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.     

GC/MS method for positive detection of Bacillus anthracis endospores

A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (~90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50,000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Single-chain variable fragment (scFv) antibodies optimized for environmental analysis of uranium

Recombinant single-chain variable fragment antibodies (scFv) were specifically generated and selected for the measurement of environmental uranium with an antibody-based sensor. These sFvs, which recognized UO(2)(2+) complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP), were produced using genetic material obtained from the spleen cells of rabbits immunized with UO(2)(2+)-DCP conjugated to keyhole limpet hemocyanin. Immunoglobulin light chain and heavy chain genes were amplified and cloned into the phagemid pSD3 for generation of a recombinant antibody library and phage-displayed antibodies. The screening process was designed to isolate antibodies that bound to a "loaded" noncovalent complex with high affinity, while selecting against binding to an "unloaded" complex. After five rounds of panning, individual positive scFv clones were used to infect E. coli TG1 and soluble scFv antibodies were purified and characterized. Binding studies showed that the best scFv bound tightly to the UO(2)(2+)-DCP complex (K(d), 19.6 nM). However, because of the depletion experiments performed on this library during the panning process, this scFv bound 1200-fold less tightly (K(d), 23.5 μM) to metal-free DCP. This scFv (clone 3A) was subsequently used to accurately determine the UO(2)(2+) concentrations in environmental water samples using a sensor based on kinetic exclusion analysis. The present studies demonstrate that recombinant scFvs with properties engineered for specific applications (i.e., biosensor-based measurement of metals in groundwater) can be prepared if the correct genetic material and techniques are employed. The phage display system permitted the generation of proteins with very specific binding properties (in this case, high affinity for a metal-chelate complex and low affinity for metal-free chelator). The recombinant scFvs isolated in these studies will be the basis for rapid and affordable assays for the detection of residual uranium in environmental water samples.     

Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min

The use of a combination of low-cost technologies to both extract and detect anthrax DNA from spores and vegetative cells in two steps within 1 min is described. In a cavity, microwave energy is highly focused using thin-film aluminum "bow-tie" structures, to extract DNA from whole spores within 20 s. The detection of the released DNA, from less than 1000 vegetative cells, without additional preprocessing steps is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. The new platform technology presented here is a highly attractive alternative method for DNA extraction and the fast detection of gram-positive bacteria and potentially other pathogenic species and cells as well.     

Silver nanoclusters films for single molecule detection using Surface Enhanced Raman Scattering (SERS)

A highly efficient Surface Enhanced Raman Scattering (SERS) substrate was prepared using a nanocluster deposition system that enabled detection of Crystal Violet molecules down to a single molecule level. The large SERS signal enhancement can be attributed to the presence of nano gaps on the surface of the nanoclusters which create abundant hot spots for electric field enhancement. Observed variations in the Raman peaks at very low molar concentrations in the range 4 × 10⁻¹⁴–3.2 × 10⁻¹⁸ M suggest that the spectra are due to a single molecule. Possible mechanisms for ultrahigh SERS sensitivity of the substrates are discussed. These substrates take the detection limit of CV down by two orders of magnitude as compared to those reported in literature.     

Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy

Germination is the process by which a dormant spore returns to its vegetative state when exposed to suitable conditions. We report on the real-time detection of kinetic germination and heterogeneity of single Bacillus thuringiensis spores in an aqueous solution by monitoring the calcium dipicolinate (CaDPA) biomarker with laser tweezers Raman spectroscopy (LTRS). A single B. thuringiensis spore was optically trapped in a focused laser beam, and its Raman spectra were recorded sequentially in time after exposure to a nutrient-rich medium, so that the CaDPA amount inside the trapped spore was monitored during the dynamic germination process. The CaDPA content in an individual spore was observed to remain almost constant in the first period and then decrease very rapidly due to its release into the medium (within approximately 2 min). The time-to-germination (t(germ)), defined as the time required for the CaDPA band intensity to decrease to the midpoint from its initial value, was found to be stochastic for individual spores with a typical value of approximately 30 min under the experimental conditions. The distribution of the time-to-germination was measured from a time lapse measurement of a population of spores. The results demonstrated that LTRS can be used to noninvasively detect the kinetic germination process at the single-cell level and explore cellular heterogeneity.     

Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores

Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.     

