Charging and Charged Species in Quantum Dot Light-Emitting Diodes

Despite recent rapid advances in improving quantum dot light-emitting diodes, many fundamental aspects of the device operating mechanism remain unresolved. Through transient electroluminescence and time-resolved photoluminescence measurements, the effects of offset voltage on charging and charge transport are examined. First, capacitive charging occurs with a time constant of ∼500 ns, followed by electron transport through quantum dots with a mobility of ∼10–5 cm2 V–1 s–1. Hole injection then initiates an electroluminescence rise that is independent of offset voltage. The photoluminescence lifetime is also unaffected by the offset voltage, indicating no injection of charges into the quantum dots or on their surfaces prior to the voltage pulse. A slower equilibration to steady-state electroluminescence is dependent on the offset voltage, indicative of another charging process. Elemental mapping shows that ZnO deposition from solution can lead to the diffusion of charged species into the quantum dot layer, which may cause the slower process

Alignment of human and mouse APP 5′UTRs with human PrP 5′UTR sequences relative to the L- and H-ferritin Iron-responsive elements (IREs).

<p><b>Panel A</b>: The human and mouse APP 5′UTR specific IRE-like RNA stem loops, the human PrP 5′UTR, and the human and mouse <i>SNCA</i> specific IRE –like stem loops each aligned adjacent to the ferritin L- and H IRE RNA stem loops. Shown, the L- and H-mRNAs encode canonical IRE RNA stem loops whereas the APP IRE in non canonical although fully iron responsive <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Cho1" target="_blank">[6]</a>. The α-synuclein IRE (<i>SNCA</i> IRE) represents a non canonical IRE traversing the central splice junction of exon-1 and exon-2 (the CAGUGN loop/splice site sequences) of <i>SNCA</i> mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Friedlich1" target="_blank">[49]</a>. Typical IRE stem loops fold to exhibit an apical AGU pseudotriloop which is depicted in red lettering at the apex of the H-ferritin and <i>SNCA</i> IREs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Goforth1" target="_blank">[28]</a> relative to an analogous AGA from the IRE–like stem loop encoded by APP mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Cho1" target="_blank">[6]</a>. <b>Panel B</b>: Maps of the 5′UTRs encoding by the human and mouse APP mRNAs, PrP mRNA, <i>SNCA</i> mRNA, and the mRNAs for L- and H-ferritin (IRE stem loops are displayed as lollipops). <b>Panel C</b>: Relative alignment of the sequences that encode the 5′UTR specific IRE-like stem loops in human APP mRNA, PrP mRNA, <i>SNCA</i> mRNA, and the L- and H-ferritin mRNAs. <b>Panel D</b>: <u>Screen and counter-screening Constructs </u><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Bandyopadhyay2" target="_blank">[<b>21</b>]</a><b>:</b> The human APP 5′UTR cassette was subcloned in front of the luciferase reporter gene in the dicistronic pCD(APP) reporter construct. The same-sized and related PrP 5′UTR was subcloned in an identical format into the pCD(PrP) reporter construct for the purpose of counter-screening, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#s2" target="_blank">materials and methods</a> section.</p

The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein (<i>SNCA</i>).

<p><b>Panels A and B</b>: Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and <i>SNCA</i> levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). <u>Right Panel:</u> Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. <b>Panel C</b>: Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p<0.01), 0.1 µM (** = p<0.015), and 1 µM (*** = p<0.01) analyzed by ANOVA (N = 5). <u>Dotted line</u>: Representative LDH assay parallel to Aβ determination for SH-SY5Y cells treated for 72 h at concentrations up to 100 µM of JTR-009 (N = 4). <b>Panel D</b>: MTS assay for cellular mitochondrial viability after treatment of SH-SY5Y cells with JTR-005 and JTR-009 at the concentrations shown. Y axis: Percent of maximal viability ± SEM after treatment of the cells with JTR-009 and JTR-005 (N = 3)). Shown are the relative trend-lines for the dose-responsive viability of JTR-005 and JTR-009 compared to untreated cells (‘poly’ = non linear polynomial regression of the data).</p

Comparative IC₅₀ of JTR-009 relative to posiphen to inhibit APP 5′UTR driven luciferase expression relative to suppression of APP and Aβ levels in SH-SY5Y cells and primary neurons.

<p>Comparative IC₅₀ of JTR-009 relative to posiphen to inhibit APP 5′UTR driven luciferase expression relative to suppression of APP and Aβ levels in SH-SY5Y cells and primary neurons.</p

RNA pulldown assay to measure the dose-dependent capacity of the cyclic benzimidazole JTR-009 to substitute for IRP1 binding to APP 5′UTR sequences in SH-SY5Y cells: correlated repression of APP translation.

<p>RNA pulldown assays were conducted as ilustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone-0065978-g006" target="_blank">Figure 6</a> and as described by Cho et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Cho1" target="_blank">[6] </a><b>Panel A and B</b>: Representative RNA binding assays in which recovered beads measured the dose-responsive capacity of JTR-009 (0 µM 0.3 µM, 3 µM and 30 µM) to inhibit IRP1 binding to 30 base biotinylated probes encoding the APP 5′UTR. In Panel B Western blots measured relative levels of IRP1 and IRP2 bound to biotinylated RNA probes for APP IRE sequences after recovery in steptavidin bead fractions. Densitomteric quantitation of bead-specific IRP1 is shown in Panel A. <b>Panel C</b>: Measurement of the dose-dependent off-target action of JTR-009 to suppress H-ferritin IRE binding to SH-SY5Y specific IRP1 and IRP2 (bead fraction). <b>Panel D and E</b>: Dose-dependent decrease of APP levels in response to JTR-009 measured in the supernatants of bead fractions (experimentalPanel E: Western blots of lysate supernatants showing APP as measured using the N terminal specific 22C11 and C-terminal specific A8717 anitibodies. <b>Panel D</b>: Densitometric quantitation of the data in Panel E to measure the extent to which JTR-009 dose dependently repressed APP expression in SH-SY5Y cells (Dunnetts test, p = 0.03). Data from 5 separate trials, each in triplicate.</p

Evaluation of the potency and selectivity of APP blocker-9.

<p><b>Panel A</b>: Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC₅₀ = 0.1 µM; posiphen: IC₅₀ = 5 µM, N = 4). <b>Panel B</b>: Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. <b>Panel C</b>: Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). <b>Panel D</b>: Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein (<i>SNCA</i>) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). <b>Panel E</b>: Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. <b>Panel F</b>: Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).</p

Relative capacity of thirteen APP 5′UTR translation blockers to reduce Aβ levels in the conditioned medium of SH-SY5Y cells.

<p>Following 48 h treatment (1 µM) for each inhibitor, the histogram shows reduction of total Aβ levels confirmed after averaging five independent samplings from the following:- JTR-009 treated</p

IC₅₀ (nM) for APP 5′UTR blockers in pIRES-APP-5′UTR transfectants (top row) and pIRES-PrP-5′UTR transfectants (bottom row).

<p>Values were calculated from inhibiton curves in 384-plate assays to reduce 5′UTR driven luciferase expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065978#pone.0065978-Bandyopadhyay2" target="_blank">[21]</a>.</p