Developments in diagnostic technologies for bioterrorism agents

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Figure 1. Basic structure of an organic light-emitting device (OLED). The total thickness of the OLED, not including the glass or plastic substrate and the sensor film, is ~0.5 µm (a). Basic “back-detection” geometry of an OLED-based O2 and glucose sensor. The OLED pixels are formed between the indium tin oxide (ITO) anode and the Al metal cathode (b) (click to enlarge).

Diagnostic technologies for bioterrorism agents have received an upwelling of interest in recent years. Driven by increased funding from government agencies, as well as by exposure from government- and industry-sponsored scientific conferences (e.g., Biotechnica America, the American Society for Microbiology's Biodefense Research Meeting, and the Army Science Conference), innovation in detection technologies has progressed at a quick pace. Many of these developments are increasingly focused on miniaturization, field deployability, capability to detect and analyze multiple analytes on a single platform, and in-line and on-the-spot analysis. In addition, several Web sites have aided collaboration by reporting ongoing research efforts on new detection methods.

At a symposium on novel diagnostics held in April 2004 at the U.S. Department of Agriculture's National Animal Disease Center in Ames, IA, a representative selection of such cutting-edge technologies were on display. These included a photoluminescence-based biological sensor, a suitcase time-of-flight (TOF) mass spectrometer, a chip-scale diagnostic platform produced by the Biochip Technology Center at Argonne National Laboratory (Argonne, IL), and advanced photonics sensors.

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Table I. Comparison of diagnostic technologies (click to enlarge).

This article will look at developments in each of these technologies, as well as those in older technologies such as gas chromatography, as they pertain to bioterrorism detection. Areas of discussion will include field application, portability, ability to provide on-the-spot analysis, and analyte detection limits (see Table I).

OLEDs

Organic light-emitting devices (OLEDs) have been developed to detect environmental pollutants (e.g., hydrazine), biomedically significant analytes (e.g., oxygen and glucose), as well as agents of bioterrorism (e.g., lethal factor produced by Bacillus anthracis).1 The basic construction of the OLED consists of a plastic or glass slide on which the substrate for the analyte is embedded (see Figure 1a). When the substrate binds its ligand, light is emitted, which is detected by a photodetector (see Figure 1b). The surface of the slide contains the bound substrate labeled with a fluorescence resonance energy transfer (FRET) pair of labels. An example of this is the synthetic peptide substrate for anthrax, which is labeled with a FRET pair consisting of a donor and acceptor label. This has the result of quenching the peptide's fluorescence. Upon cleavage of the peptide by the anthrax lethal factor (LF), the peptide fragments separate, allowing the donor-labeled peptide fragment to fluoresce.

Factors that affect the amplitude of the fluorescent signal, and hence the sensitivity of the detector, are dependent on the fluorescent FRET pairs used and the level of LF that can be detected. The physical and optical characteristics of the device may also influence the sensitivity of measurements.¹

OLEDs have inherent advantages over inorganic LEDs or laser sources in that they can be operated at an extremely high brightness (in excess of 106 Cd/m2 under pulsed operation, compared to the ~2000 Cd/m2 with a fluorescent tube).¹ These bright OLEDS are available commercially and are relatively simple to fabricate, either by spin-coating or ink-jet printing of polymer solutions.² They can be fabricated in almost any two-dimensional shape on a plastic substrate to yield flexible displays or light sources.³ Due to the nearly ideal coupling with the sensing component and the ability to operate them in an ac or pulsed mode, OLEDs consume little power and produce little heat.

Ultimately, OLEDs will be constructed as multianalyte detection systems. It is expected that as the volume of manufactured OLEDs increases, their eventual cost will drop, making such devices not only field deployable but potentially disposable. Detection limits for glucose, hydrazine, and LF are sufficient at 0.1 mg/ml, 50 µg/ml, and 1.2 pmol, respectively, and small sizes mean that such a device would be field deployable.

Suitcase TOF

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Figure 2. Suitcase time-of-flight mass spectrometer for demonstration purposes. The data system is not shown (click to enlarge).

