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 × 10⁵ 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: R² ≥ 0.994; ddLAMP: R² ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 µΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.

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.

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 (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R² ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.