Field of Invention
The present invention relates to preparation of assay substrates, and, more specifically, to methods and systems for improving detection sensitivity.
Description of Related Art
An assay substrate is a surface upon which various chemical and/or biological analyses can be performed. Examples of an assay substrate include microarray plates, glass plates, and microtiter plates. A microtiter plate is a flat plate that has multiple “wells” formed in its surface. Each well can be used as a small test tube into which various materials can be placed to perform biochemical analyses. One illustrative use of microtiter plates includes an enzyme-linked immunosorbent assay (ELISA), which is a modern medical diagnostic testing technique.
Generally, in an ELISA, a capture antibody is printed on the bottom of a well in a microtiter plate. The capture antibody has specificity for a particular antigen for which the assay is being performed. A sample to be analyzed is added to the well containing the capture antibody, and the capture antibody “captures” or immobilizes the antigen contained in the sample. A detect antibody is then added to the well, which also binds and/or forms a complex with the antigen. Further materials are then added to the well which cause a detectable signal to be produced by the detect antibody. For example, when light of a specific wavelength is shone upon the well, the antigen/antibody complexes will fluoresce. The amount of antigen in the sample can be inferred based on the magnitude of the fluorescence. In another example, a compound can be added to the well that causes the detect antibody to emit light within a predetermined wavelength (e.g., 400-500 nm). This light can be read by a charge-coupled device (CCD) camera to measure the optical brightness of the emitted light.
Currently, solid-phase 2D multiplexed protein assays use a variety of detection techniques and substrate-surface preparations. Known detection techniques include fluorescence, chemiluminescence, or colorimetric detection. Substrate-surface preparations include a glass substrate with a nitrocellulose surface coating and a plastic substrate with a plasma-treated surface. For example, in one known technique, a nitrocellulose coating is applied to a surface of a glass plate, biological materials (e.g., proteins) are bound to the coating, fluorescence reactions are performed, and the slide is imaged from the printed side.
Current substrate-surface combinations have certain weaknesses. While nitrocellulose-coated glass has excellent binding capacity, it is limited by autofluorescence, that is, emission of natural light by biological structures. The interfering emission from the nitrocellulose coating lowers the accuracy and effectiveness of the fluorescence-based detection techniques. Alternatively, plasma-treated plastic is inexpensive, has consistent surface characteristics, and can be used with a variety of detection techniques. Plasma-treated plastic, however, has low binding capacity.