7 research outputs found
Electrochemiluminescence at Bare and DNA-Coated Graphite Electrodes in 3D-Printed Fluidic Devices
Clear plastic fluidic
devices with ports for incorporating electrodes
to enable electrochemiluminescence (ECL) measurements were prepared
using a low-cost, desktop three-dimensional (3D) printer based on
stereolithography. Electrodes consisted of 0.5 mm pencil graphite
rods and 0.5 mm silver wires inserted into commercially available
1/4 in.-28 threaded fittings. A bioimaging system equipped with a
CCD camera was used to measure ECL generated at electrodes and small
arrays using 0.2 M phosphate buffer solutions containing trisÂ(2,2′-bipyridyl)ÂdichlororutheniumÂ(II)
hexahydrate ([RuÂ(bpy)<sub>3</sub>]<sup>2+</sup>) with 100 mM tri-<i>n</i>-propylamine (TPA) as the coreactant. ECL signals produced
at pencil graphite working electrodes were linear with respect to
[RuÂ(bpy)<sub>3</sub>]<sup>2+</sup> concentration for 9–900
ÎĽM [RuÂ(bpy)<sub>3</sub>]<sup>2+</sup>. The detection limit was
found to be 7 ÎĽM using the CCD camera with exposure time set
at 10 s. Electrode-to-electrode ECL signals varied by ±7.5%.
Device performance was further evaluated using pencil graphite electrodes
coated with multilayer polyÂ(diallyldimethylammonium chloride) (PDDA)/DNA
films. In these experiments, ECL resulted from the reaction of [RuÂ(bpy)<sub>3</sub>]<sup>3+</sup> with guanines of DNA. ECL produced at these
thin-film electrodes was linear with respect to [RuÂ(bpy)<sub>3</sub>]<sup>2+</sup> concentration from 180 to 800 ÎĽM. These studies
provide the first demonstration of ECL measurements obtained using
a 3D-printed closed-channel fluidic device platform. The affordable,
high-resolution 3D printer used in these studies enables easy, fast,
and adaptable prototyping of fluidic devices capable of incorporating
electrodes for measuring ECL
3D-Printed Fluidic Devices for Nanoparticle Preparation and Flow-Injection Amperometry Using Integrated Prussian Blue Nanoparticle-Modified Electrodes
A consumer-grade
fused filament fabrication (FFF) 3D printer was
used to construct fluidic devices for nanoparticle preparation and
electrochemical sensing. Devices were printed using polyÂ(ethylene
terephthalate) and featured threaded ports to connect polyetheretherketone
(PEEK) tubing via printed fittings prepared from acrylonitrile butadiene
styrene (ABS). These devices included channels designed to have 800
ÎĽm Ă— 800 ÎĽm square cross sections and were semitransparent
to allow visualization of the solution-filled channels. A 3D-printed
device with a Y-shaped mixing channel was used to prepare Prussian
blue nanoparticles (PBNPs) under flow rates of 100 to 2000 ÎĽL
min<sup>–1</sup>. PBNPs were then attached to gold electrodes
for hydrogen peroxide sensing. 3D-printed devices used for electrochemical
measurements featured threaded access ports into which a fitting equipped
with reference, counter, and PBNP-modified working electrodes could
be inserted. PBNP-modified electrodes enabled amperometric detection
of H<sub>2</sub>O<sub>2</sub> in the 3D-printed channel by flow-injection
analysis, exhibiting a detection limit of 100 nM and linear response
up to 20 ÎĽM. These experiments show that a consumer-grade FFF
printer can be used to fabricate low-cost fluidic devices for applications
similar to those that have been reported with more expensive 3D-printing
methods
Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences
Nonenzymatic, template-directed synthesis of nucleic
acids is a
paradigm for self-replicating systems. The evolutionary dynamics of
such systems depend on several factors, including the mutation rates,
relative replication rates, and sequence characteristics of mutant
sequences. We measured the kinetics of correct and incorrect monomer
insertion downstream of a primer-template mismatch (mutation), using
a range of backbone structures (RNA, DNA, and LNA templates and RNA
and DNA primers) and two types of 5′-activated nucleotides
(oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5′-phosphorimidazolides).
Our study indicated that for all systems studied, an initial mismatch
was likely to be followed by another error (54–75% of the time),
and extension after a single mismatch was generally 10–100
times slower than extension without errors. If the mismatch was followed
by a matched base pair, the extension rate recovered to nearly normal
levels. On the basis of these data, we simulated nucleic acid replication
in silico, which indicated that a primer suffering an initial error
would lag behind properly extended counterparts due to a cascade of
subsequent errors and kinetic stalling, with the typical mutational
event consisting of several consecutive errors. Our study also included
different sequence contexts, which suggest the presence of cooperativity
among monomers affecting both absolute rate (by up to 2 orders of
magnitude) and fidelity. The results suggest that molecular evolution
in enzyme-free replication systems would be characterized by large
“leaps” through sequence space rather than isolated
point mutations, perhaps enabling rapid exploration of diverse sequences.
The findings may also be useful for designing self-replicating systems
combining high fidelity with evolvability
Metformin reduces tumor hypoxia.
<p>Mice bearing subcutaneous A549 xenografts were given metformin in the drinking water to the indicated concentration. A-B: After 1 week, metformin concentrations in plasma (A) and tumor (B) were measured using HPLC. Assuming the density of tumor tissue is 1g/ml, 1ug/g corresponds to ~8uM. C: After 1 day, mice were administered EF5 to label hypoxic tumor cells. Viable EF5-positive cells were quantified from tumor sections. Circles represent the average value from two sections of a tumor; the mean value across all tumors is indicated with a bar.</p
Metformin response is associated with MATE2 and OCT3 expression.
<p>A-B: AUCs for proliferation inhibition (A) and OCR inhibition (B) were correlated with MATE2 and OCT3 expression respectively. C: Intracellular metformin concentration after 30 minutes exposure to 2mM metformin as measured by HPLC. Circles represent biological replicates, the mean value is indicated with a bar. Assuming a cell volume of 2pL, 1mM corresponds to 2.5pg/cell.</p
Expression of OCTs and MATEs across cell lines.
<p>A-E: Relative mRNA expression of OCTs and MATEs was normalized across the cell line panel and is shown after arranging cell lines in order of increased expression. Mean ± S.E.M., <i>n</i> = 3. F-G: Relative expression of OCT1 correlated significantly with OCT2 (F) between cell lines.</p
Additional file 1: of Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial
Supplementary methods and Tables S1–S4. (DOCX 148 kb