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Journal of Medical Chemical, Biological and Radiological Defense
J Med CBR Def  |  Volume 8, 2010
Submitted 21 September 2009 | Accepted 28 October 2009 | Revised 15 March 2010 | Published 12 April 2010

Ultra Fast And Sensitive Detection Of Biological Threat Agents Using Microwaves, Nanoparticles And Luminescence

K. Aslan1, L.W. Baillie2 and C.D. Geddes1,*

1,* The Institute of Fluorescence, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD, USA, 21202
Tel: 410 576 5723 | email: geddes@umbi.umd.edu

2 Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, Cardiff, Wales U.K.

Suggested citation:
          Aslan, K; Baillie, LW; Geddes, CD, “Ultra Fast And Sensitive Detection Of Biological Threat Agents Using Microwaves, Nanoparticles And Luminescence”, JMedCBR 8, 12 April 2010, http://www.jmedcbr.org/issue_0801/Geddes/Geddes_04_10.html.

 

 

abstract

In this paper, we review our recent work on the detection of biological threat agents using a combination of focused microwaves, low-power microwave heating and metal-fluorophore systems. The combination of focused microwaves and metal thin films in the proper geometric configuration enables the extraction of biological target molecules from organisms, such as bacteria, viruses and cells, within seconds. In addition, low power microwave heating drives the capture and binding of pathogen-specific molecules to fluorescence-based bioassay components located on metal nanoparticle-deposited surfaces such that the detection bioassay is completed in less than a minute. The bioassays can be conducted in biological medium such as serum and whole blood. The addition of metal nanoparticles to the sample increases the observed fluorescence emission due to a phenomenon called metal-enhanced fluorescence (MEF). The fluorescence-based bioassays can also be carried out on metal thin film surfaces, where the fluorescence emission is observed more efficiently at a certain angle from the back of the metal thin films (surface plasmon coupled luminescence, SPCL). In conclusion, MEF and SPCL are technology solutions that enable afford lower detection limits in the bioassays for biological threat agents to be realized.

Acronyms:

  • MAMEF: Microwave Accelerated Metal Enhanced Fluorescence
  • MEF: Metal-Enhanced Fluorescence
  • SPCF: Surface Plasmon Coupled Fluorescence
  • SPCL: Surface Plasmon Coupled Luminescence

INTRODUCTION

Protection of and improvement in the heath of communities, i.e., public health, includes the practice of identifying biological threat agents in order that measures can be implemented for that protection. Those on the first line of protection, the first responders, require simple, rapid and accurate identification of suspected biothreats agents in order to determine which countermeasures and practices to implement. This is similar to the requirement for a country's military, which may be involved in the same response. Currently defense communities lack robust, all purpose biodetectors capable of being scaled down to section or individual use. The current detection systems are either single use, lack sensitivity (such as Hand-Held Assays), or are designed to address specific defense missions, such as the provision of standoff or critical point monitoring for high value assets (airfields or C3I facilities) [Walt and Franz, 2000]. For the military, biodetectors need to be compatible with other CBRN detection equipment and carried along with any mission specific defense equipment; therefore any extra weight or supplies must be justified as essential to the overall detection requirements.

An ideal biodetector must be able to detect a broad spectrum of biothreat agents, ranging from preformed toxins to viruses (DNA and RNA) to bacteria and Rickettsia. It should also be inexpensive, use little power, and light weight, as well as give unambiguous results even in complex environmental samples. The technology should be robust and capable of being miniaturized. We describe here some of the new analytical techniques we have used, which we think may be adaptable to use by both military and first responders.

