Introduction: To respond to a bioterrorist incident or enemy attack, rapid, easy-to-use sensors are urgently needed. Currently, several commercially available sensors are available that are capable of detecting biological weapons (BW) and other pathogens, but most of these sensors are not designed for rapid testing of multiple biological threats at the same time. We are developing an Array Biosensor to fill this unique requirement: rapid, field-deployable detection of multiple biological threats in different kinds of samples. The Array Biosensor uses antibodies as recognition elements to detect targets with high sensitivity and selectivity. A series of different "capture" antibodies are attached to the surface of a microscope slide at specific locations (arrays) and are used to grab threat agents out of the sample. A second, fluorescent "tracer" antibody binds to the captured target, and the resulting fluorescent "sandwich" is detected using a CCD camera. If multiple "capture" or "tracer" antibodies are used, each binding to a different BW agent or pathogen, we can simultaneously detect and identify multiple different targets on the same slide. The optical components of the system include a red diode laser, like that used in a laser pointer, and a digital camera. The assays are fast (10-15 min), sensitive, and specific.

To make the array biosensor portable for field use, we have developed and optimized a novel fluidics component milled in a plastic cube (Fig. 12). The fluidics cube allows the operator to preload all the necessary solutions and perform the test while simultaneously imaging the microscope slide. The entire fluidics control system fits within a small tacklebox and can be operated in a fully automated fashion.

FIGURE 12
Ergonomically designed fluidics cube. The cube includes six reservoirs for sample and six reservoirs for fluorescent "tracer" antibodies. The solutions can be dried and stored inside the cube for extended periods. The cube unit also includes a flow channel device for passing the fluids over the microscope slide and a specially molded gasket for a fluid-tight seal. The protruding end of the unit is designed for easy handling and insertion into the optics box. The entire unit minimizes potential sources of leakage, size, and weight.

Variety of Tests: Because food and water sources are potential targets for bioterrorists, we have recently developed tests for pathogens for use in a wide variety of foods. Tests for a food poisoning agent, staphylococcal enterotoxin B (SEB), conducted on milk, homogenized ham, ground beef, cantaloupe, and eggs, were as sensitive as tests conducted in laboratory buffer. Tests for Salmonella showed equivalent results in ground cantaloupe, washes of chicken carcasses, bean sprouts, and eggs as in buffer. The presence of irrelevant bacteria, even in 1000-fold excess, did not interfere with Salmonella detection.

To increase the number of tests performed on a single slide, we used state-of-the-art technology developed during the Genome Project, an automated dispensing system, to deposit "capture" antibodies onto our slides. We have demonstrated that 32 tests can be performed in each lane, with up to six samples analyzed simultaneously. While 192 separate tests are shown (Fig. 13), this number can potentially be much greater. The limitation on the number of targets that can be screened for in each sample is primarily a function of image resolution and antibody availability.

FIGURE 13
An automated printer, designed for depositing high-density arrays of DNA, was used to create patterns of capture antibodies on microscope slides. Eight spots of each capture antibody (indicated at the top) were immobilized in each lane. Six samples (indicated to the right of the image) were analyzed simultaneously for all four targets. Thus, 32 assays were performed per sample, yielding a total of 192 assays in 12 minutes. CT: Cholera toxin; B. glob: Bacillus globigii; SEB: Staphylococcal enterotoxin B.

FIGURE 14
The portable array biosensor with fluidic cubes. To conduct a test, the cube is filled with six samples and fluorescent "tracer" antibodies. It is inserted into a spring-loaded Teflon slot. Three finger-sized peristaltic pumps draw the samples and fluorescent reagents across the microscope slide. Light emitted from a laser shines across the end of the waveguide, exciting any fluorescent "sandwiches" that form on the surface. A digital camera records the location of the fluorescent spots, and image analysis software quantifies the bound target.

Conclusions: The portable array biosensor (Fig. 14) addresses the need for rapid, sensitive, and specific analysis for multiple biohazards at the site of sample collection. We have demonstrated that the sensor is effective for rapid detection of pathogens, toxins, and clinical markers in a wide variety of sample types. Real-world testing is now underway for bioterrorism defense, infectious disease detection, food and water safety, and environmental monitoring applications.

[Sponsored by ONR, NSWC, NASA, EPA, and USDA]