Gregory Kunkel’s research while affiliated with Nanosphere, Inc. and other places

Staphylococcus aureus (SA) is one of the most important human pathogens, causing both nosocomial and community-acquired infections (1)(2). The occurrence of methicillin-resistant SA (MRSA) has increased steadily worldwide and now accounts for a substantial portion of all staphylococcal infections in US hospitals (3). To develop preventive measures, a rapid screening method, along with accurate and timely identification of MRSA, is essential. The existing techniques for doing so are either time-intensive (culturing of bacteria on selective media), relatively insensitive (use of latex agglutination), or expensive and easily susceptible to operator error (such as PCR). We describe a method designed for clinical laboratories using oligonucleotides conjugated to gold nanoparticles. We avoid radioactivity, fluorescence, or target amplification (such as PCR), and use a simple and rapid hybridization-based approach in a microarray format, with ClearRead™ technology to detect specific genomic sequences (4). The ClearRead procedure involves a two-step process: the first involves the hybridization of target to oligonucleotides conjugated to gold nanoparticles as well as oligonucleotides attached to a solid matrix; the second step involves the catalytic deposition of silver on the gold nanoparticle, providing a sixfold amplification of signal (Fig. 1A⇓ ) (4)(5). The differentiation at isothermal hybridization temperatures is a result of the sharp melting transitions characteristic of nanoparticle probes (5). Previous methods based on this property (6)(7) have required the use of PCR and have not directly assayed for genomic DNA. Our assay, on the other hand, requires minimal amounts of genomic DNA (∼500 ng, or ∼108 DNA molecules) and has been used to reliably identify MRSA from liquid cultures, based on the detection of the mecA and tuf genes. Resistance to methicillin is mediated by the presence of penicillin-binding protein 2a, encoded by the mecA gene (8)(9). We are …

The molecular probes and associated instrumentation necessary to perform genetic analyses are typically expensive, complex, and prone to error. While techniques such as real-time polymerase chain reaction (PCR) and gene expression analysis have provided a wealth of information previously unattainable, their utility in clinical diagnostics has yet to be realized due to the aforementioned limitations. Nanosphere Inc. has developed a gold nanoparticle-based platform for sequence specific DNA detection that is well-suited for clinical diagnostics due to its cost-effectiveness, simplicity, and accuracy. Thirteen nanometer gold nanoparticle probes, stabilized by a shell of oligonucleotides using proprietary attachment chemistries, enable highly sensitive and specific detection of bacterial genomic DNA sequences without synthetic amplification techniques on a glass array. After silver staining, light scattered by the nanoparticle probes is collected with robust, cost-effective instrumentation. It is the unique features of Nanosphere's detection methodology that provide the necessary steps forward to allow for genetic analyses to become part of routine clinical diagnostics.