My institution has used the Luminex 100 for the past several years for nucleic acid detection in oligonucleotide ligation assays, allele-specific primer extension assays, and allele-specific hybridization assays. We have also used it for protein applications such as receptor-ligand assays and antigen-antibody based assays. The Luminex 100 utilizes xMAP technology, which enables the simultaneous analysis of up to 100 analytes in a single reaction well. The system combines a sophisticated microfluidics path with fluorescence detectors, a photomultiplier tube, and user-friendly software. The microspheres are 5.6um in diameter and are color-coded into 100 distinct sets by the application of precise ratios of two fluorescent dyes. For protein studies, beads are coupled to labeled antibodies, peptides, or receptors. For nucleic acid detection, beads are coupled to a biotin-labeled oligonucleotide probe. The probe-bead reagent is hybridized to a nucleic acid sample, washed several times, and reacted with streptavidin-phycoerythrin.
Two lasers in the instrument excite the bead-specific classification dye and the reporter dye (phycoerythrin) as each bead passes single-file through the detection chamber. The instrument detects fluorescence from the phycoerythrin in direct proportion to the number of hybridized molecules. The detection is very fast, considering that a typical reaction well contains 2000-10,000 beads. Reaction time for a full 96-well plate is approximately 1.5 hours. Results are reported real-time during the run. When the run is complete, the results can be saved in Excel format for further statistical analysis. Inter-run reproducibility is excellent, allowing quantitative measurements by use of a standard curve.
Sample uptake volumes range from 20 – 200µL. The instrument is sensitive enough to detect as few as 100 phycoerythrin molecules and can report up to approximately 32,000 median fluorescence intensity units.
The SD (sheath delivery system) fluid pump is recommended. The Luminex 100 can be used without the pump by directly running tubing from a one liter bottle. However, the smaller volume of fluid in the supply bottle increases the risk of running out of fluid during a run and requires more frequent refilling. The pump has valves that control air flow and allow the user to eject bubbles before they can reach the instrument. An indicator light on the pump warns the user of low fluid level.
Each day before running, the instrument is warmed up. Subsequently, a series of sanitizing solution, water, and 70% ethanol washes precede several sheath fluid washes of the internal tubing. One reservoir in the XY plate is used for four different fluids, which must be changed between each washing solution. This is a minor inconvenience that would be improved by building in a set of four reservoirs. The XY plate must return to its home position before the user can initiate a step. At times the software indicates that the plate is ready, but the plate has not caught up and an error message appears.
The Luminex 100 is well designed for ease of use. The software is intuitive and prompts the user for each subsequent step. The real-time graphical display helps the user diagnose anomalies with bead signals or with individual reactions.