Our research group has recently described a series of new analytical techniques in which the combination of low-power microwave heating and metal-fluorophore interactions enables the development of ultra fast and sensitive bioassays. The first of these techniques is called microwave-accelerated metal enhanced fluorescence (MAMEF) [Aslan and Geddes, 2005], in which fluorescence-based surface assays are performed on plasmonic nanoparticles (non-continuous surfaces) in the presence of low-power microwave heating. In MAMEF, the fluorescence signatures are amplified by plasmonic nanoparticles [Geddes and Lakowicz, 2002; Aslan et al., 2005] and the biomolecular recognition events are kinetically accelerated by microwave heating. Silver nanoparticles are typically the plasmonic nanoparticles of choice for use in MAMEF-based bioassays. Silver nanoparticles serve as: 1) a platform on which biomolecular recognition events occur, 2) an amplifier of fluorescence signatures and 3) a low temperature surface (during microwave heating) to create a temperature gradient between the bulk and surface that results in mass transport of biomolecules to the surfaces.[Aslan and Geddes, 2005] The second technique is called microwave-accelerated surface plasmon coupled luminescence (MA-SPCL) [Aslan et al., 2007a; Aslan et al., 2008b], where luminescence-based bioassays are carried out on continuous metal thin films in the presence of microwave heating. The major differences between MA-SPCL and MAMEF are the type of surfaces (MA-SPCL: continuous, MAMEF: non-continuous surface) and the placement of detection optics (MA-SPCL: back of the films, MAMEF: from the sample side, front surface). In MA-SPCL, biomolecular recognition events are accelerated with low-power microwaves, and the luminescence emission is coupled to the metal thin film and is emitted at a specific angle from the back of the film. In both techniques, metals are not heated by microwaves and thus remain at a lower temperature than the water in bulk. Subsequently, a temperature gradient is created, which caused the biomolecules in the warmer bulk to move towards the colder surface. Since one of the bioassay components (i.e., DNA probe or capture antibody) is present on the surface, the biomolecular recognition events occur at the metal surface.

In this review, we summarize our recent work on the use of these technologies to detect bacterial, toxin and viral targets, specifically Bacillus anthracis the causative agent of anthrax, protective antigen (PA) a component of the anthrax tripartite toxin and Hepatitis C a major human viral pathogen. Using MAMEF we were able to detect the presence of PA in whole rat blood in 1 minute using a high-throughput screening format. Aluminum thin films when fashioned into “bow-tie” structures in combination with microwaves were employed to extract DNA from B. anthracis spores which were subsequently detected using MAMEF. The MA-SPCL technique successfully detected the presence of Hepatitis C RNA in human blood.

EXPERIMENTAL METHODS

The reader is referred to the cited articles for details of the individual experimental procedures. A brief summary for each technique is provided in this section.

Silver Island Films (SIFs)

In a typical SIFs preparation on a glass support, a solution of silver nitrate (0.5 g in 60 mL of deionized water) in a clean 100-mL glass beaker, equipped with a Teflon-coated stir bar, is prepared and placed on a stirring/hot plate. While stirring at the quickest speed, 200 μL of freshly prepared 5% (w/v) sodium hydroxide solution is added. This results in the formation of dark brown precipitates of silver particles. Approximately 2 mL of ammonium hydroxide is then added, drop by drop, to re-dissolve the precipitates. The clear solution is cooled to 5°C by placing the beaker in an ice bath, followed by soaking the amino silane-coated glass slides in the solution. While keeping the slides at 5°C, a fresh solution of D-glucose (0.72 g in 15 mL of water) is added. Subsequently, the temperature of the mixture is then warmed to 30°C. As the colour of the mixture turns from yellow-green to yellow-brown, and the colour of the slides become green, the slides are removed from the mixture, washed with water, and sonicated for 1 minute at room temperature. SIFs-deposited slides were then rinsed with deionized water several times and dried under a stream of nitrogen gas.

Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF)

In the model protein assay [Figure 1],[Aslan and Geddes, 2005] first, 10 μM biotinylated-BSA was attached to the surface of SIFs by 30 min incubation at room temperature. Then, fluorescein-labeled streptavidin was incubated on BSA-modified SIFs at room temperature (for 30 minutes) or through low-power microwave heating (for 20 seconds). Fluorescence emission spectrum was collected with a HD 2000 spectrofluorometer (Ocean Optics, Inc. USA). A 473 nm laser was used the excitation source.

In the PA assay [Figure 2], Poly-L-Lysine coated high-throughput screening (HTS) wells (Sigma-Aldrich Co. USA) are modified by overnight incubation in the presence of a solution of silver nanoparticles (60 nm in diameter, Ted-Pella, Inc, USA) to create silver island films [Aslan et al., 2006]. PA solutions with various concentrations were incubated on silver colloid-modified HTS wells through low-power microwave heating for 20 seconds. Subsequently, a PA-specific primary antibody (1 mg/ml) and Rhodamine G-labeled secondary antibody was sequentially incubated through low-power microwave heating for 20 seconds. Fluorescence emission spectrum was collected with a HD 2000 spectrofluorometer. A 532 nm laser was used the excitation source.

