Ultrasensitive and rapid endotoxin assay

LAL test is an important part of the pharmaceutical industry quality control toolkit. It is approved by FDA as a recommended endotoxin testing strategy for oral/injectable medications, USP-grade water at hospitals, and implantable devices. The LAL regent is an aqueous extract of blood cells (amoebocytes) from horseshoe crabs (Limulus polyphemus), which reacts with endotoxins and forms a semi-solid mass (coagulation) due to a clotting factor contained in LAL. This reaction establishes the basis of the three LAL test methods currently used in industry. The most basic test is termed “gel clot” test, which simply requires one to mix LAL regents with an analyte solution and check if the mixture forms gels and withstand 1800 inversion without breaking. The gel-clot test yields an end-point binary (either positive or negative) result in one hour after the mixing. The second approach, turbidimetric assay, utilizes the same enzymatic cascade as the gel-clot test, but adds a turbidity scanner to detect the change in scattered light over the reaction process to obtain kinetic analysis of the test sample. Chromogenic detection has also been developed as another approach for kinetic LAL assays. The coagulation in the final step of the enzymatic cascade is switched to a chromogenic substrate, which when cleaved by the activated clotting enzyme produces a yellow color for monitoring the coagulation. In contrast, the SAFEbiosense’s approach utilizes a fundamentally different physical parameter- refractive index (RI)- to sensitively monitor the early onset of the gel forming process in the mixture of the analyte solution and LAL regents. The changes in RI is monitored with a unique photonic crystal biosensor to sensitively quantify endotoxin concentrations. This new approach is necessary to address the sensitivity requirement for overcoming the interference issues in LAL test, a challenging issue frequently encountered in many industry applications. In addition, the new approach allows for rapid endotoxin test, which is critical for timely processing decisions in production lines that can significantly save cost for many industry applications. Rapid endotoxin test is also crucial for time-critical applications, such as for testing radiopharmaceuticals with a short half-life.