Superparamagnetic microparticles have been widely used for biological affinity separations (as described above) as they offer a simple and efficient method for the purification of cells, viruses, and macromolecules. The microparticles used in these applications need to be stable in water, uniform in size, and responsive to magnetic field gradients. The micron size paramagnetic particles that have been developed for these applications are typically composed of nanometer scale particles of maghemite (or magnetite) distributed in a polymer matrix. These particles are coated with a polymer that stabilizes them in aqueous solution and provide chemical groups to which proteins and polynucleotides can be conjugated. These magnetic particles have also been used in novel modes of drug delivery, in vivo imaging, analytical measurement, diagnosis and hyperthermia therapy.

This laboratory has developed several new techniques for synthesizing superparamagnetic particles composed of either an iron-gold nanorods and self-assembled iron oxide nanoparticles. In the latter, an emulsion templated, UV initiated polymerization scheme is used to form very high magnetization superparamagnetic microparticles. A unique property of this scheme is that the surface chemistry of the microparticles can be controlled by the adsorption of species from the aqueous phase of the emulsion into the particles before cross-linking. These particles have been commercialized by MagSense Life Sciences, Inc [http://www.magsenselifesci.com/].

2. Study of forces in single molecules and cells

The development of ultrasensitive force transducers has made it possible to directly measure inter- and intramolecular forces between and within individual macromolecules. Single molecule force measurements have been made in biomolecular systems with the atomic force microscope (AFM), micropipette, optical tweezers, flow chamber, and magnetic tweezers, with each technique having an optimal force and displacement sensitivity range. The micropipette and AFM have been used to study the physical behavior of intermolecular interactions and individual molecules under tensile load. Torque has been applied with magnetic tweezers, which has been used to characterize the influence of writhe on the interaction of proteins with DNA and the mechanical properties of F-actin networks in living cells. These measurements have provided a basis for understanding the behavior of biomolecular systems under non-equilibrium conditions and the intramolecular forces responsible for macromolecule structure. This has resulted in new insight into the important role force plays in cellular behavior.

This research group has played an active role in the study of forces in biomolecular systems. Our early work focused on using the AFM to understand the nature of specific molecular interactions under load. This included pioneering measurements of the influence of force on the bond lifetime of streptavidin-biotin and complementary strands of DNA. This work included initial work on the calibration of the AFM to allow quantitative force measurements to be made and the developed novel surface chemistries to immobilize macromolecules on the AFM probe and surface. More recently, the AFM has been applied to the study of the mechanical properties of cells, which we believe will provide new insight into the molecular mechanisms of cell motility and signaling.

As single molecule force measurements have been applied to more biomolecular systems, higher measurement sensitivity has become increasingly desirable. The magnetic tweezers technique can, in principle, be used to transduce piconewton (pN) forces to superparamagnetic microparticles. This laboratory has developed new magnetic transducers (described below) that make it possible to simultaneously measure the behavior of hundreds of ligand-receptor pairs. This technique has been used to characterize the force-bond lifetime behavior of the mouse IgG type 2a-protein A interaction and screen phage displayed libraries.

 3.  Biosensing and In Vitro Diagnostics

Early detection of diseases could greatly enhance the quality of medical care and limit the spread of diseases. Thus, there is a need for  rapid, sensitive, and inexpensive point-of-care sensors that are capable of identifying multiple pathogens in complex samples such as blood. There is also the need for new in vitro diagnostics that will allow cancers to be identified and the efficacy of therapies to be determined at an earlier stage. 

Superparamagnetic microparticles have made it possible to rapidly and efficiently separate cells from complex mixtures such as fermentation broths and culture media without complicated equipment. In this technique, the magnetic particles are coated with antibodies that react with the cell-line of interest. After a short reaction, a simple permanent magnet can be used to separate the superparamagnetic particles and specific cell type of interest. Magnetic separation is exceptionally efficient because most biological materials are not susceptible to magnetic fields and the fluidized beds of microparticles have a very high reaction rate. This research group has developed novel techniques for sensing and separating pathogens bound to superparamagnetic microparticles. Early work at the Naval Research Laboratory focused on using magnetic particles to detect pathogens bound to a surface and using force to enhance the specificity of the specific molecular interaction. Techniques were developed to detect the magnetic particles through force, optical, and magnetic field sensing.

More recently, this group has focused on identifying magnetic particle pathogen complexes through magnetophoretic separation. Magnetophoresis is a separation process in which both hydrodynamic and magnetic fields are used to separate a magnetic microparticle from an aqueous solution. We have demonstrated magnetophoretic sensing is capable of detecting type 2 Dengue virus at a concentration < 1000 virus per ml in serum, which in principle could allow this deadly disease to be accurately diagnosed when symptoms first become evident. This technique can also be applied to biomarkers such as exosomes.

A new mode of magnetophoresis has also been 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 bead.s 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. 

4.  Down Stream Processing

We have introduced a new concept for the analysis and purification of the Bowman-Birk-Inhibitor (BBI), which is bioactive protein derived from soybean whey.  By using a phage display technique we have identified a set of peptides that bind specifically to BBI for which no synthetic probes have been reported to date.  These peptides have been covalent grafted onto superparamamagnetic beads and used for the rapid isolation of functional BBI from soybean protein using high gradient magnetophoretic separation (HGMS).  The high affinity of the peptides and novel surface chemistry made it possible to purify BBI at > 90% purity from soybean whey in a single separation step in less than 60 minutes.  The peptide probes and HGMS process described in this manuscript provide a new means for isolating BBI that overcomes the speed, cost, and throughput limitations imposed by traditional liquid chromatography.  More broadly we believe that the peptide-HGMS approach can be applied to a range of bioactive compounds in foods. 

Commercialization:  U.S. and International Patent Disclosures and Patents Awarded:

Co-Founder of MagSense Life Sciences, Inc. 

Prof. Donal Brennan

Prof.. Walter Kolch 

Prof. William Hall  

Prof. Dominic Zerulla

Prof. Brian Rodriguez  

Dr. Cindi Denis
 
Dr. G. M. Willmott