Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS 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 only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

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

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.

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. As a potential application we demonstrate automated minimum residual disease (MRD) monitoring of prognostic markers in patients with acute lymphoblastic leukaemia (ALL). The disk-workflow covers all aspects of clinical gold standard MRD quantification: generation of standard curves, specificity controls, no template controls and quantification of the ALL patient sample. We integrated a highly sensitive, colorimetric 2-plex analysis of MRD targets, as well as a 2-plex analysis of reference genes, both in parallel and in a single LabDisk cartridge. For this purpose, a systematic procedure for crosstalk- and signal-to-noise-optimisation is introduced, providing a guideline for efficient multiplex readout inside microfluidic platforms. The qPCR standard curves (n = 12/12) generated on-disk reach clinically required linearity (R2 = 98.1% to R2 = 99.8%). In three consecutive MRD disk runs with an ALL patient sample containing the two representative MRD targets VH3D3D5JH3 and VkIkde, we observe high accordance between the on-disk quantifications (48 ± 6 copies/reaction and 69 ± 6 copies/reaction) and the expected concentrations (57 copies/reaction for both targets). In comparison to the clinical gold standard of manually pipetted, singleplex assays, the MRD disk yields comparable limit of quantification (1 × 10-4) in n = 6/6 analyses (vs. n = 4/4 in gold standard) and a limit of detection (1 × 10-5) in n = 6/6 analysis (vs. n = 2/4 in gold standard). The automation reduces the risk of manual liquid handling errors, making the MRD disk an attractive solution to assure reproducibility in moderate-throughput, longitudinal gene quantification applications.

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 (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.

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. All fluidic features are implemented in a monolithic fashion and disks are manufactured by foil thermoforming for scalable manufacturing. The new valving concept is demonstrated to reliably prevent valving if the diluted sample is not added to the mixing chamber (n = 30) and ensure valving if the dilution stage is completed (n = 15). The accuracy and precision of automated serial dilutions are verified by on-disk generation of qPCR standard curve dilutions and compared with manually generated reference dilutions. In a first step, the 5-log-stage standard curves are evaluated in a commercial qPCR thermocycler revealing a linearity of R2 ≥ 99.92% for the proposed LabDisk method vs. R2 ≥ 99.67% in manual reference dilutions. In a second step, the disk automated serial dilutions are combined with on-disk qPCR thermocycling and readout, both inside a LabDisk player. A 4-log-stage linearity of R2 ≥ 99.81% and a sensitivity of one leukemia associated ETV6-RUNX1 mutant DNA copy in a background of 100 000 wild-type DNA copies are achieved.

An air-breathing enzymatic cathode with extended lifetime by continuous laccase supply.

We present a novel concept of an air-breathing enzymatic biofuel cell cathode combined with continuous supply of unpurified laccase-containing supernatant of the white-rot fungus Trametes versicolor for extended lifetime. The air-breathing cathode design obviates the need for energy-intensive active aeration. In a corresponding long-term experiment at a constant current density of 50 µA cm-2, we demonstrated an increased lifetime of 33 days (cathode potential above 0.430 V vs. SCE), independent of enzyme degradation. The obtained data suggest that theoretically a longer lifetime is feasible. However, further engineering efforts are required to prevent clogging and fouling of the supply tubes. These results represent an important step towards the realization of enzymatic biofuel cell cathodes with extended lifetime and enhanced performance.

Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells.

Microbial electrochemical cells are an emerging technology for achieving unbalanced fermentations. However, organisms that can serve as potential biocatalysts for this application are limited by their narrow substrate spectrum. This study describes the reprogramming of Escherichia coli for the efficient use of anodes as electron acceptors. Electron transfer into the periplasm was accelerated by 183% via heterologous expression of the c-type cytochromes CymA, MtrA and STC from Shewanella oneidensis. STC was identified as a target for heterologous expression via a two-stage screening approach. First, mass spectroscopic analysis revealed natively expressed cytochromes in S. oneidensis. Thereafter, the corresponding genes were cloned and expressed in E. coli to quantify periplasmic electron transfer activity using methylene blue. This redox dye was further used to expand electron transfer to carbon electrode surfaces. The results demonstrate that E. coli can be reprogrammed from glycerol fermentation to respiration upon production of the new electron transport chain.

Systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1.

We present a systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1. Under anoxic conditions nanoporous activated carbon cloth is a superior anode material in terms of current density normalized to the projected anode area and anode volume (24.0±0.3 µA cm(-2) and 482±7 µA cm(-3) at -0.2 vs. SCE, respectively). The good performance can be attributed to the high specific surface area of the material, which is available for mediated electron transfer through self-secreted flavins. Under aerated conditions no influence of the specific surface area is observed, which we attribute to a shift from primary indirect electron transfer by mediators to direct electron transfer via adherent cells. Furthermore, we show that an aerated initial growth phase enhances the current density under subsequent anoxic conditions fivefold when compared to a similar experiment that was conducted under permanently anoxic conditions.