Feasibility of on-chip detection of endotoxin by LAL test

Abstract

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (labon-a-chip) prototype, 62 mm (L) x 18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2 mm (W) x 44.3 mm (L) x 100 Pm (D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PIDMS chip thickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 to ca. 4.4 muL. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.