The BARC biosensor applied to the detection of biological warfare agents.

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.

A biosensor based on magnetoresistance technology.

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.

Scanning probe microscopy.

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.

Float-polishing process and analysis of float-polished quartz.

A fluid-mechanical model is developed for the float-polishing process. In this model laminar flow between the sample and the lap results in pressure gradients at the grooves that support the sample on a fluid layer. The laminar fluid motion also produces supersmooth, damage-free surfaces. Quartz substrates for applications in high-stress environments were float polished, and their surfaces were analyzed by optical scatterometry, photoacoustic spectroscopy, and atomic force microscopy. The removal of 100 µm of material by a lapping-polishing process, with final float polishing, left low levels of subsurface damage, with a surface roughness of approximately 0.2-nm rms.

Subfibrillar structure of type I collagen observed by atomic force microscopy.

We have imaged native rat tail and reconstituted bovine dermal type I collagen by atomic force microscopy, obtaining a level of detail comparable to that obtained on the same samples by transmission electron microscopy. The characteristic 60-70 nm D periodicity consists of ridges exhibiting high tip-sample adhesion alternating with 5-15-nm-deep grooves having low adhesion. We also observe an intraperiod or "minor" band consisting of 1-nm-deep grooves, and "microfibrils" arranged parallel to or inclined approximately 5 degrees to the fibril axis. In air collagen fibrils exhibit negligible compression under the forces exerted by the tip. When immersed in water the subfibrillar features disappear and the fibrils become softer, compressing by 5% of their height under an 11-nN force. Material on the surface of the sample sometimes accumulates on the atomic force microscope tip; contrary to expectation such tip contamination can improve as well as reduce resolution.

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.