Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics

Abstract

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.

Observations on phenomenological changes in Klebsiella Pneumoniae under fluidic stresses

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KlebsiellaPneumoniae turns more virulent under flow stresses in capillary like microchannels

“…Few of the important advantages of using microfluidic technologies include easy streamlining of various assays, reduction of reagent volumes (and hence, the cost), better control over biological systems, faster reaction time 8,9 etc. The penetration of microfluidics technology in the field of biomedical research has led to various impactful innovations like lab-on-a-chip that have revolutionized point-of-care testing and have solved many laboratory challenges like easy and early detection of diseases 10,11 , modelling of diseases 12,13 , cell sorting, cell focusing and manipulation 14–19 , automation and parallelization 20,21 to name a few. Using microfluidic techniques Huh et al 22 developed the idea of organ-on-a-chip (OoC) that promises to change the traditional ways of drug development and modelling diseases.…”

Automated Centrifugal Microfluidic Chip Integrating Pretreatment and Molecular Diagnosis for Hepatitis B Virus Genotyping from Whole Blood

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