Convectively driven polymerase chain reaction thermal cycler.

We have fabricated a low-cost disposable polymerase chain reaction thermal chamber that uses buoyancy forces to move the sample solution between the different temperatures necessary for amplification. Three-dimensional, unsteady finite element modeling and a simpler 1-D steady-state model are used together with digital particle image velocimetry data to characterize the flow within the device. Biological samples have been amplified using this novel thermal chamber. Time for amplification is less than 30 min. More importantly, an analysis of the energy consumption shows significant improvements over current technology.

Field-deployable sniffer for 2,4-dinitrotoluene detection.

A field-deployable instrument has been developed to detect low-level 2,4-dinitrotoluene (2,4-DNT) vapors. The system is based on previously developed artificial nose technology and employs an array of sensory materials attached to the distal tips of an optical fiber bundle. Both semiselective and nonspecific, cross-reactive sensors were employed. Each sensor within the array responds differentially to vapor exposure so the array's fluorescence response patterns are unique for each analyte. The instrument is computationally "trained" to discriminate target response patterns from nontarget and background environments. This detection system has been applied to detect 2,4-DNT, an analyte commonly detected on the soil surface above buried 2,4,6-trinitrotoluene (TNT) land mines, in spiked soil and aqueous and ground samples. The system has been characterized and demonstrated the ability to detect 120 ppb 2,4-DNT vapor in blind (unknown) humidified samples during a supervised field test.

A minisonicator to rapidly disrupt bacterial spores for DNA analysis.

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.

Rapid pathogen detection using a microchip PCR array instrument.

An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 10(5)-10(7) organisms/L (10(2)-10(4) organisms/mL).

Novel use of a fiber-optic-based on-line trichloroethylene sensor in a column retardation experiment.

A newly developed fiber-optic-based trichloroethylene (TCE) sensor previously described [F.P. Milanovich, S.B. Brown, B.W. Colston, Jr., P.F. Daley and K. Langry, Talanta, 41 (1994) 2189], was used to provide analyses of TCE in laboratory tests of retardation of TCE in ground water. The sensor enabled inexpensive real time analyses of TCE in retardation tests conducted in a sand-filled flow-through column. The simultaneous data analysis of TCE, (18)O and Cl(-) breakthrough curves enabled the calculation of an estimated retardation coefficient which was found to be in good agreement with that predicted by the octanol/water partitioning K(d) method. The fiber-optic sensor was demonstrated to be a fast and reliable method for conducting on-line laboratory analyses of TCE at the parts per billion level in a small volume of contaminated water, thus providing excellent temporal resolution of the data as well as minimizing volatile losses during sample collection and analysis.

A fiber-optic sensor system for monitoring chlorinated hydrocarbon pollutants.

We have developed and field-tested a fiber-optic chemical sensor system for use in environmental monitoring and remediation. The system detects chlorinated hydrocarbon pollutants with colorimetry, and is based on an irreversible chemical reaction between the target compound and a specific reagent. The reaction products are detected by their absorption at 560 nm and can be monitored remotely with optical fibers. Continuous measurements are made possible by renewing the reagent from a reservoir with a miniature pumping system. The sensor has been evaluated against gas chromatography standards and has demonstrated accuracy and sensitivity (5 ppbw) sufficient for the environmental monitoring of trichloroethylene and chloroform. Successful preliminary field tests have been conducted in a variety of contamination monitoring scenarios.

Evidence of novel secondary structure in DNA-bound protamine is revealed by Raman spectroscopy.

Raman spectroscopy studies of protamine-DNA complexes are reported for samples in the solid state at 98% relative humidity. Previous reports utilizing other physical techniques have indicated the presence of B-form DNA in protamine-DNA complexes. The present Raman data support the assignment of a modified B-form which is characterized by appreciable unstacking of the bases. The quality of the present spectra has made it possible, for the first time, to obtain the Raman spectrum of DNA-bound protamine by digital spectral subtraction. The difference spectrum indicates that protamine adopts an unusual secondary structure upon binding to DNA. A dominant amide I band is observed at 1683 cm-1 which is indicative of neither an alpha-helix or beta-sheet conformation. An amide I band at this position has been associated with the 1-->3 hydrogen bond that occurs within a gamma-turn [Bandekar, J., & Krimm, S. (1985) Int. J. Pept. Protein Res. 26, 158-165]. On the basis of this assignment, as well as preliminary results obtained by computer modeling, we propose a new model for the secondary structure of DNA-bound protamine that is rich in 1-->3 hydrogen bonding. Spectral data demonstrate that this structure is absent in protamine molecules in solution. Analyses of spectra of polyarginine-DNA complexes suggest that polyarginine, although similar to protamine in primary structure, assumes a conformation when bound to DNA that is distinct from that adopted by protamine.

A fiber-optic sensor for CO(2) measurement.

A fiber-optic sensor has been prepared which responds to carbon dioxide at physiologically significant concentrations. It is based on pH modulation by dissolved carbon dioxide in a sensing layer of fluorescent dye. By use of a previously developed methodology by which the sensing chemistry is bonded directly to the glass fiber tip, the miniature size of the sensor is preserved. This method involves consecutive applications of solution polymers to the fiber tip rather than mechanical attachment of sensor reagents. Preparations of polymer-immobilized dyes and polymer membranes are described.

Escherichia coli ribosome unfolding in low Mg2+ solutions observed by laser Raman spectroscopy and electron microscopy.

Ribosomes unfolded by the removal of Mg2+ at 25 degrees C were studied by Raman spectroscopy and electron microscopy. Raman spectra showed a reduction in the 813 cm-1 phosphodiester signal of 30S and 50S ribosomes compared to intact ribosomes, suggesting that a fraction of the ribose moieties had shifted from the 3' endo (ordered) to the 3' exo (disordered) conformation. The maximum diameters of unfolded 30S and 50S ribosomes, judged by electron microscopy, were 1.8 and 2.5-fold greater, respectively, than those of intact ribosomes. Most unfolded 30S ribosomes had three distinct structural domains and appeared "Y-shaped"; whereas most unfolded 50S ribosomes had four distinct domains and appeared "X-shaped". When ribosomes were partially unfolded (by brief exposure to 0.04 mM Mg2+ or EDTA), several possible intermediates in the unfolding process were observed. Both the shapes of particles and their Raman spectra reached the same final state in 0.04 mM Mg2+, where more than 50% of the rRNA phosphates are discharged by Mg2+, as in 10 mM EDTA, where less than 1% are discharged.

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.

Dissolved yellow organics: quantitative and qualitative aspects of extraction by four common techniques.

The dissolved yellow organic matter is removed from aliquots of the same freshwater sample by four common techniques: lyophilization, ion-exchange, ultrafiltration and organic solvent. Quantitative and qualitative aspects of the recovery, including fractionation by gel filtration and total nitrogen determinations, are presented.