Suitcase TOF devices comprise a miniaturized matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer arranged in a suitcase-sized enclosure.^3,4 The instruments have four major components: a vacuum system, an optical system with a nitrogen laser, a source/analyzer, and an electronics/data system that includes high-voltage power supply modules (see Figure 2). An aerosol sample-collection system deposits particles onto a VHS magnetic recording tape. The tape is then transferred to a module for deposition of the MALDI matrix onto the aerosolized sample spot.

Following the matrix deposition and sample drying (including internal standards), the sample is moved to the focal point of the laser. The laser is fired, causing the desorbing of the matrix and sample, and a drifting of the charged molecular ions toward the detector. Data are processed by a signal-processing system that filters noise and converts the time signal to a mass/charge. The system then determines the baseline and identifies the major peaks.

This field-deployable mass spectrometer has produced good results when measuring standard proteins with a 1200–66,000 molecular weight, as well as with biological toxins and intact microorganisms (e.g., spore components from Bacillus globii).^4-7 The performance of suitcase TOF compares well with that of commercial mass spectrometers.^5-8 Limits for detecting biological materials are in the range of 4 pmol for proteins and peptides, and 1 µg of spores.

Biochip Technologies

The microarray platform developed at the Biochip Technology Center has been designed specifically for in-the-field detection of ribosomal RNA (rRNA). Using the biochip system, RNA detection can be automated and quantified; and, provided that their genome information is stored in a database, most microorganisms can be detected. Even so, the development of microarray methods for environmental microbiology has been relatively slow and is limited by the expense of microarray printing and imaging equipment.⁹ In general, microarray- and gene-detection methods are limited by the time and labor required for sample processing and nucleic acid purification; inefficient purification or concentration of nucleic acids at low target concentrations is especially prevalent for environmental samples. Another problem that sometimes arises with gene-based detection methods is the coextraction of inhibitory components that interfere with subsequent molecular manipulations for PCR. These considerations are especially important for field and point-of-use applications such as real-time microbial monitoring.^9,10

In addition, PCR amplification represents a significant bottleneck for routine application and deployment of DNA microarrays in environmental microbiology.⁹ This has necessitated the need for development of specific and sensitive direct nucleic acid detection methods. The objectives of the sRNA biochip program were to detect as many different microorganisms as possible simultaneously, and to incorporate bioanalytical detection methods that lend themselves to automation and field deployment. Among the advantages of such a technique is the potential to monitor and detect a large number of microorganisms using the multiplex array format of the detection platform. Doing so circumvents some of the trouble spots of PCR, in which fragments must be amplified before detection methods can proceed.

The detection limit of the biochip platform is currently 0.5 µg of RNA. For environmental samples, soluble substances that interfere with the detection of autofluorescence, or cause fluorescence quenching, are being addressed.

MAGIChips. The detection of rRNA using region-specific DNA probes provides another alternative to PCR-based technologies and other DNA detection methods. MAGIChips (micro arrays of gel-immobilized compounds on chips), the product of a collaboration between the Biochip Technology Center and
the Englehardt Institute of Molecular Biology (Moscow), is one such technology developed to address the need to rapidly and accurately identify
microorganisms.¹¹

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Figure 3. MAGIChips depicting the positioning of the probe-permeated gel pads (click to enlarge).

MAGIChips are three-dimensional DNA microarrays formed from oligonucleotide-impregnated gel pads (see Figure 3). A microorganism pellet is first collected from a fresh culture. After the pellet is washed and lysed, the nucleic acids are bound to a column. The RNA-containing nucleic acids are eluted, then fragmented, labeled with a fluorophore, and hybridized onto a gel-matrix chip containing DNA probes for specific and unique microbial rRNAs. Preparation of relatively small RNA fragments (fewer than 500 base pairs) permits easier diffusion through the gel matrix than larger RNA fragments.

This design increases surface area by a factor of 50, allowing for a probe density of 1012 molecules per gel pad. Specific probes can recognize unique rRNA sequences, and all together, more than 2700 probes can be loaded onto a single MAGIChip. For example, Bacillus species can be distinguished by hybridization to fewer than a hundred probes for the same target species.

