Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions

The use of digital microfluidics for a silicon photonic sensors platform

status: publishe

Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions

Evaluating 3D printing to solve the sample-to-device interface for LRS and POC diagnostics: example of an interlock meter-mix device for metering and lysing clinical urine samples

This paper evaluates the potential of 3D printing, a semi-automated additive prototyping technology, as a means to design and prototype a sample-to-device interface, amenable to diagnostics in limited-resource settings, where speed, accuracy and user-friendly design are critical components. As a test case, we built and validated an interlock meter-mix device for accurately metering and lysing human urine samples for use in downstream nucleic acid amplification. Two plungers and a multivalve generated and controlled fluid flow through the device and demonstrate the utility of 3D printing to create leak-free seals. Device operation consists of three simple steps that must be performed sequentially, eliminating manual pipetting and vortexing to provide rapid (5 to 10 s) and accurate metering and mixing. Bretherton's prediction was applied, using the bond number to guide a design that prevents potentially biohazardous samples from leaking from the device. We employed multi-material 3D printing technology, which allows composites with rigid and elastomeric properties to be printed as a single part. To validate the meter-mix device with a clinically relevant sample, we used urine spiked with inactivated Chlamydia trachomatis and Neisseria gonorrhoeae. A downstream nucleic acid amplification by quantitative PCR (qPCR) confirmed there was no statistically significant difference between samples metered and mixed using the standard protocol and those prepared with the meter-mix device, showing the 3D-printed device could accurately meter, mix and dispense a human urine sample without loss of nucleic acids. Although there are some limitations to 3D printing capabilities (e.g. dimension limitations related to support material used in the printing process), the advantages of customizability, modularity and rapid prototyping illustrate the utility of 3D printing for developing sample-to-device interfaces for diagnostics

Silicon photonic sensors incorporated in a digital microfluidic system

Label-free biosensing with silicon nanophotonic microring resonator sensors has proven to be an excellent sensing technique for achieving high-throughput and high sensitivity, comparing favorably with other labeled and label-free sensing techniques. However, as in any biosensing platform, silicon nanophotonic microring resonator sensors require a fluidic component which allows the continuous delivery of the sample to the sensor surface. This component is typically based on microchannels in polydimethylsiloxane or other materials, which add cost and complexity to the system. The use of microdroplets in a digital microfluidic system, instead of continuous flows, is one of the recent trends in the field, where microliter- to picoliter-sized droplets are generated, transported, mixed, and split, thereby creating miniaturized reaction chambers which can be controlled individually in time and space. This avoids cross talk between samples or reagents and allows fluid plugs to be manipulated on reconfigurable paths, which cannot be achieved using the more established and more complex technology of microfluidic channels where droplets are controlled in series. It has great potential for high-throughput liquid handling, while avoiding on-chip cross-contamination. We present the integration of two miniaturized technologies: label-free silicon nanophotonic microring resonator sensors and digital microfluidics, providing an alternative to the typical microfluidic system based on microchannels. The performance of this combined system is demonstrated by performing proof-of-principle measurements of glucose, sodium chloride, and ethanol concentrations. These results show that multiplexed real-time detection and analysis, great flexibility, and portability make the combination of these technologies an ideal platform for easy and fast use in any laboratory

Ring resonator based SOI biosensors

In this paper, two recent advances in silicon ring resonator biosensors are presented. First, we address the problem that due to the high index contrast, small deviations from perfect symmetry lift the degeneracy of the normal resonator mode. This severely deteriorates the quality of the output signal. To address this, we discuss an integrated interferometric approach to give access to the unsplit, high-quality normal modes of the microring resonator. Second, we demonstrate how digital microfluidics can be used for effective fluid delivery to nanophotonic microring resonator sensors fully constructed in SOI

Digital biology and chemistry

This account examines developments in “digital” biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide “analog-to-digital” signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in “digital” microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This “digitizing” offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more deeply, and advancing molecular and cellular analysis. Microfluidics will impact digital biology and chemistry and will also benefit from them if it becomes massively distributed

ELIXIR and Toxicology: a community in development [version 2; peer review: 2 approved]

