RESUMEN
The amine-containing ligand L, composed of two bidentate pyridyl-thiazole moieties linked by a 1,3-diaminophenylene unit, reacts with copper(II)â ions to form a dinuclear double helicate [Cu2 L2 ](4+) . Reaction of [Cu2 L2 ](4+) with dihydrogen phosphate (0.5â equivalents) gives the unsaturated dinuclear double helicate [Cu2 L2 (OPO3 H2 )](3+) . [Cu2 L2 (OPO3 H2 )](3+) further reacts with another 0.5â equivalents of dihydrogen phosphate to give a trinuclear circular helicate which then self-assembles into a hexameric cluster [{Cu3 L3 (OPO3 H2 )3 }](26+) .
RESUMEN
A microfluidic device was developed to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The complete aqueous chemistry associated with the second purification step yields a highly concentrated PCR-ready eluate of nucleic acids devoid of PCR inhibitors that are reagent-based (isopropanol) and sample-based (indigo dye), both of which are shown to be successfully removed using the dual-phase device but not by the traditional microfluidic SPE (muSPE). The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The same samples purified using muSPE were not all PCR amplifiable due to a smaller concentration of the DNA and the lack of PCR-compatible aqueous chemistry in the extraction method. The utility of the device for the purification of RNA was also demonstrated, by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a "closed system" that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants.
Asunto(s)
ADN/aislamiento & purificación , Análisis por Micromatrices/métodos , Técnicas Analíticas Microfluídicas/métodos , ARN/análisis , Extracción en Fase Sólida/métodos , 2-Propanol/química , Colorantes/química , ADN/sangre , Genética Forense , Carmin de Índigo , Indoles/química , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificaciónRESUMEN
A new route to luminescent derivatives of androgenic steroids containing a ketone group in the 3- or 17-position has been developed. Reaction with the fac-Re(CO)(3)Cl complex of 3,3'-diamino-2,2'-bipyridine (complex 1) afforded a cyclic aminal product with different steroids. The rate of reaction and yield varies according to the conjugation or steric hindrance around the ketone group.