In a recently published paper, it was reported that the MAGIChip is able to detect and identify a broad range of biological species in a matter of hours.¹¹ The current detection limit is 0.5 µg of RNA and, as approximately 100 unique probes per organism are used, is specific for the microorganism. Applications include the detection of a broad spectrum of pathogenic organisms, target genes necessary for drug development, and plant and animal diseases. Future developments may include protein MAGIChips for clinical diagnostics, bacterial forensics, viral identification, drug customization, pharmacogenomics, and agricultural applications.

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Figure 4. A chip-based analyzer for DNA and proteins, based on technology developed by the Center for Advanced Biomedical Photonics at Oak Ridge National Laboratory (Oak Ridge, TN) (click to enlarge).

Advanced Photonics. Developed at Argonne National Laboratory by Tuan Vo-Dinh, PhD, and licensed to HealthSpex Inc. (Knoxville, TN), this technology consists of a miniaturized laser-diode system (see Figure 4). A chip containing known DNA and antibody probes is exposed to a drop of processed blood in order to separate proteins and DNA sequences. This occurs, presumably, through some affinity-type binding and elution. DNA and protein molecules are subsequently labeled with a fluorescent dye and allowed to bind to the chip's complementary DNA and protein probes. A laser diode illuminates the array sites, resulting in fluorescence at the sites of DNA hybridization and protein binding. The signals are then sent to a microprocessor chip for analysis and comparison with probes known to be at specific locations.

In 2002, when Vo-Dinh published his research, he reported that the chips could detect the tuberculosis bacterium, an HIV gene, a cancer suppressor gene, the anthrax bacillus, and Escherichia coli found in contaminated food.¹² According to Vo-Dinh, if the detection method employed uses laser-induced fluorescence, the detection limits of the analytes are potentially at the single- molecule level. The system is constructed to be field deployable.

Gold Biosensor Chip. The development of biosensors that are capable of detecting multiple analytes in a single sample is a goal for any biosensor research program. In general, biosensors exhibit clear advantages over the more-conventional methods for analyte detection, such as enzyme immunoassays, in that they consume lower amounts of materials and therefore are less expensive to use.^13,14

Biosensors have wide application in medicinal analysis, environmental screening, and quality control. Shorter analysis time due to the reduction or elimination of sample pretreatment is another advantage. Biosensors developed using immobilized antibody are referred to as immunosensors. Antibody-antigen binding is highly specific and has high affinity constants, in the order of 108 M–1. The sensitivity for detecting DNA-adducts is at the femtomol level. The instrument is not field deployable.

An article published last year describes the construction of a monoclonal (MAb)-gold biosensor chip with low-temperature laser-induced fluorescence detection. Depending on the MAb immobilized, a number of either single or multiple analytes can be detected. In this particular study, two different MAb were immobilized in order to detect two types of DNA-benzo[a]pyrene, a potent carcinogen. To optimize detection of the analyte, the linker distance immobilizing the MAb can be increased from about 9 nm using dithiobis (succinnimidyl) proprionate (DSP) to approximately 18 nm by using a protein A-MAb/DSP binding to the gold chip. The authors conclude that this new chip methodology can be scaled up for detecting multiple luminescent antigens for which monoclonal antibodies can be generated.¹³

Gas Chromatography

Although gas chromatography is far from a novel method, the technology has been highly refined and is included here because of its importance in detecting bioterrorism microorganisms. For example, in a study led by Jeffrey D. Teska, PhD, gas chromatography was used in conjunction with the Sherlock microbial identification system (MIS) by MIDI Inc. (Newark, DE) to analyze fatty acid methyl esters and identify organisms.¹⁵ The Sherlock MIS, used together with the Sherlock Bioterrorism (BIOTER1.0) Library, can detect more than 1500 bacterial species, including six considered to be potential bioterrorism agents.

The analysis itself takes about three hours, not including the time needed for culturing the microorganisms. The sensitivity is 40 mg of microorganism. At the current time, this technology is not field deployable.

