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

Abstract

We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 ± 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for four-color fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R² = 0.99) across all targets in the LabDisk.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA

“…As the Prism3 is limited to three fluorescence color channels, for the readout of the 4-plex MP ddPCR assay, a standard fluorescence microscope (LionheartLX, Biotek, Winooski, VT, USA) was used. The workflow for droplet readout with a customized chip holder and quantification with the LionheartLX microscope followed the previously published protocol [ 22 ].…”

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.…”

Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR

“…Recently, multiplex MP PCRs with highly optimized universal reporter sets have been successfully introduced for the quantification of various cancer-associated target sequences. 15,[18][19][20] These assays already include sequences used for ALL MRD monitoring. 20 Although these multiplex MP PCRs met the criteria of the EuroMRD consortium 7 , no guidelines for successful personalized assay design have yet been described.…”