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.

Fractal LAMP: Label-Free Analysis of Fractal Precipitate for Digital Loop-Mediated Isothermal Nucleic Acid Amplification.

Nucleic acid amplification assays including loop-mediated isothermal amplification (LAMP) are routinely used in diagnosing diseases and monitoring water and food quality. The results of amplification in these assays are commonly measured with an analog fluorescence readout, which requires specialized optical equipment and can lack quantitative precision. Digital analysis of amplification in small fluid compartments based on exceeding a threshold fluorescence level can enhance the quantitative precision of nucleic acid assays (i.e., digital nucleic acid amplification assays), but still requires specialized optical systems for fluorescence readout and the inclusion of a fluorescent dye. Here, we report Fractal LAMP, an automated method to detect amplified DNA in subnanoliter scale droplets following LAMP in a label-free manner. Our computer vision algorithm achieves high accuracy detecting DNA amplification in droplets by identifying LAMP byproducts that form fractal structures observable in brightfield microscopy. The capabilities of Fractal LAMP are further realized by developing a Bayesian model to estimate DNA concentrations for unknown samples and a bootstrapping method to estimate the number of droplets required to achieve target limits of detection. This digital, label-free assay has the potential to lower reagent and reader cost for nucleic acid measurement while maintaining high quantitative accuracy over 3 orders of magnitude of concentration.

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.

Ferrodrop Dose-Optimized Digital Quantification of Biomolecules in Low-Volume Samples.

We present an approach to estimate the concentration of a biomolecule in a solution by sampling several nanoliter-scale volumes and determining if the volumes contain any biomolecules. In this method, varying volume fractions (nanoliter-scale) of a sample of nucleic acids are introduced to an array of uniform volume reaction wells (100 µL), which are then fluorescently imaged to determine if signal is above a threshold after nucleic acid amplification, all without complex instrumentation. The nanoliter volumes are generated and introduced using the simple positioning of a permanent magnet, and imaging is performed with a cellphone-based fluorescence detection scheme, both methods suitable for limited-resource settings. We use the length of time a magnetic field is applied to generate a calibrated number of nanoliter ferrodrops of sample mixed with ferrofluid at a step emulsification microfluidic junction. Each dose of ferrodrops is then transferred into larger microliter scale reaction wells on chip through a simple shift of the external magnet. Nucleic acid amplification is achieved using loop-mediated isothermal amplification (LAMP). By repeating each nanoliter dosage a number of times to calculate the probability of a positive signal at each dosage, we can use a binomial probability distribution to estimate the sample nucleic acid concentration. Using this approach we demonstrate detection of lambda DNA molecules down to 25 copies per microliter. The ability to dose separate nanoliter-scale volumes of a low-volume sample across wells in this platform is suited for multiplexed assays. This platform has the potential to be applied to a range of diseases by mixing a sample with magnetic nanoparticles.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

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.

Flexible and stretchable micromagnet arrays for tunable biointerfacing.

A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 µm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

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.