstrated high repeatability, sensitivity, and efficiency, with no evidence of sample-to-sample carryover.

Biothreat agent monitoring using a flow-through polymerase chain reaction instrument.

Extract: The 2001 anthrax letter mailings highlighted critical shortfalls in the USA's capabilities for dealing with the threats of bioterrorism: the lack of effective, reliable, low-cost detection systems for use by state and local authorities. During the few months after the letters were received, 17,000 false alarms and hoaxes were reported, and Americans everywhere were terrified to open their mail. Over 200,000 samples were processed by the Center for Disease Control (CDC) and others. In a scenario where the attack was more wide spread, the laboratory processing requirements could be far greater. In light of such statistics, the imperative for high confidence, high throughput, and inexpensive diagnostics is clear. In answer to this technical challenge, nucleic acid-based methodologies are being standardized and documented to provide acceptable mechanisms of detection. Real time PCR (polymerase chain reaction), in particular, allows analysis of aerosolized or environmental samples to occur within minutes, enabling biological defense response architectures that would otherwise be impossible. PCR is a revolutionary technique for amplifying targeted sequences of DNA. The use of a thermostable enzyme (polymerase) allows sequential dissociation, annealing, and hybridization of complimentary DNA to occur very rapidly. A pair of DNA primers, about 20 nucleotides in length, is used to uniquely identify and amplify (over a million fold) the target DNA sequence.

Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.