Electron monochromator mass spectrometry for the analysis of whole bacteria and bacterial spores

Spores from a variety of Bacillus species were analyzed with direct probe mass spectrometry using an electron monochromator to select electrons of distinct energies for ionization. Electron energies were chosen to match the electron capture energies of taxonomically important compounds such as dipicolinic acid and fatty acids. Previous negative ion interferences were not observed when the monochromator was used, and the signal-to-noise ratio of targeted compounds was significantly enhanced using this approach. To demonstrate the selectivity of the technique, the monochromator was swept over a range of electron energies while monitoring the masses of compounds with known electron capture energies. Scanning the monochromator while the mass spectrometer was operated in single-ion mode enabled dipicolinic acid to be detected in 10(5) spores. The results presented here demonstrate the utility of the electron monochromator for selectively ionizing compounds directly in bacteria and bacterial spores.     

Construction of spores for portable bacterial whole-cell biosensing systems

Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life. Specifically, we have employed spore-forming bacteria such as Bacillus subtilis and Bacillus megaterium for development of luminescent sensing systems for two model analytes, namely, arsenic and zinc. These sensing cells were converted to spores, which can then be "revived" (germinated) at a later time to generate viable and metabolically active cells. Herein, we demonstrate that these spore-based sensing systems retained their analytical performance, in terms of detection limit, dynamic range, and reproducibility, after storage at room temperature for at least 6 and 8 months, respectively, as well as after three cycles where the cells alternated between being dormant or active, i.e., sporulation-germination cycles. The ability to cycle the sensing cells between active and dormant states prolongs the cell's lifetimes and increases their robustness and ruggedness, thus making them more amenable for field applications. In addition, the small size of spores allows for their easy transport and incorporation in miniaturized portable devices. Finally, we envision that this novel strategy could expand the use of whole-cell biosensors for on-site sensing not only in mild environments but also in harsh environments and locations where there is no easy access to a laboratory, e.g., in developing countries.     

Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp. kurstaki, and Bacillus globigii Dugway) were analyzed. Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded. Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain. Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels. Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library.     

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.     

A new method for concentration analysis of bacterial endotoxins in perfluorocarbon

This communication demonstrates the feasibility of the gel-clot method for the analysis of bacterial endotoxins in water extracts of perfluorocarbon which is a water insoluble liquid medical device. Perfluorocarbon （10 mL） was shaken with 10 mL water for 15 min at 2000 r/min and the endotoxin present was extracted to the aqueous phase without interference inhibition/enhancement of the product and the recovery of endotoxin added to perfluorocarbon was determined, A validation study confirmed that endotoxins presented in perfluorocarbon pass over into the aqueous phase at concentrations of 20, 10 and 5EU/mL with recoveries from 86.8% to 96.8%. Therefore, the gel-clot test is suitable for detecting bacterial endotoxins in perfluorocarbon which is a water insoluble medical device.     

Array-based binary analysis for bacterial typing

An allele-specific oligonucleotide microarray was developed for rapid typing of pathogens based on analysis of genomic variations. Using a panel of Escherichia coli strains as a model system, selected loci were sequenced to uncover differences, such as single- or multiple-nucleotide polymorphisms as well as insertion/deletions (indels). While typical genomic profiling experiments employ specific sequences targeted to genomic DNA unique to a single strain or virulent gene, the present array is designed to type bacteria based on a patterned signature response across multiple loci. In the signature concept, all strains are interrogated by hybridizing their amplified DNA to an array containing multiple probe sequences. Allele-specific oligonucleotide probe sequences targeting each of these variable regions were synthesized and included in a custom fiber-optic array. For each locus, a set of specific probe sequences is selected, such that hybridization gives a binary signal/no signal response to each of the probes. Using this strategy for multiple loci, many pathogens or microorganisms could be classified using a limited number of probes. Because of the advantages of the fiber-optic array platform over other array formats, including sensitivity and speed, the platform described in this paper is capable of supporting a high-throughput diagnostic strategy.     