Toxicology has been an active research field for many decades, with academic, industrial and government involvement. Modern omics and computational approaches are changing the field, from merely disease-specific observational models into target-specific predictive models. Traditionally, toxicology has strong links with other fields such as biology, chemistry, pharmacology, and medicine. With the rise of synthetic and new engineered materials, alongside ongoing prioritisation needs in chemical risk assessment for existing chemicals, early predictive evaluations are becoming of utmost importance to both scientific and regulatory purposes. ELIXIR is an intergovernmental organisation that brings together life science resources from across Europe. To coordinate the linkage of various life science efforts around modern predictive toxicology, the establishment of a new ELIXIR Community is seen as instrumental. In the past few years, joint efforts, building on incidental overlap, have been piloted in the context of ELIXIR. For example, the EU-ToxRisk, diXa, HeCaToS, transQST, and the nanotoxicology community have worked with the ELIXIR TeSS, Bioschemas, and Compute Platforms and activities. In 2018, a core group of interested parties wrote a proposal, outlining a sketch of what this new ELIXIR Toxicology Community would look like. A recent workshop (held September 30th to October 1st, 2020) extended this into an ELIXIR Toxicology roadmap and a shortlist of limited investment-high gain collaborations to give body to this new community. This Whitepaper outlines the results of these efforts and defines our vision of the ELIXIR Toxicology Community and how it complements other ELIXIR activities

Micropatterning of Digital Microfluidic Chips: Applications in Bio-Assay Development and Materials Synthesis

Digital microfluidics (DMF) emerged recently as a promising liquid handling technology for a wide variety of applications. DMF chips manipulate individual fluid droplets on planar surfaces by using electrostatic actuation forces. By applying series of electrical potentials to arrays of electrodes coated with a hydrophobic insulator, droplets containing reagents and samples can be transported, split, merged, mixed, and dispensed from fluid reservoirs. DMF is capable of manipulating low volumes of scarce samples and expensive reagents with great precision, while enabling parallelization and automation of fluid handling for high-throughput applications. DMF has been applied in diagnostics, biology, and chemical synthesis for which it demonstrated great potential both in terms of miniaturization and automation. Recent research demonstrated that the introduction of hydrophilic, functional zones in the fluorocarbon chip surfaces has the potential to enable a new fluidic operation called passive dispensing. In passive dispensing, tiny fluid volumes are deposited onto these hydrophilic elements when a droplet passes over them due to their selective wettability compared to the fluorocarbon layer. However, the exploration of this technique is hindered by the lack of a suitable microfabrication technique that allows accurate micropatterning of the fluorocarbon chip surface on a biocompatible way. First, a novel microfabrication technique is introduced in order to micropattern hydrophobic fluorocarbon surfaces on a biocompatible way. This novel microfabrication technique facilitates the fabrication of patterns down to low micrometer dimensions inside the fluorocarbon chip surface, while biomolecules can be selectively deposited inside these micropatterns with great precision. As such, DMF devices containing hydrophilic cell adhesion islands were fabricated for performing automated and miniaturized cell-based applications. When droplets containing suspended mammalian cells were transported over these cell adhesion islands, nanoliter droplets are split off from the original droplet and remain on the hydrophilic islands. This facilitates fully automated seeding, culturing, treating, and staining of cells and demonstrates DMF's great potential for automated drug screening applications. Subsequently, a DMF device is patterned with hydrophilic-in-hydrophobic microwells designed to trap and seal single magnetic microbeads. These microbeads are used to capture single protein or nucleic acid molecules before being printed and sealed inside the microwells on the DMF chip. When a fluorescent reaction is initiated due to the presence of an enzyme or an enzyme-labeled nucleic acid molecule, the fluorophores accumulate inside the small microwells on the chip and ensure sensitive detection of single molecules with fluorescence microscopy. Next, it is shown how micropatterned DMF chips present exciting new ways of performing miniaturized materials synthesis. This is exemplified by the on-chip synthesis of metal-organic frameworks (MOFs), an emerging class of crystalline, porous materials. By transporting droplets of MOF synthesis solutions over hydrophilic-in-hydrophobic micropatches on the DMF chip surface, MOFs can be patterned as arrays of single crystals or crystalline membranes. DMF proved to be an excellent tool for such highly controlled crystal patterning purposes, which will benefit the future applicability of these materials in functional devices. Finally, it is demonstrated that micropatterning the fluorocarbon chip surface enables the integration of optical components on DMF chips for integrated, on-chip sensing of droplet contents. Therefore, silicon microring resonator sensors were integrated in the DMF chip and were directly contacted by fluid droplets where the fluorocarbon chip surface was locally removed. This facilitated integrated optical sensing of droplet content in an automated and plug-and-play setting.status: publishe

Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection

status: publishe