Conclusion

The list of diagnostic technologies described above is by no means comprehensive. Newer and improved versions of chip and PCR technology are continually being developed. For example, last summer, a greatly improved PCR technology was described in IVD Technology.¹⁶

These new diagnostic technologies are based on a number of novel platforms. Although some platforms are still under development and not yet commercially available, published articles indicate their utility for detecting single or multiple agents at high sensitivities. If current trends continue, the next several years will witness an influx of new field-deployable instrumentation and applications.

References

01. R Shinar et al., “Structurally Integrated Organic Light-Emitting Device-Based Sensors for Oxygen, Glucose, Hydrazine, and Anthrax,” Proceedings of the International Society for Optical Engineering 5588 (2004): 59–69.

02. G Gustafson et al., “Flexible Light-Emitting Diodes Made from Soluble Conducting Polymers,” Nature 357 (1992): 447.

03. Johns Hopkins University, Applied Physics Laboratory, “Miniature Time-of-Flight Mass Spectrometry,” in Applied Physics Laboratory Home Page [online]—[cited 16 February 2005]; available from Internet: http:// jhuapl.edu/programs/rtdc/SensorTechnology/MiniTOF.html.

04. SA Ecelberger et al., “Suitcase TOF: A Man-Portable Time-of-Flight Mass Spectrometer,” Johns Hopkins APL Technical Digest 25, no. 1 (2004): 14–19.

05. PR Hardwidge, “Applications of Quantitative Mass Spectrometry in Bacterial Pathogenesis,” American Biotechnology Laboratory (December 2004): 22–25.

06. MD Antoine, “Mass Spectral Analysis of Biological Agents Using the BioTOF Mass Spectrometer,” Johns Hopkins APL Technical Digest 25, no. 1 (2004): 20–26.

07. TJ Cornish, S Ecelberger, and W Brinckerhoff, “Miniature Time-of-Flight Mass Spectrometer Using a Flexible Circuitboard Reflector,” Rapid Communications in Mass Spectrometry 14 (2000): 2408–2411.

08. PA Demirev, AB Feldman, and JS Lin, “Bioinformatics-Based Strategies for Rapid Microorganism Identification by Mass Spectrometry,” Johns Hopkins APL Technical Digest 25, no. 1 (2004): 27–37.

09. CJ Murray, “Protein Chips Could Speed Cancer Detection,” in Embedded.com [online] October 16, 2003 [cited 16 February 2005]; available from Internet: http://embedded.com/ showArticle.jhtml?articleID=15306197 [10].

10. J Small et al., “Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotides Microarrays,” Applied and Environmental Microbiology 67 (2001): 4708–4716.

11. ML Theodore, J Jackman, and WL Bethea, “Counterproliferation with Advanced Microarray Technology,” Johns Hopkins APL Technological Digest 25, no. 1 (2004): 38–43.

12. S Henkel, “One Blood Drop Provides DNA and Disease Data,” in Sensors Online [online] September 2002 [cited 16 February 2005]; available from Internet: www. sensorsmag.com/articles/0902/10/main.shtml.

13. NM Grubor et al., “Novel Biosensor Chip for Simultaneous Detection of DNA-Carcinogen Adducts with Low-Temperature Fluorescence,” Biosensors and Bioelectronics 19 (2004): 547–556.
14. SD Duhacheck et al., “Monoclonal Antibody-Gold Biosensor Chips for Detection of Depurinating Carcinogen-DNA Adducts by Fluorescence Line-Narrowing Spectroscopy,” Analytical Chemistry 72 (2000): 3709–3716.

15. JD Teska, SR Coyne, and JW Ezzell, “Identification of Bacillus anthracis Using Gas Chromatographic analysis of Cellular Fatty Acids and a Commercially Available Database,” in Agilent Technologies Applications [online] May 22, 2003 [cited 16 February 2005]; available from Internet: www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=33573 [11].

16. S Chen, G Selecman, and B Lemieux, “Expanding Rapid Nucleic Acid Testing,” IVD Technology 10, no. 6 (2004): 51–59.

Louisa B. Tabatabai is a research chemist at the National Animal Disease Center (Ames, IA) and a professor of biochemistry at Iowa State University (Ames, IA). She can be contacted at lbt@iastate.edu [12].

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