The BARC biosensor applied to the detection of biological warfare agents

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Abstract

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.

Introduction

The threat of biological warfare is an increasing concern. Advances in microbiology and genetic engineering have made it possible to create extremely dangerous microorganisms. Because methods for detection of these agents and treatments for infection are currently limited, the development of highly sensitive sensors for early detection of biological warfare agents is crucial.

There are many ways to detect biological molecules, however, most biosensors rely on specific molecular recognition events such as antibody–antigen, DNA–DNA, or other ligand–receptor interactions between molecules. These recognition events are usually detected indirectly by a variety of labeling techniques that employ radioactive, enzymatic, or fluorescent labels, followed by scintillation counting, recording color changes, or monitoring light emission. Each method has its advantages and limitations. For example, radioimmunoassays, once an industry standard, require expensive instrumentation and the handling and disposal of radioactive wastes. Recently, several methods have been developed for direct measurement of DNA hybridization without the need for labeling: for instance, DNA hybridization has been detected directly using evanescent wave sensors (Watts et al., 1995, Abel et al., 1996), a quartz crystal microbalance (Okahata et al., 1992), and optical interferometric measurements of porous silicon (Lin et al., 1997). Other important advances have been in the area of DNA array technology. Methods have been developed for in situ synthesis of high density DNA arrays on glass and silicon substrates using photolithography (Fodor et al., 1991, Chee et al., 1996) and semiconductor photoresists (McGall et al., 1996). Devices are also being built that can deliver pre-synthesized oligonucleotides in small, well-defined spots onto solid substrates using ink-jet delivery (Blanchard et al., 1996) and micro-contact pen (also called pin, tip, or quill) spotting (Graves et al., 1998, Bowtell, 1999). Array technology is a significant advancement for biosensor development since it makes multianalyte analysis feasible. However, current readers and detectors for DNA array chips typically use radioisotope or fluorescence measurements, requiring relatively large and expensive instrumentation.

We are developing a revolutionary bioassay that uses DNA arrays, magnetic microbeads, and GMR sensors to detect biomolecules. A significant advantage of our sensor over more traditional detection methods is that we apply a magnetic field gradient to test the bonding between interacting molecular species. The applied force discriminates between species that are specifically and non-specifically bound, resulting in increased selectivity (i.e. lower false positives and negatives) and increased sensitivity (i.e. lower backgrounds).

Development of the BARC biosensor has been enabled by recent advances in two areas: magetoresistive materials and magnetic microbeads. Over the past few years, magnetoresistive materials have been discovered which allow the microfabrication of magnetic field sensors with high sensitivity and micrometer-scale size. These giant magnetoresistive (GMR) materials are typically thin-film metal multilayers, the resistance of which changes in response to magnetic fields. By passing a current through a strip of GMR material and measuring its resistance, local magnetic fields can be measured. The BARC sensor uses this technology to detect the presence of magnetic microbeads, which a number of companies have independently developed for biological separations and purification. The beads are typically paramagnetic particles (they are only magnetic in the presence of a magnetic field), containing an iron oxide core and a polymer coating onto which proteins or antibodies are attached, and we are evaluating several types for use in the BARC biosensor.

The prototype BARC instrument, currently under development, is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet (Fig. 1) (Baselt et al., 1998). Thiolated DNA probes will be patterned onto a gold layer on the solid substrate chip directly above the GMR sensors using micro-contact pen spotting. Prior to analysis with BARC, sample DNA will be biotin labeled during PCR amplification. When a sample is injected into the instrument, it will hybridize with probes on the chip surface if the complementary sequence is present. Streptavidin-labeled paramagnetic beads will then be added that specifically bind to the biotinylated sample DNA on the chip surface. Any beads that are not specifically bound will be removed by applying a magnetic field gradient with the electromagnet. The GMR sensors will detect the beads remaining on the surface, and the intensity and location of the signal will indicate the concentration and identity of pathogens present in the sample (Fig. 2). In this manuscript, we describe our current progress in the development of each of the following BARC components: DNA patterning and the hybridization assay; the GMR sensor chip, magnetics, and signal detection; and fluidics.

Section snippets

DNA patterning and hybridization assay

Although BARC will be capable of detecting a wide variety of molecular recognition reactions, we are initially using DNA hybridization to detect the following biological warfare agents: Bacillus anthracis, Yersinia pestis, Brucella suis, Francisella tularensis, Vibrio cholerae, Clostridium botulinum, Campylobacter jejuni, and Vaccinia virus. In order to avoid expending our limited supply of sensor chips, we have been developing the DNA immobilization chemistry and optimizing the hybridization

Conclusions and future directions

We have shown here our results and progress in the development of each of the components for the BARC biosensor. The current prototype is a table-top instrument designed to detect several biological warfare agents simultaneously. The total active area of our 64-element GMR arrays is limited because each sensor has a separate connection to the off-chip circuitry. To increase the active area of the chip and thereby achieve the full multi-analyte potential of BARC, we will eventually need to add

Acknowledgements

This work was supported in part by the Defense Advanced Research Projects Agency. CRT is grateful for an American Society for Engineering Education postdoctoral fellowship, and PES for a National Research Council postdoctoral fellowship. We would like to thank O. Millard for her work on the optical assay and patterning, K. Lee for technical assistance on the electronics, and G. Long and C. Drombetta from NMRI for helpful discussions.

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