RESUMO
We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 µL/reaction to 9.6 µL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.