Fluorescently cationic conjugated polymer as an indicator of ligase chain reaction for sensitive and homogeneous detection of single nucleotide polymorphism

Ligase chain reaction (LCR) offers a simple and robust alternative platform for nucleic acid amplification, but its application has been limited because the LCR products are mostly detected by gel electrophoresis separation or heterogeneous analysis. In this paper, we report a novel homogeneous LCR assay by using cationic conjugated polymers (CCPs) as an indicator for detection of single-nucleotide polymorphism (SNP). For LCR, we design two pairs of unique target-complement probes. Each pair of probes contains two adjacent probes, in which one probe is designed with phosphorothioate modification at its 3'-end, and the other probe is labeled with fluorescein at its 5'-end. After the LCR, the two adjacent probes are ligated to form one DNA strand with a fluorescein label at its 5'-end and phosphorothioate modification at its 3'-end, which is resistant to the exonuclease I and exonuclease III degradation. When the CCP is added, because of the strong electrostatic interactions between CCP and DNA, effective fluorescence resonance energy transfer (FRET) from the CCP to the fluorescein-labeled DNA can be observed. In contrast, the unligated fluorescein-labeled probes are degraded to the mononucleotides by exonuclease I and exonuclease III. Introduction of CCP leads to inefficient FRET results because much weaker electrostatic interactions between the fluorescein-labeled mononucleotides and CCP keep the fluorescein far away from CCP. Accordingly, homogeneous LCR for SNP detection is performed successfully. The method is sensitive and specific enough to detect 1 fM (600 zmol) DNA molecules. It is possible to quantify SNP and accurately determine the allele frequency as low as 1.0%. This proposed assay strategy extends the application of LCR and provides a new platform for homogeneous detection of SNP.     

Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO(2)) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO(2) introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO(2) was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO(2) levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO(2) emissions below the limit. Numerous lessons were learned in the field trials of this ClO(2) decontamination technology.     

利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究

研究了检测斑节对虾(Penaeus monodon)的主要致病病原——对虾白斑综合征病毒(white spot syndrome virus,WSSV)及斑节对虾杆状病毒(monodon baculovirus,MBV)的技术。用多重PCR检测方法,设计了两对特异性引物,从不同虾池中收集斑节对虾,提取DNA模板,同时检测两种对虾病毒。研究结果表明：该方法检测灵敏度高、特异性好,可检测至每毫克组织100个病毒粒子;从对虾组织中提取的DNA模板对病毒DNA的扩增无抑制,适合于对虾中两种病毒的同时检测。     

Crystal growth and concentration change of (Mn,Ni,Zn)-ferrite single crystals by the flame fusion method

Single crystals of (Mn,Ni,Zn)ferrite are grown by the flame fusion method. The chemical analysis of composition reveals that ZnO shows the largest change in concentration among the components, for example, from 26.7 mol% to 17.3 mol% in a crystal of 50 mm in length and 25 mm in diameter. This inhomogeneity is only observed along the growth direction and cannot be detected by analysis at this level of accuracy along the direction perpendicular to the growth direction, except at the periphery to a depth of 1 mm. This inhomogeneity may be explained as a result of the evaporation of Zn from the molten zone during growth. The equation $\text{C}{}_{\mathrm{o}}\text{C}\text{= 1 +}\text{K}\text{V}$ is derived (C /2b Zn concentration, V = crystal growth rate and C_o, K = const). Crystal free from cracks can be grown successfully by using an after-furnace made of SiC heater. A new apparatus to feed the crystal constantly with raw material powder is proposed, and by using this the change in concentration can be reduced to less than 1 mol% throughout the crystal of the above size. These crystals have also been investigated for the Micro-Vicker's hardness.     

Realizing fragment spawning and fragment growth in the parallelized Discrete Element Method (DEM) during modelling of comminution

The particle based Discrete Element Method (DEM) can be applied to examine comminution processes. In this study, a DEM framework has been extended to model particle breakage without mass loss. After a breakage event occurs, spherical particles, as often considered in the DEM, are replaced by size reduced spherical fragments. During the following time steps, the fragments grow to their desired sizes, so that the mass loss can be counterbalanced. Previously defined overlaps with adjacent unbroken and broken particles (fragments) as well as walls are allowed. The breakage model has been realized in a parallelized DEM framework because comminution processes are often attributed to large numbers of particles and by parallelization the computational time can be reduced efficiently. An oedometer (one-dimensional compression in axial direction of a confined particle bed) has been modelled to investigate the parallelization efficiency and the influence of the permitted overlaps during the growth process on the growth duration. A simplified roller mill has been considered to examine the applicability of the breakage procedure considering parallelization. The results show that parallelization reduces computational time considerably. The breakage procedure is suitable to model comminution processes involving even densely packed particle systems and is superior to existing approaches.     

