Generic Reporter Sets for Colorimetric Multiplex dPCR Demonstrated with 6-Plex SNP Quantification Panels.

Digital PCR (dPCR) is a powerful method for highly sensitive and precise quantification of nucleic acids. However, designing and optimizing new multiplex dPCR assays using target sequence specific probes remains cumbersome, since fluorescent signals must be optimized for every new target panel. As a solution, we established a generic fluorogenic 6-plex reporter set, based on mediator probe technology, that decouples target detection from signal generation.

Nanobead handling on a centrifugal microfluidic LabDisk for automated extraction of cell-free circulating DNA with high recovery rates.

cfDNA is an emerging biomarker with promising uses for the monitoring of cancer or infectious disease diagnostics. This work demonstrates a new concept for an automated cfDNA extraction with nanobeads as the solid phase in a centrifugal microfluidic LabDisk. By using a combination of centrifugal and magnetic forces, we retain the nanobeads in one incubation chamber while sequentially adding, incubating and removing the sample and pre-stored buffers for extraction.

Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification.

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by permeabilization, hybridization, and signal detection.

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

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types ( and ) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments.

Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of Point Mutations.

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel.

The MRD disk: automated minimal residual disease monitoring by highly sensitive centrifugal microfluidic multiplex qPCR.

We present a proof-of-principle study on automated, highly sensitive and multiplexed qPCR quantification by centrifugal microfluidics. The MRD disk can be used for standardisation of repetitive, longitudinal assays with high requirements on reproducibility and sensitivity, such as cancer monitoring. In contrast to high-throughput qPCR automation by bulky and expensive robotic workstations we employ a small centrifugal microfluidic instrument, addressing the need of low- to mid-throughput applications.

Automated serial dilutions for high-dynamic-range assays enabled by fill-level-coupled valving in centrifugal microfluidics.

We introduce a new concept for centrifugal microfluidics that enables fully automated serial dilution generation without any additional means besides temperature control. The key feature is time-independent, serial valving of mixing chambers by fill-level-coupled temperature change rate (FLC-TCR) actuated valving. The automated dilution is realized under continuous rotation which enables reliable control of wetting liquids without the need for any additional fabrication steps such as hydrophobic coating.