Detection method needs only a minuscule amount of target DNA.
October 14, 2004 | Detecting minute amounts of DNA is a critical tool in today's basic and clinical research labs. It has also become a crucial component of the move toward "bedside medicine" and efforts to enhance bioterror surveillance. Rapid detection of potential pathogens is key to mounting an effective response to a bioterror attack — the ability to detect the presence of a drug resistance gene in a natural or weaponized microbe could be a matter of life or death.
PCR has been the foundation for most tests that detect minute amounts of nucleic acid. PCR amplifies the molecules of DNA exponentially until a sufficient quantity is present to allow detection by fluorescence or other techniques. However, the need for enzymatic amplification of the DNA target adds a measure of complexity and cost to the assay — neither of which is ideal for a quick and simple assay that can be used in the field.
Recently, scientists at Nanosphere developed a colorimetric method for DNA detection that obviates the need for target or signal amplification. The method uses the change in optical properties of 50nm gold nanoparticles that occurs when two or more nanoparticles are brought into close proximity. The wavelength of light scattered from the surface of the particles is within the visible range. The particles can be engineered to bind to specific target DNA molecules to create DNA-modified gold nanoparticles (DNA-GNPs) so that they are densely packed with DNA oligonucleotides that hybridize to the target DNA.
EASY TARGET: Researchers using Nanosphere technology observed a green-to-orange color change that indicated presence of a targeted sequence. |
To create the assay, two or more individual nanoparticles are designed to bind to distinct but adjacent regions within a target sequence (see figure). Hybridization of the DNA-GNPs to the target sequences aggregates the nanoparticles; this changes the wavelength of scattered light from their surface compared to the light scattered by unbound particles. A minuscule amount of target DNA can be detected by examining the wavelength of the scattered light — in this case from green to orange — either visually or with detection instrumentation. The method enables the detection of zeptomole (10-21) quantities of nucleic acid.
Although not yet commercially available, Nanosphere is developing the assay, along with related assays to detect antibodies, proteins, and SNPs. The assay kits will be compatible with Nanosphere's Verigene ID optical detection system, which is already on the market.
In August, Nanosphere received an NIAID grant to fund the development of Verigene-based tests for anthrax and plague, as well as methicillin-resistant Staphylococcus aureus (MRSA). Nanosphere will also integrate DNA bar-code technology and automated microfluidics into its platform. Further funding comes from the U.S. government's Technical Support Working Group (TSWG) to adapt the Verigene platform into a field-deployable system to enable emergency first responders and hospital triage personnel to identify biological toxins.
Nanosphere may find competition from IatroQuest, which is developing nanostructured silicon materials that can serve as recognition elements for a variety of molecules. When targets bind to the recognition elements, surface energy perturbations alter the photoluminescence response, detected as an increase in green light intensity. In addition to demonstrating real-time detection in the picogram (10-12) range, the system offers many of the advantages of the Nanosphere approach: speed, portability, and simplicity. IatroQuest has received U.S. and Canadian government funding, including $1.5 million from In-Q-Tel, a private enterprise funded by the CIA to identify and invest in technologies that serve national security interests.
Both technologies will compete with more traditional diagnostic platforms, such as Cepheid's SmartCycler and Roche's LightCycler technology, which use real-time PCR detection; non-amplified fluorescent techniques such as those developed by US Genomics; mass spec-based techniques from Bruker Daltonics and others; and surface plasmon resonance devices by Biacore. 
Robert M. Frederickson is a biotech writer based in Seattle. E-mail: rfreder@yahoo.com.
Storhoff, J.; Lucas, A.; Garimella, V.; Bao, Y.; Müller , U. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nature Biotechnology 22, 883-7; 2004.