Gil U. Lee

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.

Biosensor based on force microscope technology

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.

A high-sensitivity micromachined biosensor

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.

Atomic Force Microscope Image Contrast Mechanisms on Supported Lipid Bilayers

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.

Characterization of the physical properties of model biomembranes at the nanometer scale with the atomic force microscope

Interaction forces and topography of mixed phospholipid-glycolipid bilayers were investigated by atomic force microscopy (AFM) in aqueous conditions with probes functionalized with self-assembled monolayers terminating in hydroxy groups. Short-range repulsive forces were measured between the hydroxy-terminated probe and the surface of the two-dimensional (2-D) solid-like domains of distearoyl-phosphatidylethanolamine (DSPE) and digalactosyldiglyceride (DGDG). The form and range of the short-range repulsive force indicated that repulsive hydration/steric forces dominate the interaction at separation distances of 0.3-1.0 nm after which the probe makes mechanical contact with the bilayers. At loads < 5 nN the bilayer was elastically deformed by the probe, while at higher loads plastic deformation of the bilayer was observed. Surprisingly, a short-range repulsive force was not observed at the surface of the 2-D liquid-like dioleoylphosphatidylethanolamine (DOPE) film, despite the identical head groups of DOPE and DSPE. This provides direct evidence for the influence of the structure and mechanical properties of lipid bilayers on their interaction forces, an effect which may be a major importance in the control of biological processes such as cell adhesion and membrane fusion. The step height measured between lipid domains in the AFM topographic images was larger than could be accounted for by the thickness and mechanical properties of the molecules. A direct correlation was observed between the repulsive force range over the lipid domains and the topographic contrast, which provides direct insight into the fundamental mechanisms of AFM imaging in aqueous solutions. This study demonstrates that chemically modified AFM probes can be used in combination with patterned lipid bilayers as a novel and powerful approach to characterize the nanometer scale chemical and physical properties of heterogeneous biosurfaces such as cell membranes.

Traveling wave magnetophoresis for high resolution chip based separations

A new mode of magnetophoresis is described that is capable of separating micron-sized superparamagnetic beads from complex mixtures with high sensitivity to their size and magnetic moment. This separation technique employs a translating periodic potential energy landscape to transport magnetic beads horizontally across a substrate. The potential energy landscape is created by superimposing an external, rotating magnetic field on top of the local fixed magnetic field distribution near a periodic arrangement of micro-magnets. At low driving frequencies of the external field rotation, the beads become locked into the potential energy landscape and move at the same velocity as the traveling magnetic field wave. At frequencies above a critical threshold, defined by the beads hydrodynamic drag and magnetic moment, the motion of a specific population of magnetic beads becomes uncoupled from the potential energy landscape and its magnetophoretic mobility is dramatically reduced. By exploiting this frequency dependence, highly efficient separation of magnetic beads has been achieved, based on fractional differences in bead diameter and/or their specific attachment to two microorganisms, i.e., B. globigii and S. cerevisiae.

Structure, force, and energy of a double-stranded DNA oligonucleotide under tensile loads

Abstract The end-to-end stretching of a duplex DNA oligonucleotide has been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) simulations and atomic force microscopy (AFM) experiments. Near quantitative agreement between the calculations and experiments was obtained for both the extension length and forces associated with strand separation. The PMF calculations show that the oligonucleotide extends without a significant energetic barrier from a length shorter than A-DNA to a length 2.4 times the contour length of B-DNA at which the barrier to strand separation is encountered. Calculated forces associated with the barrier are 0.09±0.03 nN, based on assumptions concerning tip and thermal-activated barrier crossing contributions to the forces. Direct AFM measurements show the oligonucleotide strands separating at 2.6±0.8 contour lengths with a force of 0.13±0.05 nN. Analysis of the energies from the MD simulations during extension reveals compensation between increases in the DNA-self energy and decreases in the DNA-solvent interaction energy, allowing for the barrierless extension of DNA beyond the canonical B form. The barrier to strand separation occurs when unfavorable DNA interstrand repulsion cannot be compensated for by favorable DNA-solvent interactions. The present combination of single molecule theoretical and experimental approaches produces a comprehensive picture of the free energy surface of biological macromolecular structural transitions.

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 +/- 33, 690 +/- 274, and 1322 +/- 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.

Development and characterization of surface chemistries for microfabricated biosensors

The high cost and harsh processing conditions associated with microfabricated biosensors demand a new approach to receptor immobilization. We have grafted biotin labeled, 3400 molecular weight poly(ethylene glycol) (PEG) to silicon surfaces to produce a dense PEG monolayer with functionally active biotin. These surfaces have been activated with antibodies through the strong streptavidin-biotin interaction by simply incubating the surfaces with antibody-streptavidin conjugates. The stability of the biotinylated PEG monolayers produces a sensing element that can be regenerated by removal of the streptavidin conjugate and stored in a dry state for extended periods of time.

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.