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Concerns about the use of anthrax spores as a weapon of mass destruction have motivated the development of portable instruments capable of detecting and monitoring a suspected release of the agent. Optimal detection of bacterial spores by PCR requires that the spores be disrupted to make the endogenous DNA available for amplification. The entire process of spore lysis, PCR, and detection can take several hours using conventional methods and instruments. In this report, a minisonicator and prototype spore lysis cartridge were built to disrupt Bacillus spores in 30 s for rapid, real-time PCR analysis. Utilization of the minisonicator improved PCR analysis by decreasing the limit of detection, reducing the time of detection, and increasing the signal amplitude. Total time of spore disruption and detection using the minisonicator and a microchip PCR instrument was less than 15 min.

This report describes real-time 5[prime] nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.

In this paper, we describe a miniature analytical thermal cycling instrument (MATCI) to amplify and detect DNA via the polymerase chain reaction in real-time. The MATCI is an integrated, miniaturized analytical system that uses silicon-based, high-efficiency reaction chambers with integrated heaters and simple, inexpensive electronics to precisely control the reaction temperatures. Optical windows in the silicon and solid-state, diode-based detection components are employed to perform real-time fluorescence monitoring of product DNA production. The entire system fits into a briefcase and runs on rechargeable batteries. The applications of this miniaturized nucleic acid analysis system include clinical, research, environmental, and agricultural analyses as well as others which require rapid, portable, and accurate analysis of biological samples for nucleic acids. This paper describes the MATCI and presents results from ultrafast thermal cycling and real-time PCR detection. Examples include human genes and pathogenic viruses and bacteria.

Thin film shape memory alloys (SMA's) have the potential to become a primary actuating mechanism for mechanical devices with dimensions in the micron-to-millimeter range requiring large forces over long displacements. The work output per volume of thin film SMA microactuators exceeds that of other microactuation mechanisms such as electrostatic, magnetic, thermal bimorph, piezoelectric, and thermopneumatic, and it is possible to achieve cycling frequencies on the order of 100 Hz due to the rapid heat transfer rates associated with thin film devices. In this paper, a quantitative comparison of several microactuation schemes is made, techniques for depositing and characterizing Ni-Ti-based shape memory films are evaluated, and micromachining and design issues for SMA microactuators are discussed. The substrate curvature method is used to investigate the thermo-mechanical properties of Ni-Ti-Cu SMA films, revealing recoverable stresses up to 510 MPa, transformation temperatures above 32 [degrees] C, and hysteresis widths between 5 and 13 [degrees] C. Fatigue data shows that for small strains, applied loads up to 350 MPa can be sustained for thousands of cycles. Two micromachined shape memory-actuated devices - a microgripper and microvalve - also are presented.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 [degrees] C/s heating, 2.5 [degrees] C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photo-lithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an 'electrophoretic valve' to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a [Beta]-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the [Beta]-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 107 and 4 x [10.sup.5] copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.

Cell disruptions using ultrasonic energy transmitted through a flexible interface into a liquid region has limitations because the motion of the vibrating tip is not completely transferred into the liquid. To ensure that some degree of contact will be maintained between the ultrasonic horn tip and the flexible interface, the liquid must be pressurized. The pressure conditions that yield consistent coupling between the ultrasonic horn tip and the liquid region were explored in this study by using an analytical model of the system and test fixture experiments. The nature of the interaction between the horn tip and the flexible interface creates pulses of positive pressure rises, increase in temperature, streaming flow, and almost no cavitation in the liquid. There was sufficient energy to create a cloud of microspheres, or beads, that maintain a consistent pattern of ballistic motion in the liquid. The sonication was found to be repeatable by studying video recordings of bead motion and was shown to be statistically consistent using measurements of temperature rise. Sonication of bacterial spores to obtain measurements of released nucleic acid and SEM images of damaged spores were used to verify the effects of liquid pressure on the horn--interface--liquid coupling.

A compact, real-time PCR instrument was developed for rapid, multiplex analysis of nucleic acids in an inexpensive, portable format. The instrument consists of a notebook computer, two reaction modules with integrated optics for four-color fluorescence detection, batteries, and a battery-charging system. The instrument weighs 3.3 kg, measures 26 x 22 x 7.5 cm, and can run continuously on the internal batteries for 4 h. Independent control of the modules allows differing temperature profiles and detection schemes to be run simultaneously. Results are presented that demonstrate rapid (1) detection and identification of Bacillus subtilis and Bacillus thuringensis spores and (2) characterization of a single nucleotide polymorphism for the hereditary hemochromatosis gene.

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