We describe the electrogenerated chemiluminescence (ECL) processes of the Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl)/ tripropylamine (TPrA) system at glassy carbon, platinum, and gold electrodes. The electrochemical behavior of TPrA on different electrode materials and its influence on the ECL process are demonstrated. At glassy carbon electrodes, the direct oxidation of TPrA began at approximately 0.6 V vs SCE and exhibited a broad irreversible anodic peak. Two ECL waves were observed, one in the potential region more negative than 1.0 V vs SCE and one at more positive potentials. The first ECL process apparently occurs without the electrogeneration of Ru(bpy)3(3+), in contrast to that of the second ECL wave. At Pt and Au electrodes, however, the formation of surface oxides significantly blocked the direct oxidation of TPrA. An ECL wave below 1.0 V did not appear at Pt and was very weak at gold. The ECL peaks at potentials of 1.1-1.2 V were also much weaker than those observed at the glassy carbon electrode. These results showed that the direct oxidation of TPrA played an important role in the ECL processes. Therefore, the enhancement of the TPrA oxidation current might lead to an increase in the ECL intensity. Small amounts of halide species were found to inhibit the growth of surface oxides on Pt and gold electrodes and led to an obvious increase of TPrA oxidation current. The anodic dissolution of gold in halide-containing solution was also important in activating the gold electrode surface. The electrochemical catalytic effect of bromide further promoted the oxidation of TPrA. A halide effect on ECL at Pt and Au electrodes was also evident. The most effective enhancement of ECL was observed at Au electrode in a bromide-containing solution. This effect was also found in an commercial flow-through instrument (IGEN) and provided a simple way to improve the detection sensitivity at low concentrations of Ru(bpy)3(2+).

The electrochemistry, UV-vis spectrophotometry, photoluminescence, and electrogenerated chemiluminescence (ECL) of perylenedicarboxylic imide, perylenetetracarboxylic diimide (PDI), terrylenetetracarboxylic diimide (TDI), and quaterrylenecarboxylic diimide (QDI) were investigated. All compounds undergo two reversible one-electron reductions and one reversible one-electron oxidation reaction. The first reduction potential shifts to less negative values and the potential for oxidation to less positive values for the diimide series with increasing size of the aromatic core. These changes in potential correlate well with orbital energies from molecular orbital calculations. The difference in potential between the first and second reduction waves decreased with increasing distance between the imide groups, so that TDI and QDI show only a single reduction wave, equivalent to a two-electron reduction. These reduction potentials provide estimates for the equilibrium constant for disproportionation of the radical anion. Very stable ECL spectra of PDI or TDI generated by sequential production of the radical cation and radical anion at an electrode were observed; these were identical to the photoluminescence spectra. A consideration of the energetics of the electron transfer reaction and the singlet energy leads to the conclusion that emission occurs mainly via generation of triplets followed by triplettriplet annihilation (the T-route).

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.

We report gene targeting via homologous recombination in zebrafish. We co-injected fertilized eggs with transcription activator-like effector nuclease mRNAs and a donor vector with long homologous arms targeting the tyrosine hydroxylase (th) locus, and we observed effective gene modification that was transmitted through the germ line. We also successfully targeted two additional genes. Homologous recombination in zebrafish with a dsDNA donor expands the utility of this model organism.