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Luminex Assays: How it Works

The Luminex System is a flexible analyzer based on the principles of flow cytometry that is designed to meet the needs of any size research laboratory. The system enables you to multiplex (simultaneously measure) up to 100 analytes in a single microplate well, using very small sample volumes. At Affymetrix though, we offer multiplexed solutions of up to 40 different analytes in a single well. The system delivers fast and cost-effective bioassay results on many assay formats that Affymetrix offers which include: gene expression, transcription factor profiling, and immunoassay profiling.

The Luminex System is the combination of three core xMAP technologies. The first is xMAP microspheres, a family of 100 fluorescently dyed 5.6 micron-sized polystyrene microspheres that act as both the identifier and the solid surface to build the assay. The second is a flow cytometry-based instrument, the Luminex analyzer, which integrates key xMAP detection components such as lasers, optics, advanced fluidics and high-speed digital signal processors. The third component is the assays that are designed around the microspheres.

xMAP Technolgy

The xMAP technology uses 5.6 micron polystyrene microspheres which are internally dyed with red and infrared fluorophores. Using different amounts of the two dyes for different batches of microspheres, up to 100 different microsphere sets can be created. Each bead is unique with a spectral signature determined by a red and infrared dye mixture. The bead is filled with a specific known ratio of the two dyes. As each microsphere carries a unique signature, the xMAP detection system can identify to which set it belongs. Therefore, multiplexing up to 100 tests in a single reaction volume is possible.

Luminex Reader Design

The Luminex reader combines two lasers, fluidics, and real-time digital signal processing to distinguish up to 100 different sets of color-coded polystyrene beads, each bearing a different assay. The Luminex reader is an essential tool that performs the key functions of this multiplex technology:

Fluidics  The reader detects individual beads by flow cytometry. The fluidics system of the reader aligns the beads into single file as they enter a stream of sheath fluid and then enter a flow cell. Once the beads are in single file within the flow cell, each bead is individually interrogated for bead color (analyte) and assay signal strength (PE fluorescence intensity).

Lasers  The reader uses a 532 nm green laser ("assay" laser) is used to excite the PE dye of the assay (streptavidin-PE). The 635 nm solid state laser (red "classify" laser) is used to excite the dyes inside the beads to determine their "color" or "region" and is also used for doublet discrimination by light scatter.

Detectors  The reader has four detectors, one for each of the optical paths shown in the figure below. Detectors are used to measure the fluorescence of the assay, to make bead determination (1-100) and the last to discriminate between single and aggregate beads.