When target amplification is not needed, northern blotting and other methods based on nucleic acid hybridization are convenient for detecting and measuring specific RNAs. Such methods include the Quantikine range for measuring specific cytokine mRNAs from R&D Systems of Minneapolis, Minnesota. The problem with direct hybridization is generating a strong signal from a few target molecules.

Chad Mirkin is barcoding proteins.

Chad Mirkin, director of the Northwestern University Institute for Nanotechnology, at Evanston, Illinois, thinks he has cracked the problem. He is co-founder of Nanosphere in Northbrook, Illinois, which produces BioBarcode, a sensitive detection system that can detect DNA sequences, but is proving to be just as good at spotting specific proteins. “PCR is amazing because it takes what you are trying to detect and duplicates a portion of it so that you have enough to detect. If you could do that for proteins, I would argue that you could have as big or a bigger impact, especially if you could do it without needing to use enzymes,” he says.

Mirkin hopes to make that impact with BioBarcode, which works as a two-component immunological sandwich assay with some novel twists. Antibody molecules specific for one site on the target protein are tagged with 13-nm diameter gold nanoparticles, while larger paramagnetic particles are coated in a second antibody that recognizes a separate site on the target. If present, the target protein will grab both probes, and the complexes can be isolated by magnetic separation.

So far, not too unconventional. In Mirkin's system, however, the gold nanoparticle is smothered in hundreds or thousands of identical oligonucleotides, each strand hybridized to a short sequence that acts as a molecular barcode. Nanoparticles with different antibodies carry different barcodes, allowing multiplexing. After separation, the DNA barcodes are released and their identity determined by scanning on a biochip. The initial signal is thus amplified hundreds to thousands of times, allowing as few as 10 molecules of protein to be detected.

Randy Lewis at the University of Wyoming is using BioBarcodes to search for prions associated with the elk version of mad cow disease. “BioBarcodes give us an assay that is 1,000 times more sensitive than anything else,” he says. “We should now be able to detect early onset and environmental contamination at lower levels than could be done before.”

P.M.