Seashell Technologies, a small biotechnology incubator in San Diego, hopes to commercialize an assay that uses silver plasmon resonant particles as reporter labels in SNP detection, a company official told SNPtech Reporter.
David Schultz, a co-founder of the company, said the PRP assay would remove fluorophores from SNP-analysis experiments, and can be read using a traditional microscope.
If commercialized — and Schultz said one of Seashell's goals is to find a commercial partner to commercialize it — the technology will become yet another oligoligation-based, PCR-obviating platform that may eventually rival high-throughput SNP-genotyping plays (see SNPtech Reporter 10/9/2003 and 2/28/03).
The PRP technology was developed at the University of California, San Diego, by Sheldon Schultz, a research professor in physics and co-founder and president of Seashell Technologies. Eventually spun out of the school and into Seashell, which sought life-science applications for it, the technology has already been studied as a method of analyzing chromosomal DNA and RNA in situ.
These days, Seashell is investigating ways to use the technology for SNP detection. The company has already published an article in the October 2002 Analytical Biochemistry in which PRPs were used as reporter labels in a microarray-based assay that screened for known SNPs in the BRCA1 gene. In the study, researchers used a standard microscope outfitted with dark-field illumination to observe PRPs between 40 and 100 nm in diameter as "diffraction-limited points of light. … In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 times greater … [than can be achieved] using fluorescent labels," the scientists, led by Sheldon Schultz, concluded.
Research performed at UCSD demonstrated that PRPs, when illuminated with white light, can preferentially scatter light of specific colors depending on the size, shape, and material of the PRP. "So you're going to be illuminating with a typical white light source — it could be a halogen light bulb — and depending on the size and shape and material of the particles you can actually scatter light of a certain color," he said. "Depending on those parameters, the light can only be red, green, or blue, or anywhere in between."
Researchers at UCSD found that triangular-shaped silver particles approximately 60 nm in diameter will preferentially scatter red light, whereas a spherical silver particle of similar size will scatter blue light. The amount of light that is scattered is around a million times more than the light produced by a single fluorophore. Since the amount of scattered light is proportional to the intensity of the incident light, increasing the intensity of the illumination light will cause the particles to scatter more light. "So you can easily see them in your microscope," said David Schultz, who is a project scientist in biophysics at UCSD and Sheldon Schultz’s son.
"It turns out that these particles scatter light very, very effectively," he said. "As far as we know, they are the brightest scattering object for their size that exist[s] in the world."
Researchers at UCSD and Seashell bind antibodies or DNA to the surface of the PRPs, and use these labels to bind to various biological targets. The PRP technol-ogy can be used to replace fluorescenct labels in applications such as protein microarrays or DNA microarray-based SNP detection. PRPs have several advantages over fluorophores — they never bleach, they can be archived indefinitely, and they don't blink. However, they are larger than single fluorophores, and thus cannot be readily incorporated into DNA, such as in a primer-extension experiment. To get around this, Seashell uses oligoligation assays for SNP detection.
In traditional OLA-based SNP-detection assays, if a SNP is identified and there is a perfect match in the discrimination oligo, the ligase will link the two oligos. If there's a mismatch they won't. Researchers at UCSD and Seashell used a discrimination oligo that has a Zip Code sequence extension, while the detection oligo has a biotin or some other ligand modification. If there is a perfect match, the Zip Code will bind to a particular site on the microarray, where there is an immobilized oligo that is complementary to the Zip Code oligo. "If in your sample you had some DNA which allows the ligation to occur, you now have a little biotin flopping in the breeze at the microarry spot," said Schultz.
The researchers next apply a goat-anti-biotin-coated PRP to the microarray, wash away the excess, unbound PRPs, and then place the array under an optical microscope configured for darkfield illumination "and literally count the number of PRPs that are bound to any microarray spot," the younger Schultz told SNPtech Reporter. "Each PRP seen is indicative of one detection oligo that has been linked to a discrimination oligo."
"By doing this particle counting, rather than having to get a large number of fluorophores down in your microarray spot, we can literally see one," he said. "So the idea is to get to the sensitivity that obviate[s] the need for PCR." He said Seashell "already does better in a straight comparison" of the sensitivity reached with a "typical" microarray-based fluorescence reader.
"We're not trying to make — and we certainly do not make — an instrument for high-throughout screening," he explained. "But we believe that at some future date, a commercial partner could develop the technology into a very high-throughput method."