This critical review is motivated by an increasing interest of the microfluidics community in developing complete Lab-on-a-Chip solutions based on thin and flexible films (Lab-on-a-Foil). Those implementations benefit from a broad range of fabrication methods that are partly adopted from well-established macroscale processes or are completely new and promising. In addition, thin and flexible foils enable various features like low thermal resistance for efficient thermocycling or integration of easily deformable chambers paving the way for new means of on-chip reagent storage or fluid transport. From an economical perspective, Lab-on-a-Foil systems are characterised by low material consumption and often low-cost materials which are attractive for cost-effective high-volume fabrication of self-contained disposable chips. The first part of this review focuses on available materials, fabrication processes and approaches for integration of microfluidic functions including liquid control and transport as well as storage and release of reagents. In the second part, an analysis of the state of Lab-on-a-Foil applications is provided with a special focus on nucleic acid analysis, immunoassays, cell-based assays and home care testing. We conclude that the Lab-on-a-Foil approach is very versatile and significantly expands the toolbox for the development of Lab-on-a-Chip solutions.
The generation of mixtures with precisely metered volumes is essential for reproducible automation of laboratory workflows. Splitting a given liquid into well-defined metered sub-volumes, the so-called aliquoting, has been frequently demonstrated on centrifugal microfluidics. However, so far no solution exists for assays that require simultaneous aliquoting of multiple, different liquids and the subsequent pairwise combination of aliquots with full fluidic separation before combination. Here, we introduce the centrifugo-pneumatic multi-liquid aliquoting designed for parallel aliquoting and pairwise combination of multiple liquids. All pumping and aliquoting steps are based on a combination of centrifugal forces and pneumatic forces. The pneumatic forces are thereby provided intrinsically by centrifugal transport of the assay liquids into dead end chambers to compress the enclosed air. As an example, we demonstrate simultaneous aliquoting of 1.) a common assay reagent into twenty 5 μl aliquots and 2.) five different sample liquids, each into four aliquots of 5 μl. Subsequently, the reagent and sample aliquots are simultaneously transported and combined into twenty collection chambers. All coefficients of variation for metered volumes were between 0.4%-1.0% for intra-run variations and 0.5%-1.2% for inter-run variations. The aliquoting structure is compatible to common assay reagents with a wide range of liquid and material properties, demonstrated here for contact angles between 20° and 60°, densities between 789 and 1855 kg m(-3) and viscosities between 0.89 and 4.1 mPa s. The centrifugo-pneumatic multi-liquid aliquoting is implemented as a passive fluidic structure into a single fluidic layer. Fabrication is compatible to scalable fabrication technologies such as injection molding or thermoforming and does not require any additional fabrication steps such as hydrophilic or hydrophobic coatings or integration of active valves.
We investigate TMMF photopolymer as a cost-efficient alternative to glass for the leak-tight sealing of high-density silicon microchannels. TMMF enables low temperature sealing and access to structures underneath via lamination and standard UV-lithography instead of costly glass machining and anodic bonding. TMMF is highly transparent and has a low autofluorescence for wavelengths larger than 400 nm. As the photopolymer is too thin for implementing bulky world-to-chip interfaces, we propose adhesive bonding of cyclic olefin copolymer (COC) modules. All materials were tested according ISO 10993-5 and showed no cytotoxic effects on the proliferation of L929 cells. To quantify the cost efficiency of the proposed techniques, we used an established silicon/Pyrex nanoliter dispenser as a reference and replaced structured Pyrex wafers by TMMF laminates and COC modules. Thus, consumable costs, manpower and machine time related to sealing of the microchannels and implementing the world-to-chip interface could be significantly reduced. Leak tightness was proved by applying a pressure of 0.2 MPa for 5 h without delamination or crosstalk between neighboring microchannels located only 100 μm apart. In contrast to anodic bonding, the proposed techniques are tolerant to surface inhomogeneities. They enable manufacturing of silicon/polymer microfluidics at lower costs and without compromising the performance compared to corresponding silicon/glass devices.
We present a detailed analysis of microthermoforming by soft lithography (μTSL) for replication of foil-based microfluidic substrates. The process was systematically optimized by design of experiments (DOE) enabling fabrication of defect-free lab-on-a-chip devices. After the assessment of typical error patterns we optimized the process toward the minimum deviation between mold and thermoformed foil substrates. The following process parameters have most significant impact on the dimensional responses (p < 0.05): critical temperature before start of evacuation, molding temperature, pressure of pre-stretching and duration of pre-stretching as well as duration of molding pressure. The most relevant parameter is molding temperature with >40% relative impact. The DOE results in an empirical process model with a maximum deviation between the prediction and experimental proof of 2% for the optimum parameter set. Finally, process optimization is validated by the fabrication and testing of a microfluidic structure for blood plasma separation from human whole blood. The optimized process enabled metering of a nominal volume of 4.0 μl of blood plasma with an accuracy deviation of 3% and a metering precision of ±7.0%. The μTSL process takes about 30 min and easily enables the replication of 300 μm wide microchannels having vertical sidewalls without any draft angles in a well-controllable way. It proves to be suitable for multiple applications in the field of microfluidic devices.
We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7-4.4% CV of protein concentration).We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (R g ) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.
A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclo olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time polymerase chain reaction (real-time PCR), confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes.
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.
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