The concept of multi-analyte sensing using array based sensors (Chem. Rev. 100, 2595-2626, (2000)) has opened up a wide field of technologies in detecting and analyzing specific components (analytes) in a mixture of unknowns. Such technologies benefit industries ranging across the medical, biological, environmental, and consumer sectors. For example, the medical industry depends on analysis for the detection of metabolites, drugs, and glucose; the biological sector needs to detect amino acids, cell components; environmentalists have a need to know the level of gaseous components in water or air; while consumers may want to regularly test for levels of carbon monoxide in houses, airborne allergens, or hardness of water.
The basic principles of microarray assays were already described by the end of the eighties (J. Pharm Biomed Anal 7, 155-168, (1989)). Interest increased dramatically with the development of DNA chip technology. The invention and demonstration in the early 1990s (Science, 251, 767-773, (1991)) of high-density arrays formed by spatially addressable deposition of sensors on a two-dimensional solid support has enhanced and simplified the process of array based sensor technologies. The key to current microarray technology is the placement of receptors at predetermined locations on a microchip in a “spatially addressable” manner. The presence or absence of an analyte is then discerned by monitoring a specific location on a sensor array of receptors. All of these systems require preparing a sensor array with a plurality of receptors at predetermined sites that involve complex and expensive processing steps.
Recent technologies have used various approaches to fabricate microarrays. For example, U.S. Pat. Nos. 5,412,087, and 5,489,678 demonstrate the use of a photolithographic process for making peptide and DNA microarrays. These patents teach the use of photolabile protecting groups to prepare peptide and DNA microarrays through successive cycles of deprotecting a defined spot on a 1 cm×1 cm chip by photolithography, then flooding the entire surface with an activated amino acid or DNA base. Repetition of this process allows construction of a peptide or DNA microarray with thousands of arbitrarily different peptides or oligonucleotide sequences at different spots on the array. This method is expensive. Park et al. (Science 276:1401 (1997)) have disclosed a lithographic method for producing an array of nanometer-sized holes using polystyrene-polybutadiene copolymer masks in reactive ion etching of silica nitride. This multi-step method is capable of producing arrays of picoliter-sized holes that are typically 20 nanometers in diameter and 20 nanometers deep with a spacing of 40 nanometers. Hole densities of up to 1011 holes/cm2 are disclosed. The range of sizes and spacings of the holes produced by this method is limited by the size of the copolymer microdomains. Uniformity of hole size and spacing is difficult to maintain with this method due to difficulties in controlling the etching method employed to form the holes.
Because the number of bioactive probes to be placed on a single chip usually runs anywhere from 1,000 to 100,000 probes, the spatially addressable method is intrinsically expensive regardless of how the chip is manufactured. An alternative approach is the use of fluorescent dye-incorporated polymeric beads to produce biological multiplexed arrays. U.S. Pat. No. 5,981,180 discloses a method of using color coded beads in conjunction with flow cytometry to perform multiplexed biological assay. Microspheres conjugated with DNA or monoclonal antibody probes on their surfaces were dyed internally with various ratios of two distinct fluorescence dyes. Hundreds of “spectrally addressable” microspheres were allowed to react with a biological sample and the “liquid array” was analyzed by sequentially passing microspheres through a flow cytometry cell to decode sample information. U.S. Pat. No. 6,023,540 discloses the use of fiber-optic bundles with pre-etched microwells at the distal ends to assemble dye loaded microspheres. The surface of each spectrally addressed microsphere was attached with a unique bioactive agent and thousands of microspheres carrying different bioactive probes combined to form “beads array” on pre-etched microwells of fiber optical bundles. More recently, a novel optically encoded microsphere approach was accomplished by using different sized zinc sulfide-capped cadmium selenide nanocrystals incorporated into microspheres (Nature Biotech. 19, 631-635, (2001)). Given the narrow spectral band width demonstrated by these nanocrystals, this approach significantly expands the spectrally addressable barcoding capacity in microspheres.
Even though the “spectrally addressed microsphere” approach does provide an advantage in terms of its simplicity over the old fashioned “spatially addressable” approach in microarray making, there are still needs in the art to render the manufacture of microarrays less difficult and less expensive.
U.S. patent application Ser. No. 09/942,241 discloses a microarray that is less costly and easier to prepare than those previously disclosed, because the support need not be modified, even though the microspheres remain immobilized on the substrate. The disclosed microarray includes microspheres dispersed in a fluid containing a gelling agent or a precursor to a gelling agent, wherein the microspheres are immobilized at random positions on the substrate. The substrate is free of receptacles designed to physically or chemically interact with the microspheres. Disclosed are a unique coding composition and technology to prepare a microarray on a substrate that does not require placement of microspheres at predetermined locations. Various coating methods are taught, but there is exemplified machine coating, whereby a support is layered with a fluid coating composition comprising microspheres dispersed in gelatin. Immediately after coating, the support is passed through a chill-set chamber in the coating machine where the gelatin undergoes rapid gelation and the microspheres are immobilized.
Although the disclosure of this patent application provides a manufacturing advantage over other existing technologies, some limitations need to be overcome. By moving from spatially addressable to randomly positioned microspheres, the information content contained within each bead necessarily must be extracted using a new analysis technology that is not preset-positionally dependent. Furthermore, the colors and color levels need to be accessed uniquely to correlate the tag to the analyte.
It is also known (Nature Biotech. 19, 631-635, (2001)) that the number of different color codes in spectrally addressable microspheres for use in multi-analyte sensing follows the relationship:Number of optical codes=(nm−1), where                m=color types, and        n=color intensity levels        
For example, 2 colors, with 4 intensity levels each should result in 42−1=15 codes. Hence, in order to sense a large number of analytes using several color types and numerous color levels, there exists a need for analysis methods to differentiate small changes in color types and color levels, on a micrometer scale.