Miniaturized markers and indicators have found utility in a wide variety of areas, but they are of particular interest in biological and chemical assays. The development of multiplexing and miniaturization of chemical and biochemical assays has improved the analysis of samples in such areas as biomedical analysis, environmental science, pharmaceutical research, food and water quality control. For example, in the area of genomics, DNA arrays allow multiplexing and miniaturization of tests by providing a unique DNA target a unique address in the form of a position on the array in a small area (typically less than 100 microns in diameter) on the array surface. The total size of the surface and the spacing and size of the individual target determines the number of addresses available.
Microtiter plates also allow multiplexing and miniaturization of samples by providing many individual wells, each at a unique position. It is possible for each well to have a unique target and to be tested with a unique sample, which allows for the multiplexing of both targets and samples.
Another way of achieving miniaturization and multiplexing is through the use of miniaturized devices such as nanoparticles or labeled beads. These nanoparticles and labeled beads can be provided with a unique label that can be identified through appropriate instrumentation such as a flow-through cell, a bead sorter, or an imaging system. Nanoparticles and labeled beads can be used as substrates which can be functionalized with a variety of chemical and biochemical groups, including, but not limited to nucleic acids, proteins and small molecules. These functionalized nanoparticles or beads, which actually range in size from the hundreds of microns to nanometers, can be placed in a suspension, and binding and/or interaction events can be quantified by optical techniques such as fluorescence using conventional fluorescent markers such as Cy3 and Cy5.
The use of nanoparticles and labeled beads in the analysis of biochemical binding events offers several advantages over conventional microarrays. Since binding studies can be carried out using suspensions of particles, issues related to local probe depletion encountered with microarrays can be minimized. The use of nanoparticles or labeled beads together with multiwell microtiter plates facilitates the design of highly multiplexed assays involving the binding of many different probes to many different particle types within individual wells. Although the use of nanoparticles or labeled beads offers many advantages, the manufacture of such miniaturized devices has proven difficult. More specifically, the mass production of such devices in large quantities and at a low cost is particularly problematic.
One way of manufacturing these miniature devices has been developed by SurroMed, Incorporated, Mountain View, Calif., and involves making cylindrical metal nanoparticles of which the composition along the particle length can be varied in a stripe-like manner. Varying the number of stripes, the width of stripes, the identity of the metals, and the overall particle shape enables the production of a wide variety of unique labels. These “nanobarcode” tags can be identified using conventional optical microscopy, based on the pattern of differential reflectivity of adjacent metal stripes. These identification tags facilitate multiplexing of assays in various media. However, the manufacture of these metallic nanoparticles can be technically challenging and expensive. In addition, reflectance measurements generally have a high signal to noise ratio and poor sensitivity.
A method for manufacturing microparticles is described in U.S. Pat. No. 6,268,222. U.S. Pat. No. 6,268,222 describes a core particle having on its surface smaller polymeric particles stained with different fluorescent dyes. One limitation of the use of fluorescent dyes to identify the particles is that the excitation and emission spectra of these dyes may interfere or overlap with conventional dyes such as Cy3 and Cy3 that are used for tagging reporter molecules used in bioanalysis. In addition, the fluorescence intensity of dyes tends to deteriorate over time upon prolonged or repeated exposure to light. Still another limitation of dyes is that the degradation products of these dyes are organic compounds that may interfere with biological processes and molecules being evaluated.
It would be advantageous to provide miniaturized labels that could be encoded with a large number of unique identification tags and could be utilized for sensing interaction and binding of molecules. Moreover, it would be desirable if the devices could be mass produced easily and inexpensively. Furthermore, it would be useful if the devices could be identified using conventional optical detection techniques, for example, those using fluorescence detection.