Janay Elise Kong

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.

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 sub-nanoliter 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 three orders of magnitude of concentration.

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.

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.