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. As the recovery rate of the typically low concentration of cfDNA is of high importance to attain sufficient amounts for analysis, optimal beadhandling is paramount. The goal is that the cfDNA in the sample adsorbs to the solid phase completely during the binding step, is retained during washing and completely removed during elution. In this work, we improved beadhandling by optimizing the incubation chamber geometry and both frequency and temperature protocols, to maximize recovery rates. For characterization of the extraction performance, synthetic mutant DNA was spiked into human plasma samples. The LabDisk showed better reproducibility in DNA recovery rates with a standard deviation of ±13% compared to a manual approach using spin-columns (±17%) or nanobeads (±26%). The extraction of colorectal cancer samples with both the developed LabDisk and a robotic automation instrument resulted in comparable allele frequencies. Consequently, we present a highly attractive solution for an automated liquid biopsy cfDNA extraction in a small benchtop device.

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.

Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics.

We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 µL/reaction to 9.6 µL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.

Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics.

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.

Eliminating viscosity bias in lateral flow tests.

Despite the widespread application of point-of-care lateral flow tests, the viscosity dependence of these assay results remains a significant challenge. Here, we employ centrifugal microfluidic flow control through the nitrocellulose membrane of the strip to eliminate the viscosity bias. The key feature is the balancing of the sample flow into the cassette of the lateral flow test with the air flow out of the cassette. A viscosity-independent flow rate of 3.01 ± 0.18 µl/min (±6%) is demonstrated for samples with viscosities ranging from 1.1 mPas to 24 mPas, a factor greater than 20. In a model human IgG lateral flow assay, signal-intensity shifts caused by varying the sample viscosity from 1.1 mPas to 2.3 mPas could be reduced by more than 84%.

Automated library preparation for whole genome sequencing by centrifugal microfluidics.

Next generation sequencing is evolving from a research tool into a method applied in diagnostic routine. The complete sequencing workflow includes sample pre-processing, library preparation, sequencing and bioinformatics. High quality in each of these steps is necessary to obtain excellent sequencing results. The tedious and error-prone library preparation poses a significant challenge for smaller laboratories, where high throughput pipetting robots are not cost-effective. Here we present an automated library preparation for whole genome sequencing using centrifugal microfluidics. Two samples can be run per cartridge. Precise metering of reagents allows the required liquid volumes to be reduced by 40% and the amount of sample used by 60%. The functionality of the cartridge is demonstrated with bacteria and DNA extracted from a human FFPE sample. For the bacterial sample, mean sequencing depths from 140 to 183 reads and a coverage of 99.8% of the reference genome were detected. For the human DNA, mean sequencing depths of 4.4-5.7 reads and a coverage of 78.2% of the effective reference genome were observed.

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.

UMIc: A Preprocessing Method for UMI Deduplication and Reads Correction.

A recent refinement in high-throughput sequencing involves the incorporation of unique molecular identifiers (UMIs), which are random oligonucleotide barcodes, on the library preparation steps. A UMI adds a unique identity to different DNA/RNA input molecules through polymerase chain reaction (PCR) amplification, thus reducing bias of this step. Here, we propose an alignment free framework serving as a preprocessing step of fastq files, called UMIc, for deduplication and correction of reads building consensus sequences from each UMI. Our approach takes into account the frequency and the Phred quality of nucleotides and the distances between the UMIs and the actual sequences. We have tested the tool using different scenarios of UMI-tagged library data, having in mind the aspect of a wide application. UMIc is an open-source tool implemented in R and is freely available from https://github.com/BiodataAnalysisGroup/UMIc.

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.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

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.

Flow control for lateral flow strips with centrifugal microfluidics.

Lateral flow strips (LFSs) are widely used for clinical diagnostics. The restricted flow control of the current designs is one challenge to the development of quantitative and highly sensitive LFSs. Here, we present a flow control for LFSs using centrifugal microfluidics. In contrast to previously presented implementations of lateral flow membranes into centrifugal microfluidic cartridges, we direct the flow radially outwards through the membrane. We control the flow using only the centrifugal force, thus it is independent of membrane wetting properties and permeability. The flow rate can be decreased and increased, enabling control of incubation times for a wide variety of samples. We deduced a formula as a guideline for the integration of chromatographic membranes into centrifugal microfluidic disks to ensure that all the sample liquid flows through the membrane, hence safely avoiding bypass flow around the membrane. We verified the calculated operation conditions using different membranes, different flow rates, and different sample viscosities.

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.