In the Anthrax DNA assay [Figure 7], [Aslan et al., 2007b] a single stranded oligonucleotide (anchor probe, 5’-SH-ctttttaccgttatatataag) with sequence specific to Anthrax DNA was attached to the surface of SIFs by overnight incubation at room temperature. Solutions of various concentrations of target oligonucleotide (3’-tggcaatatatattcattagggttaatattccatttacatatacgac-5’) and a 1 μM fluorophore (TAMRA)-labelled oligonucleotide (5’-TMR-tataaggtaaatgtatatgctg) were mixed and incubated on SIFs containing the anchor probe through low-power microwave heating for 20 seconds. Fluorescence emission spectrum was collected with a HD 2000 spectrofluorometer. A 532 nm laser was used the excitation source.

Deposition of Aluminum Triangles on Glass Substrates to Lyse Bacillus anthracis Spores and Vegetative Organisms

Glass microscope slides were covered with a mask (an aluminum foil cut with a razor blade, 12.5 mm in size) to leave a triangular bowtie region exposed [Aslan et al., 2008a]. Equilateral aluminum triangles of 12.5-mm size (thickness 75 nm) were deposited onto a glass microscope slide using a BOC Edwards 306 vacuum deposition system. A self-adhesive silicon isolator (1 cm in diameter) was placed on top of the aluminum triangles’ bow-tie region.

Procedure for DNA Extraction with Focused Microwaves for the MAMEF Assays

Fifty microliters of the spores (107 spores/mL of spores) or vegetative organisms (various concentrations) was placed inside the silicon isolator and was covered with a thin cover slip. These samples were then heated up to 20 s in a microwave cavity (a 0.7 cu ft, GE Compact microwave model JES735BF, maximum power 700 W, duty cycle of 2 is used).

Microwave-Accelerated Surface Plasmon Coupled Luminescence (MA-SPCL)

Gold thin films (25 nm thickness) were prepared by thermal evaporation technique [Aslan et al., 2008b]. A single stranded oligonucleotide (anchor probe, 5’-SH-cttttttgatgcacg gtctacgaga ccgg gggg tcctgg aggctgcacga) with sequence specific to Hepatitis C was attached to the surface of gold thin film by overnight incubation at room temperature. Solutions of various concentrations of target oligonucleotide labelled with a fluorophore (TAMRA) (ctacgtgc cagatgctct ggcc cccc aggacc tccgacgtgct-TMR-5’) were incubated on separate gold thin films containing the anchor probe through low-power microwave heating for 20 seconds. Fluorescence emission spectrum was collected with a HD 2000 spectrofluorometer using an optical setup built in-house. A 532 nm laser was used the excitation source.

RESULTS COMPARISONS

Figure 1 shows the proof-of-principle demonstration of MAMEF technique, which couples the benefits of MEF (enhanced fluorescence signatures, increased photostability of fluorophores) [Geddes and Lakowicz, 2002; Aslan et al., 2005] with low power microwave heating (kinetically accelerated biomolecular recognition events). A model bioassay exploiting the well-known interactions of biotin-avidin was constructed on glass surface (figure 1A). In the model assay biotin, one of the binding partners in the bioassay, is attached to the glass surface via bovine serum albumin (BSA) molecules (BSA forms a monolayer on SIFs). The other binding partner, avidin, is labeled with a fluorophore (e.g., fluorescein) is incubated on the biotinlyated-BSA surface. While this bioassay typically takes 30 minutes to complete at room temperature, the application of low-power microwaves can reduce the run time to 20 seconds. A possible explanation for this observation is depicted in figure 1B. The application of microwaves results in the selective heating of the assay medium (water and biomolecules) while the silver nanoparticles remain virtually at room temperature, resulting in a temperature gradient between silver and the assay medium. Subsequently, the biomolecules in the warmer medium are forced to move towards the colder silver nanoparticles, where the biotinlyated-BSA molecules are located. Due to the significantly larger thermal conductivity of silver, the temperature gradient is maintained for the duration of microwave heating. After the microwave heating is turned off, thermal equilibrium is reached, at which point the bioassay has been completed.

