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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 ADDS reporting a false positive is extremely low.

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.

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 pre-ablation 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. OCIS codes: 140.3440, 300.2140, 300.6210.

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. OCIS codes: 140.3440, 300.2140, 300.6210.

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 inter-pulse 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. OCIS code: 140.3440.

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.

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.

Optical coherence tomography (OCT) may be very useful for imaging teeth. This technique is similar to CT imaging except that it uses light rays instead of X-rays. It creates a two-dimensional image of a structure without all the surrounding structures to distort the view. One advantage of OCT over dental X-rays is that OCT can image soft tissues, which most X-rays cannot do.

We introduce a novel method for determining analyte concentration as a function of depth in a highly scattering media by use of a dual-wavelength optical coherence tomography system. We account for the effect of scattering on the measured attenuation by using a second wavelength that is not absorbed by the sample. We assess the applicability of this technique by measuring the concentration of water in an Intralipid phantom, using a probe wavelength of 1.53 [[micro]meter] and a reference wavelength of 1.31 [[micro]meter]. The results of our study show a strong correlation between the measured absorption and the water content of the sample. The accuracy of the technique, however, was limited by the dominance of scattering over absorption in the turbid media. Thus, although the effects of scattering were minimized, significant errors remained in the calculated absorption values. More-accurate results could be obtained with the use of more powerful superluminscent diodes and a choice of wavelengths at which absorption effects are more significant relative to scattering.

We have developed a prototype optical coherence tomography (OCT) system for the imaging of hard and soft tissue in the oral cavity. High-resolution images of in vitro porcine periodontal tissues have been obtained with this system. The images clearly show the enamel-cementum and the gingiva-tooth interfaces, indicating OCT is a potentially useful technique for diagnosis of periodontal diseases. To our knowledge, this is the first application of OCT for imaging biologic hard tissue.

We demonstrate cross-sectional birefringence- and polarization-independent backscatter imaging of laser-induced thermal damage in porcine myocardium in vitro, using a polarization-sensitive optical coherence tomography system. We compare the generated images with histological sections of the tissue and demonstrate that birefringence is a more sensitive indicator of thermal damage than is backscattered light. Loss of birefringence in thermally damaged regions is quantified and shown to have significant contrast with undamaged sections of the tissue. A detailed theoretical analysis of the birefringence measurements is provided, including a calculation of the systematic errors associated with background noise, system imperfections, and tissue dichroism.

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.

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.

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.

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., (Copyright 2004 S. Karger AG, Basel)

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.