Benjamin Sullivan

Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions

In biomedical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells and potential biomarkers in blood, plasma and other clinical samples. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanoparticles, which now will also have to be identified and separated from blood and plasma. Alternating current (AC) electrokinetic techniques such as dielectrophoresis (DEP) offer a particularly attractive mechanism for the separation of cells and nanoparticles. Unfortunately, present DEP techniques require the dilution of blood/plasma, thus making the technology less suitable for clinical sample preparation. Using array devices with microelectrodes over‐coated with porous hydrogel layers, AC electric field conditions have been found which allow the separation of DNA nanoparticles to be achieved under high‐conductance (ionic strength) conditions. At AC frequencies in the 3000 Hz to 10 000 Hz range and 10 volts peak‐to‐peak, the separation of 10‐µm polystyrene particles into low field regions, and 60‐nm DNA‐derivatized nanoparticles and 200‐nm nanoparticles into high‐field regions was carried out in 149 mM 1×PBS buffer (1.68 S/m). These results may allow AC electrokinetic systems to be developed that can be used with clinically relevant samples under physiological conditions.

Directed hybridization of DNA derivatized nanoparticles into higher order structures.

Electric field directed hybridization was used to produce twenty layer nanostructures composed of DNA derivatized nanoparticles. Using an electronic microarray device, DNA nanoparticles could be directed and concentrated such that rapid and specific hybridization occurs only on the activated sites. Nanoparticle layers were formed within 30 s of activation and twenty layer structures completed in under an hour. Results demonstrate a unique combination of bottom-up and top-down techniques for nanofabrication.

Parallel Assisted Assembly of Multilayer DNA and Protein Nanoparticle Structures Using a CMOS Electronic Array

A CMOS electronic microarray device was used to carry out the rapid parallel assembly of functionalized nanoparticles into multilayer structures. Electronic microarrays produce reconfigurable DC electric fields that allow DNA, proteins as well as charged molecules to be rapidly transported from the bulk solution and addressed to specifically activated sites on the array surface. Such a device was used to carry out the assisted self‐assembly DNA, biotin and streptavidin derivatized fluorescent nanoparticles into multilayer structures. Nanoparticle addressing could be carried out in about 15 seconds, and forty depositions of nanoparticles were completed in less than one hour. The final multilayered 3D nanostructures were verified by scanning electron microscopy.

Automated Combinatorial Process for Nanofabrication of Structures Using Bioderivatized Nanoparticles

A fully automated electronic microarray control system (Nanochip 400 System) was used to carry out a combinatorial process to determine optimal conditions for fabricating higher order three-dimensional nanoparticle structures. Structures with up to 40 layers of bioderivatized nanoparticles were fabricated on a 400-test site CMOS microarray using the automated Nanochip 400 System. Reconfigurable electric fields produced on the surface of the CMOS microarray device actively transport, concentrate, and promote binding of 40 nm biotin- and streptavidin-derivatized nanoparticles to selected test sites on the microarray surface. The overall fabrication process including nanoparticle reagent delivery to the microarray device, electronic control of the CMOS microarray and the optical/fluorescent detection, and monitoring of nanoparticle layering are entirely controlled by the Nanochip 400 System. The automated nanoparticle layering process takes about 2 minutes per layer, with 10–20 seconds required for the electronic addressing and binding of nanoparticles, and roughly 60 seconds for washing. The addressing and building process is monitored by changes in fluorescence intensity as each nanoparticle layer is deposited. The final multilayered 3D structures are about 2 μm in thickness and 55 μm in diameter. Multilayer nanoparticle structures and control sites on the microarray were verified by SEM analysis.

Next-Generation DNA Hybridization and Self-Assembly Nanofabrication Devices

The new era of nanotechnology presents many challenges and opportunities. One area of considerable challenge is nanofabrication, in particular the development of fabrication technologies that can evolve into viable manufacturing processes. Considerable efforts are being expended to refine classical top-down approaches, such as photolithography, to produce silicon-based electronics with nanometer-scale features. So-called bottom-up or self-assembly processes are also being researched and developed as new ways of producing heterogeneous nanostructures, nanomaterials and nanodevices. It is also hoped that there are novel ways to combine the best aspects of both top-down and bottom-up processes to create a totally unique paradigm change for the integration of heterogeneous molecules and nanocomponents into higher order structures. Over the past decade, sophisticated microelectrode array devices produced by the top-down process (photolithography) have been developed and commercialized for DNA diagnostic genotyping applications. These devices have the ability to produce electric field geometries on their surfaces that allow DNA molecules to be transported to or from any site on the surface of the array. Such devices are also able to assist in the self-assembly (via hybridization) of DNA molecules at specific locations on the array surface. Now a new generation of these microarray devices are available that contain integrated CMOS components within their underlying silicon structure. The integrated CMOS allows more precise control over the voltages and currents sourced to the individual microelectrode sites. While such microelectronic array devices have been used primarily for DNA diagnostic applications, they do have the intrinsic ability to transport almost any type of charged molecule or other entity to or from any site on the surface of the array. These include other molecules with self-assembling properties such as peptides and proteins, as well as nanoparticles, cells and even micron-scale semiconductor components. Microelectronic arrays thus have the potential to be used in a highly parallel electric field pick and place fabrication process allowing a variety of molecules and nanostructures to be organized into higher order two- and three-dimensional structures. This truly represents a synergy of combining the best aspects of top-down and bottom-up technologies into a novel nanomanufacturing process.