Figure 1C shows the fluorescence emission spectrum of fluorescein collected after the bioassay is run at room temperature (figure 1B-Left) or with low-power microwave heating (figure 1B-Right). The fluorescein emission intensity from the silvered surfaces (SIFs) is typically about 6-fold larger than the glass surfaces (due to MEF phenomenon) for the bioassay run at room temperature. The identical assay run with 20 second microwave heating shows that emission intensity from the silvered surfaces is similar to that of room temperature assay, which implies that the assay is completed in a similar manner. Indeed, an added benefit of microwave heating in the presence of silver is an approximately 9-fold larger increase in emission intensity compared to glass, which is thought to be due to the lack of temperature gradient on the glass surfaces (figure 1B). In the control experiments, where biotinlyated-BSA is omitted, no fluorescence emission were detected from either silvered or glass surfaces (data not shown). These observations imply that the non-specific binding of fluorescein-labeled avidin to the surfaces was minimal.

Toxin Detection

The majority of toxins, especially bacterial toxins, found in nature are proteins and as such lend themselves to antibody-based detection methods. Figure 2 shows the experimental design used in the MAMEF-based anthrax toxin detection assay. Poly-L-Lysine coated HTS wells are modified by overnight incubation in the presence of a solution of silver nanoparticles to create silver island films. The assay components comprised a primary detection antibody, recombinant anthrax Protective Antigen (PA) (The Biological Defense Research Directorate (BDRD), Naval Medical Research Center) and a fluorophore (Rhodamine G)-labeled secondary reporter antibody (Fab fragment). Each microwave-heating step is carried out for 20 seconds. Control experiments are carried out in a similar fashion to the assay except that PA was omitted. The fluorescence emission spectrum from each well is measured after the assays are completed.

Figure 3A and 3B show the emission spectrum of Rhodamine G for MAMEF-based assay and the control assay for PA in buffer, respectively. The emission intensity at 575 nm is increased as the concentration of PA is increased from 10 to 2400 mg/ml. On the other hand, the emission intensity for the same concentration range remained below ≈220 arbitrary units, which implies that the MAMEF-based assay has a lower detection limit of >10 mg/mL [Aslan et al., 2007b]. Figure 3C shows the emission intensity at 575 nm for both the assay and the control assay.

The results of identical assays for PA mixed with rat blood are shown to demonstrate the effectiveness of the MAMEF technique in the detection of PA in real-life samples. Figure 4A shows the concentration-dependent emission spectrum for PA that was mixed with blood obtained from a sample rat (BDRD). The emission intensity at 575 nm increases with the concentration (figure 4B). It is interesting to note that the emission intensity values obtained from blood samples are significantly less than those obtained from buffer samples. This may be due to less effective heating of blood samples as compared to buffer samples. Thus, while this assay is not as sensitive as other recently described PA specific antibody assays that report detection limits ranging from 10 to 1000 pg per mL of blood, it is considerably faster because it avoids the time consuming separation and preparation stages, and results can be generated in seconds rather than hours [Peruski and Peruski, 2003; Mabry et al., 2006; Tang et al., 2009]. Given the central role of the capture and detection of antibodies, it should be possible to lower the detection limit of this method by engineering the binding characteristics of these antibodies.

Bacterial Detection

B.anthracis presents a major challenge to researchers seeking to develop real-time assays, due to its ability to form resistant spores. Indeed, in the past the extraction of proteins and DNA from B. anthracis spores has required a complex, lengthy procedure. Our research laboratory has recently described an alternative method to create spore fragments that uses aluminum antenna to focus microwaves to a specific location, where spores are placed [Previte et al., 2007]. Focused microwaves generate intense electric fields capable of inducing structural changes in spores, which result in the release of biological components, such as DNA, into their surroundings [Kim et al., 2009]. Indeed, it has been proposed that the spore fragmentation occurs due to the heating and expansion of water trapped within the body of the spore [Perani et al., 1998].

Figure 5A shows the schematic depiction of the aluminum triangles fashioned in to a “bow-tie” shape to focus microwaves. Theoretical calculations shown in figure 5A predict that electric field of the incident microwaves are focused in between the two triangles. To experimentally substantiate the predictions of theoretical calculations, a temperature sensitive chemical solution is placed over the two triangles and the temperature change in the chemical solution is monitored by a thermal imaging camera. Figure 5B shows the thermal images of aluminum triangles with the chemical solution before and after their exposure to microwave heating. After a 2-second microwave heating the temperature of the chemical solution in between the triangles is increased about 12°C. These thermal images confirm the prediction that microwaves can be focused to a desired location using aluminum antenna. In addition to the control over the location of focused microwaves, the control of change in temperature of the solution at a specific location is also useful. Figure 5C shows a plot of intensity of temperature sensitive chemical solution versus temperature that was measured at different locations on aluminum triangles. The temperature of the chemical solution varied between 30 and 93°C, depending on the different aluminum triangle configurations used.

