The electrocatalytic oxidation of guanine in DNA and oligonucleotides by Ru(bpy) 3 3+/2+ was investigated using cyclic voltammetry (CV) and chronoamperometry (CA) (bpy ) 2,2′-bipyridine). Oxidation of Ru(bpy) 3 2+ to the Ru(III) form at tin-doped indium oxide (ITO) electrodes in the presence of DNA produces catalytic current due to the oxidation of guanine by Ru(III). CA traces of Ru(bpy) 3 2+ with calf thymus DNA at high salt concentration (50 mM sodium phosphate + 700 mM NaCl) give a single kinetic process with a rate constant of 3500 ( 300 M -1 s -1 that is independent of DNA concentration and similar to values determined previously by fitting CV data under the same conditions. Under low salt conditions (50 mM sodium phosphate + 0 mM NaCl), the CA data show two linear regions that give rate constants of 2.7 × 10 4 and 6 × 10 5 M -1 s -1 . Digital simulation of CV data at low salt requires a careful accounting for the binding of the metal complex to the DNA polyanion, which can be accomplished using binding constants that are independently determined. This analysis gives rate constants that are independent of DNA concentration and range from 2.3 × 10 5 to 1.4 × 10 6 M -1 s -1 as the scan rate is increased from 25 to 250 mV/s. The variation in rate constant with scan rate can be attributed to the two kinetic processes observed in the CA results. Resolution of Ru(bpy) 3 2+ into the ∆ and Λ stereoisomers showed that the two kinetic processes were not due to the stereoisomerism. Satisfactory fitting of the CV data requires addition of a second electron-transfer step from the oxidized guanine to Ru(III); this rate constant was always less than 1% of the rate constant for the first homogeneous electron transfer. In addition, the fitting at low salt requires accounting for the density of guanines in the DNA sequence. Calf thymus DNA is 20% guanine; however, the fitting shows that binding of the mediator by at least 60% of the nucleotides produces catalytic turnover. Oligonucleotides containing a single guanine gave similar rate constants to those observed by CV and CA on calf thymus DNA, and the fitting suggested that binding of the mediator by 5-10 of the 30 nucleotides (defined as "active binding sites") in the oligomer produced catalyst cycling. Thus, the electron is able to transfer to a mediator that is bound in a region that spans 2.5-5 base pairs and contains the oxidized guanine. The number of "active binding sites" increased predictably with the number of guanines in the sequence, ranging from 15% to 33% of the total nucleotides for a 15-mer duplex with one guanine to 75-100% for a 15-mer duplex with six guanines. Decreasing the salt concentration enhances the catalytic current both by increasing the number of active binding sites by a factor of 5-10 and by increasing the intrinsic oxidation rate by an order of magnitude.

Metal-to-ligand charge-transfer (MLCT) photolyses (lambda > or = 395 nm) of copper complexes of cis-1,8-bis(pyridin-3-oxy)oct-4-ene-2,6-diyne (bpod, 1), [Cu(bpod)(2)]PF(6) (2), and [Cu(bpod)(2)](NO(3))(2) (3) yield Bergman cyclization of the bound ligands. In contrast, the uncomplexed ligand 1 and Zn(bpod)(2)(CH(3)COO)(2) compound (4) are photochemically inert under the same conditions. In the case of 4, sensitized photochemical generation of the lowest energy (3)pi-pi state, which is localized on the enediyne unit, leads to production of the trans-bpod ligand bound to the Zn(II) cation by photoisomerization. Electrochemical studies show that 1, both the uncomplexed and complexed, exhibits two irreversible waves between E(p) values of -1.75 and -1.93 V (vs SCE), corresponding to reductions of the alkyne units. Irreversible, ligand-based one-electron oxidation waves are also observed at +1.94 and +2.15 V (vs SCE) for 1 and 3. Copper-centered oxidation of 2 and reduction of 3 occur at E(1/2) = +0.15 and +0.38 V, respectively. Combined with the observed Cu(I)-to-pyridine(pi) MLCT and pyridine(pi)-to-Cu(II) ligand-to-metal charge transfer (LMCT) absorption centered near approximately 315 nm, the results suggest a mechanism for photo-Bergman cyclization that is derived from energy transfer to the enediyne unit upon charge-transfer excitation. The intermediates produced upon photolysis degrade both pUC19 bacterial plasmid DNA, as well as a 25-base-pair, double-stranded oligonucleotide. Detailed analyses of the cleavage reactions reveal 5'-phosphate and 3'-phosphoglycolate termini that are derived from H-atom abstraction from the 4'-position of the deoxyribose ring rather than redox-induced base oxidation.

Clinical laboratories are recognizing the importance of implementing sensitive and specific molecular diagnostic tests. However, widespread adoption of these tests requires simplified workflows without requiring expensive supporting instrumentation. To enable microarray-based analysis to meet these requirements, we describe a valveless flow cell for disposable use that supports PCR coupled with microarray hybridization in the same chamber. The flow cell assembly consists simply of double-faced tape, a plastic microarray substrate, an absorbent, and a commercially-available hydrophilic thin film. The simple construction lends itself to low-cost and ease of manufacturing, yet several features reduces the complexity of the standard microarray workflow. First, there is no requirement for custom instrumentation. Second, the hydrophilic thin film allows uniform filling of a microfluidic chamber. Third, a geometric capillary stop design confines liquid to the microarray chamber during PCR, and thus eliminates the need for a valve or hydrophobic surface treatment. And fourth, imbibition drives the uniform removal of liquid reagents from the array chamber. Three hundred genomic copies of methicillin-resistant Staphylococcus aureus (MRSA) are detected in a flow cell with gel drop microarrays printed on an unmodified plastic substrate. This sensitivity is shown to be comparable to conventional methods (i.e., PCR in a tube, with separate hybridization in a microarray chamber, where amplicon is exposed to the workspace before and after hybridization). However, the flow cell combines these multiple steps into a simple, compact workflow without the need for complex valves or custom instrumentation and is less susceptible to contamination of the workspace than conventional methods because the amplicon is confined to the device.

There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.