Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification

Abstract

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s-4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 10 5 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R 2 ≥ 0.994; ddLAMP: R 2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance. resistance than the sample supply channel (ii), as long as the viscosity of the sample remains ≥ 4 mPa·s. Details of the fluidic design calculations and channel and chamber dimensions are listed in ESI 2, S1.Molecules 2020, 25, x 3 of 11

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

“…We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units. In contrast to the state of the art, where until now only one-plex ddPCR assays have been automated by centrifugal microfluidics [9,[17][18][19], we present an increased multiplexing degree of four. This elevated degree of multiplexing is particularly relevant for cancer diagnostics, where a comprehensive overview of the mutation status of a patient must be generated from a limited sample amount [3].…”

“…In the recent past, centrifugal microfluidics developed into a mature technology which enabled the efficient miniaturization, parallelization and integration of assays for both sample preparation and analysis [13][14][15]. In the past, several groups have presented centrifugal, microfluidic cartridges that were able to emulsify a reaction mix [6,9,[16][17][18][19][20]. We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units.…”

Low Cost, Easily-Assembled Centrifugal Buoyancy-Based Emulsification and Digital PCR

“…Homogenous water-in-oil emulsion has become increasingly essential to many biomedical applications, such as isolation of cells or enzymes [ 1 , 2 , 3 ], synthetization of micro-particles [ 4 , 5 ], and digital PCR or digital LAMP [ 6 , 7 ]. In the past decades, microfluidic-based droplet generation approaches have been proved to be robust and efficient methods that can produce uniform droplets with polydispersity lower than 10% [ 8 , 9 ].…”

“…Lab-on-a-disc (LOD) technology overcomes these shortcomings of conventional microfluidic technology by using centrifugal force instead of traditional pumps to pump fluid. Step emulsification [ 6 , 7 , 19 , 20 , 21 , 22 ] and crossflow [ 23 , 24 , 25 , 26 ] are commonly used in lab-on-a-disc (LOD) devices. Schuler et al developed a high-throughput droplet preparation method by using centrifugal step emulsification technology.…”

High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification

“…The, herein, presented approach overcomes these limitations by combining centrifugal step emulsification − with centrifugal microfluidic structures for sample metering, resulting in the automated generation of two-droplet populations on a single chip. A comparatively small amount ( n = 2,5k) of large droplets (Ø = 213.9 ± 7.5 μm) is used for covering the lower detection limit (LDL), whereas a large amount ( n = 176k) of small droplets (Ø = 31.6 ± 1.4 μm) is used for covering the dynamic range up to the upper limit of quantification (ULQ) with high resolution.…”