Biochemical reactions involve hundreds, sometimes thousands, of unique molecules. Fluorescent markers can be targeted toward specific molecules, but only a handful of spectrally distinct markers can feasibly be identified simultaneously in solution. New submicron "bar codes" may enable investigators to perform complex bio- chemical assays simultaneously.Metallic structures called Nanobarcodes, each a series of electrodeposited metallic particles in a membrane template, display unique reflective signatures, making them suitable for the simultaneous detection of hundreds of biological molecules. Courtesy of SurroMed Inc.A research team led by Christine Keating of Pennsylvania State University in University Park produced the patterned bar codes, trademarked as Nanobarcodes, by electrodepositing metal ions through pores in an Al2O3 membrane. The pattern of a Nanobarcode is established by the sequence of metal ions introduced into solution and by the charge passed in a given step of production.Nanobarcodes are a few microns in length and have a diameter of approximately 300 nm. To date, the researchers have employed seven metals, in segments varying from 10 nm to several microns in length. Despite their diameter, the reflectivity of the metal segments agrees well with that of the bulk material, enabling the identification of a Nanobarcode by standard light microscopy.For example, silver and gold are nearly indistinguishable when imaged under 600-nm illumination, but the reflectance of silver is approximately 2.5 times greater than that of gold at 430 nm. To read a code, therefore, a user need only cycle through a filter set. Similar variations in reflectivity for the other particle components allow the metal sequences to be uniquely identified.Thousands of specimens?For 6.5-µm-long particles featuring 500-nm-long segments, well above the diffraction limit, more than 4000 distinct bar codes can be produced with only two metals. By introducing a third metal, more than 80,000 distinct patterns are possible, enabling a user to monitor thousands or tens of thousands of species simultaneously.The Penn State researchers, in collaboration with scientists from SurroMed Inc. of Mountain View, Calif., bound a 12-nucleotide capture sequence to the surface of a Nanobarcode. They introduced a 24-nucleotide analyte into the solution along with a fluorescently tagged, 12-nucleotide probe sequence. When the particles captured an analyte and fluorescent marker, they showed both the optical reflectance signature of the bar code and the fluorescence signature of the marker."Because the particle identity (and, hence, analyte identity) is encoded via the metal striping pattern, fluorescence is only required for detection," Keating explained. "In contrast, other 'solution array'-type approaches use fluorescence for both encoding and detection."Besides using Nanobarcodes for biological assays, Keating is investigating the optical properties of nano-structured metallic particles and is contemplating experiments in which the particles would be assembled into electronic circuitry using DNA hybridization.