Performance investigation of a variable speed vapor compression system for fault detection and diagnosis

An experimental study has been performed to investigate the effect of four artificial faults on the performance of a variable speed vapor compression system. Experimental setup to test several artificial faults was made by modifying the conventional vapor compression test rig. Four major faults of compressor fault, condenser fault, evaporator fault, and refrigerant leakage, were implemented by observing the variation of cooling capacity. Two different rule-based modules for constant and variable speed operations were organized for an easy diagnosis of system faults. These two modules were applied differently as the cooling capacity satisfies the necessary air conditioning load. As a result, COP degradation due to the fault in a variable speed system is severer than that in a constant speed system.     

A transient (subsecond) technique for measuring heat of fusion of metals

A transient technique is described for measuring the heats of fusion of metals with melting temperatures above 1500 K. The specimen configuration consists of a strip of the metal under study sandwiched between two strips of another metal with a higher melting temperature. The basic method consists of rapidly heating the composite specimen by passing a subsecond-duration electrical current pulse through it and simultaneously measuring the radiance temperature of the containment metal surface, as well as the current through and voltage drop across the specimen. The melting of the metal under study is manifested by a plateau in the temperature versus time function for the containing metal surface. The time integral of the power absorbed by the specimen during melting yields the heat of fusion. Measurements on several tantalum-niobium-tantalum specimens yield a value of 31.5 kJ · mor^–1 for the heat of fusion of niobium, with an estimated maximum inaccuracy of ± 5%.     

面向供应链合作的信任维系协调体系的研究

为了保证节点企业合作中信任协调的合作关系,以有效地实现企业经营目标,从信任的视角提出了一个包括目标层、合作层、信任协调层、支撑层在内的供应链合作(SCC)信任维系协调的解决思路.把节点企业内、外部的供应链运作、合作行为、业务目标集成起来,给出了整体框架模型,并对基于企业目标的信任协调机制、信任金字塔和实现协调层的具体方式等关键问题进行了探讨.信任协调体系框架模型对供应链合作运作提供了一定的参照.     

Supply chain resilience for single and multiple sourcing in the presence of disruption risks

This paper investigates the use of sourcing strategies to achieve supply chain resilience under disruptions. The coping strategies considered are single and multiple sourcing, backup supplier contracts, spot purchasing, and collaboration and visibility. Collaboration and visibility, which affect suppliers’ recovery capabilities and a buyer’s warning capability, have not been similarly modelled in the past. A scenario-based mathematical model is developed such that it considers objectives under uncertainties including disruption risks and operational risks. A broad numerical study examines its output for various risk attitudes in a decision-maker, ranging from risk neutral to risk averse. The sensitivity of procurement strategies to other key parameters such as recovery and warning capabilities is examined. One of the major findings is that buyer’s warning capability plays a vital role in enhancing supply chain resilience. We seek to build on these efforts to further support disruption planning and mitigation and to obtain a deeper understanding of the relationship between supply chain characteristics and resilience.     

Sequential eluent injection technique as a new approach for the on-line enrichment and speciation of Cr(III) and Cr(VI) species on a single column with FAAS detection

This paper introduces a sequential eluent injection (SEI) technique combined with an on-line preconcentration/separation system for a fast and sensitive FAAS determination of trace amounts of Cr(III) and Cr(VI) species. The method is based on the simultaneous retention of Cr(III) and Cr(VI) on a single mini-column packed with a chloromethylated polystyrene functionalized with N,N-bis(naphthylideneimino)diethylenetriamine (PS-NAPdien) at pH 6.7. The retained chromium species was eluted by sequential injection of HCl for desorption of Cr(III), and NH₃ and NH₄NO₃ buffer solutions for desorption of Cr(VI). All the chemical and flow injection variables were optimized for the quantitative preconcentration and speciation of Cr(III) and Cr(VI). Under the optimum conditions, the calibration graph obtained is linear over the concentration range of 2.0-60.0 μg L⁻¹ for Cr(III), and 8.0-180.0 μg L⁻¹ for Cr(VI). The preconcentration factors for Cr(III) and Cr(VI) were 70 and 30, respectively. The 3σ detection limits were 0.6 μg L⁻¹ and 2.5 μg L⁻¹ for Cr(III) and Cr(VI), respectively. The relative standard deviations were 2.55% and 0.8%, respectively, for 6 replicate determinations of Cr(III) and Cr(VI) at the 40.0 μg L⁻¹ level. The proposed method was applied for determination of Cr(III) and Cr(VI) in different water samples with satisfactory results.     

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.