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.

We are developing a novel biosensor-the Bead Array Counter (DARC)-which has the potential to simultaneously detect thousands of analytes. BARC is an adaptation of atomic force microscope (AFM) experiments in which the interaction forces that bind DNA-DNA, antibody-antigen, or ligand-receptor pairs together are measured. Using magnetic microbeads to exert force and numerous magnetoresistive sensors to detect the microbeads, it is possible to perform thousands of such experiments simultaneously. Thus, with BARC, it is possible to detect individual molecular bonds and test their strength. For our preliminary investigation, we are developing BARC as a hybridization assay in which DNA probes are covalently attached to the sensors by inkjet patterning techniques. We are also:developing miniature fluidic devices through modeling and testing of prototype designs that will assure the delivery of microliter volumes of fluid and the even dispersion of microbeads, at the site of detection. Operational idiosyncrasies of BARC require special considerations in the construction and actuation of the miniaturized pumps and valves, such:as non-magnetic components and attention to particulate matter. Other objectives include thermoplastic replication of the fluidic channels, modularity of the fluidic and electronic components, ease of sample delivery and assay preparation, reduction of contamination between assays, and materials compatibility with the assay chemistry.

We are developing a biosensor that will measure, at the level of single molecules, the forces that bind DNA–DNA, antibody–antigen, or ligand-receptor pairs together. The Bead Array Counter (BARC) will use these interaction forces to hold magnetic microbeads to a solid substrate. Microfabricated magnetoresistive transducers on the substrate will indicate whether or not the beads are removed when pulled by magnetic forces. By adapting magnetoresistive computer memory technology, it may be possible to fabricate millions of transducers on a chip and detect or screen thousands of analytes. The multi-analyte capability of this portable sensor would be ideal for on-site testing, while the potential to directly gauge intermolecular interaction strengths suggests drug discovery applications.

This paper reviews work conducted at the Naval Research Laboratory to measure surface forces, adhesion and nanomechanical properties of materials using an Atomic Force Microscope (AFM). The article portrays the AFM as an enabling technology, evolving fr om a qualitative picturing-taking tool to a quantitative probe of material!, properties. For example, surface forces can be used to reduce contact forces during imaging, improve image resolution, and change image contrast. Force-distance curves and maps can be used to correlate surface topography with the adhesion, surface energy, frictional and mechanical properties of materials. Applications presented include imaging phase-segregated Langmuir-Blodgett films, measuring molecular recognition forces between individual molecules, determining the absolute modulus of materials from nanoindentation, and studying the nucleation of dislocations. Finally, the evolution of the AFM towards, dedicated micromachined instruments and sensors for on-line process control and sensing applications is introduced.

The Force Amplified Biological Sensor (FABS) is a desktop or portable instrument currently under development at the Naval Research Laboratory. FABS will use a rapid automated immunoassay to detect analytes such as proteins, viruses, and bacteria. The assay uses forces produced by micron-sized magnetic particles to pull on antibody-antigen bonds. Microfabricated piezoresistive cantilevers measure the resulting piconewton-level forces with sufficient sensitivity to detect single antibody-antigen bonds. These forces also serve to characterize the bonds, allowing FABs to distinguish specific antibody-antigen bonds from nonspecific interactions.

During the past year, scanning probe microscopy, especially atomic force microscopy (AFM), has taken root in the biological sciences community, as is evident from the large number of publications and from the variety of specialized journals in which these papers appear. Furthermore, there is a strong indication that the technique is evolving from a qualitative imaging tool to a probe of the critical dimensions and properties of biomolecules and living cells. The next stage of the evolution involves the development of microinstruments for process control and sensing applications. Recent advances have been reported in AFM instrumentation and method. For example, the tapping mode of operation is becoming the method of choice to image biological molecules; work to extend tapping-mode operation in liquids has been reported. Biological molecules can also be imaged at low temperature in a cryo-AFM with improved resolution. The measurement of recognition forces between individual molecules continues to attract much attention and has spawned new concepts for ultra-sensitive biosensors. The AFM is being used increasingly for property measurements such as determining the viscoelastic properties of biological molecules. Finally, structural studies using the AFM abound. Some specific highlights include the mapping of DNA using restriction enzymes, imaging during DNA transcription and determining the mode of drug binding to DNA.

We are developing a sensor capable of detecting biological species such as cells, proteins, toxins, and DNA at concentrations as low as 10−18 M. The force amplified biological sensor will take advantage of the high sensitivity of force microscope cantilevers to detect the presence of as little as one superparamagnetic particle bound to a cantilever by a sandwich immunoassay technique. The device, which will ultimately be small enough for hand-held use, will perform an assay in about 10 min. Lock-in detection and use of a reference cantilever will provide a high degree of vibration immunity. An array of ten or more cantilevers will provide greater sensitivity and the capability to detect multiple species simultaneously. The force amplified biological sensor also offers the potential of distinguishing and studying chemical species via its ability to measure binding forces.