Subsequently, the aluminum triangles were employed to extract biological material from B. anthracis spores and vegetative cells (separate experiments) that had been placed between the triangles and exposed to microwaves for 20 seconds. Changes in the structure of the spores and vegetative cells were observed using transmission electron microscopy (TEM). B. anthracis spores are approximately 1000 nm in length and are surrounded by a semi-transparent structure called exosporium as shown in figure 6A-(Left). Microwave heating of spores on glass without aluminum triangles caused no obvious structural changes to the spores (figure 6A-Middle). In contrast, microwave heating between aluminum triangles resulted in significant spore structural changes (figure 6A-Right). In a similar fashion, exposure of vegetative cells to microwave heating also resulted in significant structural changes to the cells (figure 6B). The release of DNA as a consequence of microwave-induced structural damage was confirmed by gel electrophoresis, (figure 6C). The gel results (lane 3 and 4) showed that a commercially available DNA extraction protocol (available in the Gentra Puregene DNA extraction kit) and the microwave heating technique yielded similar results. In addition, microwave heating of spores between aluminum triangles also released DNA (data not shown here).

To build on these promising results, a MAMEF-based three-piece DNA hybridization assay was developed for the detection of B.anthracis DNA released from microwave treated spores and vegetative organisms. [Aslan et al., 2007b] Figure 7 shows the experimental design depicting the organization of oligomers on the silver surface with both anchor and detection probes designed to recognize B.anthracis specific nucleotide sequences within the gene encoding PA.

The results of room temperature assay and MAMEF assays for DNA from B.anthracis spores and vegetative organisms are shown in figure 8. First, the level of background DNA present on the surface of the spores was determined by incubating spores on silver island films (SIFs) at room temperature in the absence of microwave heating (figure 8A). When the spores were exposed to microwave heating on glass without aluminum triangles, emission intensity values were obtained similar to those without microwave heating (figure 8B). This implies that simple microwave heating of spores on glass does not yield detectable DNA. However, when the spores placed between the aluminum are exposed to microwave heating, the emission intensity values were significantly larger, suggesting that the microwave heating DNA extraction protocol released DNA that was detected by the MAMEF-based assay. In control experiments the anchor probes were omitted from the surface, the emission intensity values were then significantly smaller.

The ability of the MAMEF technique to detect DNA originating from vegetative cells was also investigated. Figure 8D shows the concentration-dependent emission intensity for assays run with DNA obtained from vegetative cells using the microwave heating DNA extraction protocol. We examined the vegetative form of two B.anthracis variants: 1) PA-proficient, and 2) PA-deficient. The emission intensity values for PA-proficient cells were significantly larger compared to values for PA-deficient cells and control experiments. Using the MAMEF technique, we achieved a lower detection limit of 1,000 vegetative cells/mL, which is comparable to the median instrument limit of detection seen with real time polymerase chain reaction (RT-PCR), the most sensitive method identified in a recent open source literature review of methods for the detection of B. anthracis [Herzog et al, 2009]. It should be noted that, while the level of sensitivity of MAMEF is comparable to RT-PCR, the time to complete the assay (60 seconds) is considerably shorter and less labor-intensive, particularly for environmental samples, which can inhibit PCR. Indeed the median detection limit of spores in soil using current technologies is 1.2 x 104 CFU/g of soil with the exact figures being influenced by the type of soil and the extraction technique employed [Herzog et al, 2009].

VIRUSES

The microwave heating technique has been applied to the detection of a nucleic acid sequence derived from the Hepatitis C (HCV) virus [Aslan et al., 2008b]. This organism is a small (50-nm), enveloped, single-stranded, positive sense RNA virus whose viral RNA is identical to messenger RNA (mRNA) and can thus be immediately translated by the host cell.

