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 tris­(2,2′-bipyridyl)­dichlororuthenium­(II) hexahydrate ([Ru­(bpy)₃]²⁺) 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 [Ru­(bpy)₃]²⁺ concentration for 9–900 μM [Ru­(bpy)₃]²⁺. The detection limit was found to be 7 μM 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 poly­(diallyldimethylammonium chloride) (PDDA)/DNA films. In these experiments, ECL resulted from the reaction of [Ru­(bpy)₃]³⁺ with guanines of DNA. ECL produced at these thin-film electrodes was linear with respect to [Ru­(bpy)₃]²⁺ concentration from 180 to 800 μM. 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 poly­(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 μm × 800 μm 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 μL min^–1. 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 H₂O₂ in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 μM. 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