Design for DNA separation medium using bacterial cellulose fibrils

In this paper, we present a novel DNA separation medium using bacterial cellulose fibrils. Bacterial cellulose has an intrinsic three-dimensional micrometer- to nanometer-scale network structure. Addition of this material to a low-concentration polymer solution (<5 cP) enables high-resolution electrophoretic separation of DNA, even for fragments of 10-100-bp or single-nucleotide polymorphism. The newly designed medium consists of a double mesh: a 10-nm flexible mesh derived from a conventional polymer medium containing 10-nm to 1-microm rigid pores made up of 10-microm bacterial cellulose fragments.     

An introduction to DNA microarrays for gene expression analysis

This tutorial presents a basic introduction to DNA microarrays as employed for gene expression analysis, approaching the subject from a chemometrics perspective. The emphasis is on describing the nature of the measurement process, from the platforms used to a few of the standard higher-level data analysis tools employed. Topics include experimental design, detection, image processing, measurement errors, ratio calculation, background correction, normalization, and higher-level data processing. The objective is to present the chemometrician with as clear a picture as possible of an evolving technology so that the strengths and limitations of DNA microarrays are appreciated. Although the focus is primarily on spotted, two-color microarrays, a significant discussion of single-channel, lithographic arrays is also included.     

A floating-window algorithm for detecting certain power line faultsthat disrupt sensitive electronic loads

AC power is generally distributed in a voltage waveform that closely approximates a sinusoidal function with respect to time. Sensitive electronic loads, including computers and medical equipment, may be disrupted by substantial deviations from this sine wave approximation. A floating-window algorithm that uses a triggering function to detect changes in the voltage waveform that may disrupt sensitive electronic loads is described. This triggering function can be used to capture digitally sampled power line disturbances so that their cause can be identified and corrective measures taken. The algorithm can be applied in other situations where deviation from a repetitive waveform must be detected, such as electrocardiograms     

Hybridization-modulated ion fluxes through peptide-nucleic-acid- functionalized gold nanotubes. A new approach to quantitative label-free DNA analysis

The inner walls of gold nanotubes, prepared by template synthesis in the nanopores of polycarbonate track etch membranes, have been chemically modified with peptide nucleic acid (PNA) and used for label-free quantification of complementary DNA sequences. Selective binding of DNA to the PNA-modified nanotubes is shown to decrease the flux of optically detected anionic markers through the nanotubes in a concentration-dependent manner. The strong dependence of the biorecognition-modulated ion transport through the nanopores on the ionic strength suggests a dominantly electrostatic exclusion mechanism of the ion flux decrease as a result of DNA binding to the PNA-modified nanopores.     

Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.     

A miniature biochip system for detection of aerosolized Bacillus globigii spores

The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a compact biochip detection system, which includes a miniature diode laser for excitation. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air. Moreover, the incorporation of the miniature diode laser with the self-contained biochip design allows for a compact system that is readily adaptable to field use. In addition, these studies have included investigations into the tradeoff between assay time and sensitivity.     

Applying cognitive work analysis to the design of rapidly reconfigurable interfaces in complex networks

The objective of this paper is to illustrate the interconnections between the different phases (or tools) within the cognitive work analysis framework; the benefits of extending an analysis across each of the five phases are highlighted through these interconnections. The paper uses a command and control micro-world example to describe how each of the five phases can be used to describe the constraints within the micro-world domain from a different perspective. Based upon the social organisation and cooperation analysis, design requirements are extracted in order to develop role specific customisable interfaces for use within the micro-world. The interfaces have been specifically developed to communicate real time reconfiguration of the network through each of the individual interfaces; the reallocations of functions or roles are communicated to the actors through changes to the interface.     

Video analysis of DNA sequence homologies.

A method for the rapid quantitative analysis of dot blot assays is presented. A video camera, an NTSC compatible frame grabber board, and an AT personal computer are used to read photographic exposures of the assay plate. Image processing and image analysis techniques are used to calculate the orientation of the dot raster and then to compensate for the effect of variations in field illumination on measurements of local contrast. Local contrast (between dots and background) is an exponential function of the amount of hybridization between blotted DNA and complimentary oligonucleotide probes. The amount of hybridization between blotted DNA and oligonucleotide probes of known sequence is the criteria used to establish HLA-DR tissue types. Although the assay described here utilizes a chemiluminescent reaction, this algorithm may be used to read any assay that produces a rectangular raster of dots.     

Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.     

Cationic comb-type copolymers for DNA analysis

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.