To detect the hepatitis C RNA sequence, a thiolated-anchor probe specific to HCV target RNA was attached to the surface of a gold thin film (figure 9A). The thiol groups (6- methylene chain) on the anchor probe interact (chemisorption) with the gold surface, which results in the formation of a monolayer of anchor probe on the gold surface [Whitesides and Laibinis, 1990]. The fluorophore (TAMRA)-labeled target RNA in whole blood was placed on the gold thin film and microwave heated for 1 min. The detection of HCV was achieved by surface plasmon coupled-luminescence (SPCL) technique (figure 9B). In this detection scheme, the emission spectrum from the assay is measured at a predetermined angle from the back of the gold thin film as shown in Figure 9B. It is important to note that one can also make the measurements at the same side as the excitation beam. However, the fluorescence signal from fluorophores within 200 nm (as in the assay here) is detected more efficiently from the back of the film [Liebermann and Knoll, 2000; Liebermann et al., 2000]. Figure 9C shows the emission spectrum of TAMRA increases as the concentration of HCV target RNA increases. A lower detection limit 100 nM HCV is achieved using the MA-SPCL technique (figure 9D). It is also important to note that synthetic target Hep C RNA was used in this study. One could thus employ aluminum thin films and microwave heating to extract target RNA from real HCV viruses and quantify the nucleic acid using both MAMEF and MA-SPCL techniques [Aslan et al., 2008b]. Further work is required to optimize this approach and will be reported in the future.

CONCLUSIONS

We have summarized our efforts to examine the technologies in an all purpose bio-detector that can be used to assist service personnel and first responders to make real-time, mission-critical decisions. We have shown that by combined use of microwave heating with metal-enhanced fluorescence we can rapidly detect the presence of both toxins and nucleic acid in less than 60 seconds. While the technology as currently configured offers comparable or slightly lower sensitivity than laboratory based assays, its rapidity (minutes versus hours), ability to operate in the presence of a range of environmental matrices and potential for miniaturization makes it a strong candidate for further development as a multiplex portable detector.

ACKNOWLEDGMENTS

The authors would like to acknowledge the Henry M. Jackson Foundation for financial support.

 

Figures

Figure 1: (A) Schematic representation of the effect of microwave heating on the protein detection surface assay; while water is selectively heated with microwaves silver nanoparticles remain at the same temperature before the microwave heating is initiated. (B) Emission spectra of a fluorophore (FITC) for a model protein assay run with microwave heating for 20 seconds at room temperature for 30 minutes. Real-color photographs taken through an emission filter are also shown as visual conformation of enhanced fluorescence observed due to the presence of silver nanoparticles. RT: Room Temperature. SIFs- silver island films.

 

Figure 1: (A) Schematic representation of the effect of microwave heating on the protein detection surface assay; while water is selectively heated with microwaves silver nanoparticles remain at the same temperature before the microwave heating is initiated. (B) Emission spectra of a fluorophore (FITC) for a model protein assay run with microwave heating for 20 seconds at room temperature for 30 minutes. Real-color photographs taken through an emission filter are also shown as visual conformation of enhanced fluorescence observed due to the presence of silver nanoparticles. RT: Room Temperature. SIFs- silver island films. [go to text reference]

Figure 2: Measurement of Lipid Peroxidation.

 

Figure 2: Experimental procedure for the MAMEF-based detection of Protective Antigen (PA) in High Throughput Screening (HTS) Wells. PA assay and control assay are shown separately. Mw-Microwave; Rhod G-Rhodamine G; 1° Ab-Primary capture antibody; 2° Ab Fab-Secondary Antibody Fab fragment. [go to text reference]

 

Figure 3: The effect of foam application on CWA spreading? Application of CASCAD foam from a backpack to a layer of 0.2 mm of methyl salicylate, MS, in a 60 cm x 60 cm x 20 cm tub from a distance of 3 m. Wind direction is left to right. The Swiss CAM is positioned upwind and measures the average MS concentration in the climate chamber.

 

Figure 3: Concentration-dependent emission spectra of Rhodamine G obtained during MAMEF-based detection of (A) PA and (B) Control Assay. (C) The emission peak of Rhodamine G at 575 nm as a function of PA concentration (range:10-2400 ug/ml). [go to text reference]

 

Figure 4: Concentration of phosphorus in the vapor space above the foam as a function of time for GB and CASCAD foam with foam layers of 3, 5, 7 and 10 cm.

