The requirement for analysis of large numbers of biomolecules for drug discovery and clinical diagnostics has driven the development of low-cost, flexible and high-throughput methods for simultaneous detection of multiple molecular targets in a single sample (multiplexed analysis). The technique that seems most likely to satisfy all of these requirements is the multiplexed suspension (bead-based) assay, which offers a number of advantages over alternative approaches such as ELISAs and microarrays. In a bead based assay, different probe molecules are attached to different beads (of a few tens of microns in size), which are then reacted in suspension with the target sample. After reaction, the beads must be identifiable in order to determine the attached probe molecule, and thus each bead must be labelled (encoded) with a unique identifier. A large number of techniques have been proposed for encoding beads. This critical review analyses each technology on the basis of its ability to fulfil the practical requirements of assays, whilst being compatible with low-cost, high-throughput manufacturing processes and high-throughput detection methods. As a result, we identify the most likely candidates to be used for future integrated device development for practical applications.

Microparticles incorporating micrometer-sized diffractive bar codes have been modified with oligonucleotides and immunoglobulin Gs to enable DNA hybridization and immunoassays. The bar codes are manufactured using photolithography of a chemically functional commercial epoxy photoresist (SU-8). When attached by suitable linkers, immobilized probe molecules exhibit high affinity for analytes and fast reaction kinetics, allowing detection of single nucleotide differences in DNA sequences and multiplexed immunoassays in <45 min. Analysis of raw data from assays carried out on the diffractive microparticles indicates that the reproducibility and sensitivity approach those of commercial encoding platforms. Micrometer-sized particles, imprinted with several superimposed diffraction gratings, can encode many million unique codes. The high encoding capacity of this technology along with the applicability of the manufactured bar codes to multiplexed assays will allow accurate measurement of a wide variety of molecular interactions, leading to new opportunities in diverse areas of biotechnology such as genomics, proteomics, high-throughput screening, and medical diagnostics.

We describe a new non-contact high capacity optical tagging technique based on the use of nanostructured barcodes. The tags are generated from a number of superimposed diffraction gratings. With one-dimensional diffraction, capacity for up to 68,000 distinguishable tags has been demonstrated, with a theoretical capacity of up to 10 9 tags. Extension into two dimensions increases this theoretical limit to 10 21 tags.

A nano-imprint lithography technique is described for fabrication of optically encoded microparticles (diffractive barcodes). The particles are fabricated from SU8 -a material which can be processed lithographically, and which can be used for attachment of molecular tags. The barcodes are identified by their unique diffraction patterns.