Miniaturization of the Luminescent Oxygen Channeling Immunoassay (LOCITM) for Use in Multiplex Array Formats and Other Biochips

摘要

Many of the emerging technologies in clinical chemistry and research require the ability to perform hundreds or thousands of measurements on a single sample such as amplified DNA, typically by contacting the sample with an array of different probes or other reagents. If these array approaches are to be practical, the underlying technology must be simple, robust, inexpensive, and amenable to automation. The Luminescent Oxygen Channeling Immunoassay (LOCITM) is a recently developed homogeneous assay method that should be suitable for arrays because of its simplicity. However, to perform large numbers of measurements on reasonable sample sizes (e.g., 500 different measurements on aliquots of a 50-μL volume), it must be possible to detect LOCI signals from very small volumes. Miniaturization has also become a central theme in other areas of clinical chemistry (1). Accordingly, we have constructed a LOCI microscope and used it to demonstrate sensitive detection of analytes in small volumes for three types of assays of potential interest in arrays: detection of a single-stranded DNA fragment, detection of a double-stranded DNA amplicon, and an immunoassay for a protein.LOCI is a sensitive (femtomolar) detection method that uses chemiluminescence to quantify latex agglutination (2). This technique utilizes one latex particle dyed with a photosensitizer and a second dyed with a chemiluminescent dye, both having binding ligands on their surfaces. Particle suspensions are mixed with the sample, and cross-linking by any analyte present leads to formation of bead pairs or higher aggregates. When the suspension is then illuminated, singlet oxygen is generated by the sensitizer particle, migrates to the chemiluminescent particle, and generates light. Nonspecific signals are low because singlet oxygen decays before it can reach unpaired particles.A small-volume LOCI reader was constructed by modifying a fluorescence microscope to allow sample illumination with a 678 nm laser and monitoring …