 

Figure 4: (A) Concentration-dependent emission spectra of Rhodamine G obtained during MAMEF-based detection of PA that was present in a blood obtained from a sample rat. (B) The emission peak of Rhodamine G at 575 nm as a function of PA concentration (range:10-2400 ug/ml). [go to text reference]

 

Figure 5: Breakthrough times as a function of foam layer thickness.

 

 

Figure 5: (A) Normalized FDTD simulation of field intensity distributions for equilateral aluminum triangles in a simulated 2.45 GHz transverse electric (TE) polarized total-field scattered-field (TFSF) that propagates across the geometries from bottom to top. A schematic representation of the experimental setup used to release DNA from B anthracis spores and the vegetative organisms is also shown. (B) Thermal images of bow-tie aluminum structures before (t = 0 sec) and after (t = 2 sec) microwave heating. Duty cycle of microwave cavity is 1 second. (C) Arrhenius plot and fit for the chemiluminescence reaction on glass slides and the estimated temperature increase for the different sample geometries after exposure to low-power microwave pulses. Sample geometries are shown (insets). [go to text reference]

 

Figure 6: Correlation of dehydration times D<sub>90</sub> and breakthrough times for GB for a choice of foams for a layer of 5 cm.

 

 

Figure 6: (A) Transmission Electron Micrograph images of B. anthracis spores before, on glass and on aluminum triangles after 20 seconds of low power microwave heating. (B) TEM images of vegetative organisms both before after a 20 second exposure to microwave heating from between the aluminum triangles. (C) Gel electrophoresis study of DNA extracted vegetative cells by microwave heating and by using a commercially available procedure (labeled as No Mw). No Mw- no microwave heating; Mw-microwave heating. [go to text reference]

 

Figure 2: Measurement of Lipid Peroxidation.

 

 

Figure 7: (A) Experimental design depicting the organization of the DNA oligomers on SIFs used for the detection of Bacillus anthracis. Emission spectra of a fluorophore (TAMRA) labeled-ssDNA as a function of concentration (B) of a mixture of sample containing B. anthracis and B. cereus after 30 sec low power microwave heating and (C) the corresponding control experiment where the anchor probe is omitted from the surface. (D) Semi-logarithmic plot of the fluorescence emission intensity at 585 nm for the TAMRA-ssDNA (from (b) and (c) as a function of the target DNA concentration. TAMRA- Tetramethylrhodamine. ss-single stranded. SIFs- silver island films. [go to text reference]

 

Figure 8: Concentration of the simulant MS in the air of the climate chamber as a function of time for the 5 foams CASCAD, Reheal, FC 602, Jomos and MXOL as well as for the uncovered tub.

 

 

Figure 8: (A) Room temperature assay and (B-C) MAMEF assays for B. anthracis spores. B. anthracis spores (10^5 spores / mL) were microwave heated for 20 seconds on the MAMEF assay, and the same number of spores were also incubated on the assay platform for 2 hours without microwave heating in the room temperature assay. (D) MAMEF assays for vegetative organisms; PA-proficient strain and and for PA-deficient strain. Vegetative organisms were microwave heated for 20 seconds and were used in the assay. The anchor probe was omitted on the control assays. The anchor probe is omitted in the control assays. A.U.- arbitrary fluorescence units. [go to text reference]

Figure 8: Concentration of the simulant MS in the air of the climate chamber as a function of time for the 5 foams CASCAD, Reheal, FC 602, Jomos and MXOL as well as for the uncovered tub.

 

 

Figure 9: (A) Experimental design depicting the organization of the DNA oligomers on gold disks used for the detection of the model Hepatitis C (Hep C) assay. The lower panel shows the structures of the DNA oligomers used (conserved sequence for Hep C is shown). (B) Optical set-up for microwave-accelerated surface plasmon coupled luminescence (MA-SPCL) bioassays. (C) The emission spectra (measured at 315 degrees) for varying concentrations of TAMRA-labeled target DNA used in the MA-SPCL assay run in whole blood. (D) The calibration curve, semi-logarithmic plot of intensity at 585 nm vs. concentration of the target Hep C DNA. Control experiment: 2500 nM. [go to text reference]

 

references


Aslan, K. and Geddes, C.D. (2005) Microwave-accelerated metal-enhanced fluorescence: Platform technology for ultrafast and ultrabright assays. Analytical Chemistry 77, 8057-8067.

