6 research outputs found
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
Altered expression of heparan sulfate in <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> mammary epithelia.
<p>(<b>A</b>) Frozen sections of mammary glands were incubated with biotinylated FGF2, which binds to heparan sulfate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bai1" target="_blank">[55]</a>. Binding of FGF2 was detected with streptavidin-HRP (brown stain). In wildtype <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>–</sup> glands FGF2 binds to the basement membrane surrounding the epithelial ducts (arrowheads). The right panel magnifies the boxed region, revealing the sharp staining of the basement membrane underlying the epithelial cells. FGF2 also binds to the matrix surrounding the fat pad adipocytes (white arrowheads). Mutant <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> glands retain FGF2 binding around fat pad adipocytes, but binding to the basement membrane was greatly reduced. The lower right panel magnifies the boxed region. The bar in the left panels = 50 µm, right panels 12.5 µm. (<b>B</b>) Heparan sulfate was isolated from [6-<sup>3</sup>H]glucosamine labeled mammary epithelial cells derived from <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>–</sup> and <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> mice and degraded with nitrous acid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Shively1" target="_blank">[58]</a>. The individual oligosaccharides were separated by gel filtration chromatography and the area under the peaks was used to determine the extent of N-sulfation of the chains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bame1" target="_blank">[59]</a>. dp, degree of polymerization. Inset: Graph of comparison of areas under the curves.</p
Hematoxylin/eosin stained sections of mammary glands.
<p>Mammary glands from D8P, D15P, and D1L <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>−</sup> (<b>A–C, respectively</b>) and <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> (<b>D–F, respectively</b>) mice were stained with hematoxylin/eosin. No difference in glandular morphology was noted at D8P, but differences in ductal density occurred at D15P, which is quantified in (<b>G</b>). The difference in density between mutant and wildtype increased dramatically by D1L. Bars = 125 µm.</p
Characterization of the lactational defect in <i>Ndst1</i>-deficient mammary glands.
<p>Quantitative RT-PCR and Western blotting were used to characterize lactational capacity of <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> females. (<b>A</b>) RNA isolated from d1L <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>–</sup> and <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> mammary glands was analyzed by quantitative RT-PCR for the expression of whey acidic protein (WAP), β-casein, and keratin 18, genes specifically expressed by mammary epithelial cells. Data was normalized to the expression of GAPDH transcripts present in the sample. (<b>B</b>) Western blotting with antibodies to keratin 7 in d1L glands further confirmed that the <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> mammary glands have a reduced population of epithelia. (<b>C</b>) Western blotting with antibodies to mouse milk from d1L glands shows that milk production was diminished in <i>Ndst1</i><sup>f/f</sup><i>MMTVCre</i><sup>+</sup> glands. Blotting of d14P extracts showed that the antibodies were selective for milk protein except for a minor band at 67 kDa.</p
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
Amphiphilic Particle-Stabilized Nanoliter Droplet Reactors with a Multimodal Portable Reader for Distributive Biomarker Quantification
Compartmentalization,
leveraging microfluidics, enables highly
sensitive assays, but the requirement for significant infrastructure
for their design, build, and operation limits access. Multimaterial
particle-based technologies thermodynamically stabilize monodisperse
droplets as individual reaction compartments with simple liquid handling
steps, precluding the need for expensive microfluidic equipment. Here,
we further improve the accessibility of this lab on a particle technology
to resource-limited settings by combining this assay system with a
portable multimodal reader, thus enabling nanoliter droplet assays
in an accessible platform. We show the utility of this platform in
measuring N-terminal propeptide B-type natriuretic peptide (NT-proBNP),
a heart failure biomarker, in complex medium and patient samples.
We report a limit of detection of ∼0.05 ng/mL and a linear
response between 0.2 and 2 ng/mL in spiked plasma samples. We also
show that, owing to the plurality of measurements per sample, “swarm”
sensing acquires better statistical quantitation with a portable reader.
Monte Carlo simulations show the increasing capability of this platform
to differentiate between negative and positive samples, i.e., below
or above the clinical cutoff for acute heart failure (∼0.1
ng/mL), as a function of the number of particles measured. Our platform
measurements correlate with gold standard ELISA measurement in cardiac
patient samples, and achieve lower variation in measurement across
samples compared to the standard well plate-based ELISA. Thus, we
show the capabilities of a cost-effective droplet-reader system in
accurately measuring biomarkers in nanoliter droplets for diseases
that disproportionately affect underserved communities in resource-limited
settings