Methylation-Sensitive Loop-Mediated Isothermal Amplification (LAMP): Nucleic Acid Methylation Detection through LAMP with Mobile Fluorescence Readout.

The emergence of epigenetic gene regulation and its role in disease have motivated a growing field of epigenetic diagnostics for risk assessment and screening. In particular, irregular cytosine DNA base methylation has been implicated in several diseases, yet the methods for detecting these epigenetic marks are limited to lengthy protocols requiring bulky and costly equipment. We demonstrate a simple workflow for detecting methylated CpG dinucleotides in synthetic and genomic DNA samples using methylation-sensitive restriction enzyme digestion followed by loop-mediated isothermal amplification. We additionally demonstrate a cost-effective mobile fluorescence reader comprising a light-emitting diode bundle, a mirror, and optical fibers to transduce fluorescence signals associated with DNA amplification. The workflow can be performed in approximately 1 h, requiring only a simple heat source, and can therefore provide a foundation for distributable point-of-care testing of DNA methylation levels.

Research highlights: translating chips.

Microfluidic and microfabricated systems are providing key functionalities in diagnostic and therapeutic scenarios, translating beyond the research laboratory to pre-clinical animal studies and clinical studies with patients. Here, we highlight a recent study making use of miniaturization and automation in the development of a smartphone-integrated point-of-care diagnostic to detect antibodies to infectious diseases in a global health setting. We also review an intraocular implanted diagnostic system for glaucoma that relies on imaging the location of a fluid meniscus in a microchannel to readout pressure within the eye. Developments in low-cost and highly functional consumer electronic systems (e.g. smartphones in both highlighted works) has led to a continuing trend to incorporate such technologies with microfluidic fluid handling capabilities to achieve complete diagnostic solutions. We conclude with another implanted microdevice that delivers drug locally to tumors through electroosmotic flow and electromigration of charged drug species, which allows high drug concentrations near a tumor or resected tumor site while preventing high systemic levels associated with significant side-effects. The maturity of microsystem components are now allowing integration into fully functional systems that are poised to reach the clinic in a variety of forms - diagnostic to therapeutic.

Research highlights: digital assays on chip.

The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

Research highlights: micro-engineered therapies.

Lab on a chip systems have often focused on diagnostic, chemical, and cell analysis applications, however, more recently the scale and/or precision of micro-engineered systems has been applied in developing new therapies. In this issue we highlight recent work using microfluidic and micro-engineered systems in therapeutic applications. We discuss two approaches that use microfluidic precision to address challenges in filtering blood--to both remove unwanted pathogens and toxins and isolate rare cells of interest that have therapeutic potential. In both cases chemically-modified surfaces, a bioengineered mannose binding lectin on magnetic particles and antibody-functionalized reversibly degradable alginate film, provide the functionality to remove (or isolate) target cells of interest. The third paper we highlight generates microscale gels as protective niches for cell-based therapies. Importantly, the microgels are designed to have controlled porosity but also mechanical rigidity to protect housed therapeutic cells, like mesenchymal stem cells. We expect continued progress in micro- & nano-enabled therapies facilitated by the fabrication of new microstructured materials, precise separations, and closed-loop sensing and drug delivery.

Advances in the production and handling of encoded microparticles.

Here we highlight emerging technologies in the synthesis, handling, and application of encoded microparticles for multiplexed assays. Traditionally, in drug discovery and life sciences research, multiple reactions will be conducted in parallel using microwell plate formats or microfluidic implementations, in which volumes are confined and reactions annotated by knowledge of what reagents were added to each volume. Microparticle-based information carriers provide an alternative approach to performing such multiplexed reactions, in which reactions and events are instead annotated with unique codes associated with the solid-phase particle. One challenge has been in creating a unique and large enough code set that is also easily readout, and we highlight two approaches that have brought orthogonal optical tagging techniques to bear. Another challenge has been that in such approaches, reactions have usually been confined to the surface of, or within the bulk of the specifically-tagged particle. We also highlight a creative approach and strategy for multiplexing - called "partipetting"- in which the coded particle can be a carrier of a unique fluid reagent.