Aslan, K., Gryczynski, I., Malicka, J., Matveeva, E., Lakowicz, J.R. and Geddes, C.D. (2005) Metal-enhanced fluorescence: an emerging tool in biotechnology. Current Opinion in Biotechnology 16, 55-62.

Aslan, K., Holley, P. and Geddes, C.D. (2006) Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) with silver colloids in 96-well plates: Application to ultra fast and sensitive immunoassays, High Throughput Screening and drug discovery. Journal of Immunological Methods 312, 137-147.

Aslan, K., Malyn, S.N. and Geddes, C.D. (2007a) Microwave-Accelerated Surface Plasmon-Coupled Directional Luminescence: Application to fast and sensitive assays in buffer, human serum and whole blood. Journal of Immunological Methods 323, 55-64.

Aslan, K., Previte, M.J., Zhang, Y., Gallagher, T., Baillie, L. and Geddes, C.D. (2008a) Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min. Anal Chem 80, 4125-32.

Aslan, K., Previte, M.J., Zhang, Y. and Geddes, C.D. (2008b) Microwave-accelerated surface plasmon-coupled directional luminescence 2: A platform technology for ultra fast and sensitive target DNA detection in whole blood. J Immunol Methods 331, 103-113.

Aslan, K., Zhang, Y., Hibbs, S., Baillie, L., Previte, M.J. and Geddes, C.D. (2007b) Microwave-accelerated metal-enhanced fluorescence: application to detection of genomic and exosporium anthrax DNA in <30 seconds. Analyst 132, 1130-8.

Geddes, C.D. and Lakowicz, J.R. (2002) Metal-enhanced fluorescence. Journal of Fluorescence 12, 121-129.

Herzog, A.B., McLennan, S.D., Pandey, A.K., Gerba, C.P., Haas, C.N., Rose, J.B. and Hashsham, S.A. (2009 mplications of limits of detection of various methods for Bacillus anthracis in computing risks to human health. Appl Environ Microbiol. 75, 6331-9.

Kim, S.Y., Shin, S.J., Song, C.H., Jo, E.K., Kim, H.J. and Park, J.K. (2009) Destruction of Bacillus licheniformis spores by microwave irradiation. J Appl Microbiol 106, 877-85.

Liebermann, T. and Knoll, W. (2000) Surface-plasmon field-enhanced fluorescence spectroscopy. Colloids and Surfaces A-Physicochemical and Engineering Aspects 171, 115-130.

Liebermann, T., Knoll, W., Sluka, P. and Herrmann, R. (2000) Complement hybridization from solution to surface-attached probe-oligonucleotides observed by surface-plasmon-field-enhanced fluorescence spectroscopy. Colloids and Surfaces A-Physicochemical and Engineering Aspects 169, 337-350.

Mabry, R., Brasky, K., Geiger, R., Carrion, R., Jr., Hubbard, G.B., Leppla, S., Patterson, J.L., Georgiou, G. and Iverson, B.L. (2006) Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays. Clin Vaccine Immunol 13, 671-7.

Perani, M., Bishop, A.H. and Vaid, A. (1998) Prevalence of beta-exotoxin, diarrhoeal toxin and specific delta-endotoxin in natural isolates of Bacillus thuringiensis. FEMS Microbiol Lett 160, 55-60.

Peruski, A.H., and Peruski, L.F. (2003) Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clin Diag Lab Immunol 10, 506–513

Previte, M.J., Aslan, K. and Geddes, C.D. (2007) Spatial and temporal control of microwave triggered chemiluminescence: a protein detection platform. Anal Chem 79, 7042-52.

Tang, S., Moayeri, M., Chen, Z., Harma, H., Zhao, J., Hu, H., Purcell, R.H., Leppla, S.H. and Hewlett, I.K. (2009) Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles. Clin Vaccine Immunol 16, 408-13.

Walt, D.R. and Franz, D.R. (2000) Biological warfare detection. Anal Chem 72, 738A-746A.

Whitesides, G.M. and Laibinis, P.E. (1990) Wet Chemical Approaches to the Characterization of Organic-Surfaces - Self-Assembled Monolayers, Wetting, and the Physical Organic-Chemistry of the Solid Liquid Interface. Langmuir 6, 87-96.