For large-scale analysis of complex protein mixtures, liquid chromatography – tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity...
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.
Microfluidics allows the miniaturization of biochemical analyses. Small dimensions reduce sample and reagent consumption and enhance reaction rates. A downside is that high surface-to-volume ratios increase the unspecific binding of proteins to the substrate material. The resulting sample loss and reagent depletion decrease the sensitivity and specificity of protein-based assays, especially if low concentrations are analyzed. Here, we introduce the hydrophobin coating of microfluidic chips made of cyclic olefin copolymers (COC). The recombinant hydrophobin H*Protein B self-assembles into stable monolayers on hydrophobic surfaces, making them hydrophilic and thus reducing hydrophobic interactions between the chip surfaces and proteins. The substrate and sealing layers of the microfluidic chip were simply dip-coated and subsequently assembled by thermodiffusion bonding, which renders our coating procedure compatible with mass fabrication. Contact angle measurements and atomic force microscopy were used to evaluate the effect of high temperatures (107 °C) on COC substrates coated with H*Protein B. The efficiency of the protein-repellent coating was evaluated by depletion experiments with bovine serum albumin, human serum, and cerebrospinal fluid in microfluidic chips. Protein recovery was investigated down to protein concentrations of 0.3 μg/mL. Recoveries of 90% were observed with total protein amounts of 10 ng, even for microfluidic channels up to 835 mm in length and with a cross section of 80 μm × 230 μm in a COC 6013/8007 foil. For comparison, only 30 and 60% of the protein was recovered in uncoated microfluidic channels with lengths of 835 and 128 mm, respectively. The longterm stability of the hydrophobin-coated chips for 8 weeks was demonstrated.
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