RESUMO
A condensation nucleation light scattering detector (CNLSD) was adapted for use as a detector for supercritical fluid chromatography. The performance of the CNLSD was evaluated and compared to evaporative light-scattering detector (ELSD) using a well-defined equimass mixture of uniform poly(ethylene glycol) oligomers and a certified reference material of poly(ethylene glycol) 1000. The CNLSD was able to detect a 10 times less concentrated solution of uniform oligomers compared to the ELSD. The quantitativeness of CNLSD was high enough to obtain the molecular mass distribution of poly(ethylene glycol) 1000 without any calibrations; on the other hand, the original data measured by ELSD was about 4% smaller than the certified value of poly(ethylene glycol) 1000. The CNLSD was suitable for supercritical fluid chromatography as a mass concentration detector.
Assuntos
Cromatografia com Fluido Supercrítico/instrumentação , Cromatografia com Fluido Supercrítico/métodos , Luz , Polietilenoglicóis/análise , Espalhamento de Radiação , Peso Molecular , Polietilenoglicóis/química , Temperatura , VolatilizaçãoRESUMO
SELEX (systematic evolution of ligands by exponential enrichment) is a process for isolating DNA or RNA sequences with high affinity and selectivity for molecular targets from random sequence libraries. These sequences are commonly referred to as aptamers. The process typically requires 10-15 cycles of enrichment, PCR amplification and nucleic acid purification to obtain high-affinity aptamers. We have demonstrated that using capillary electrophoresis (CE) as an enrichment step greatly improves the efficiency of the process. CE-SELEX is capable of isolating high-affinity aptamers in as little as 2-4 rounds of selection, shortening the process time from several weeks to as little as a few days.
Assuntos
Aptâmeros de Nucleotídeos/isolamento & purificação , Eletroforese Capilar/métodos , Técnica de Seleção de Aptâmeros/métodos , Precipitação Química , Cromatografia em Agarose , Eletroforese Capilar/economia , Etanol/química , Etanol/metabolismo , Ligantes , Reação em Cadeia da Polimerase , Técnica de Seleção de Aptâmeros/economia , Estreptavidina/química , Estreptavidina/metabolismo , Fatores de TempoRESUMO
The high affinity and specificity of aptamers make them ideal reagents for a wide range of analytical applications. It is not surprising that they are finding application in microfluidics as well. CE has proven to be an efficient technique for isolating aptamers. Aptamers have been used as affinity reagents in CE assays. Aptamer-based chromatography stationary phases have demonstrated unique selectivities. Possibly the application that holds the highest potential is aptamer microarrays for screening proteomic samples.
Assuntos
Aptâmeros de Nucleotídeos/isolamento & purificação , Microfluídica/métodos , Técnica de Seleção de Aptâmeros , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas/análiseRESUMO
Capillary electrophoresis-SELEX (CE-SELEX) was used to select ssDNA aptamers with affinity for HIV reverse transcriptase (HIVRT). A library of ssDNA was incubated with HIVRT. Sequences bound to HIVRT were isolated using CE, PCR amplified, and purified, yielding an enriched ssDNA pool suitable for further rounds of selection. Aptamers with dissociation constants as low as 180 pM were isolated after four rounds of selection. This is the first report of aptamers isolated by CE-SELEX with higher affinity than those obtained for the same target using conventional selection techniques. No sequence motifs were identified in the 27 clones sequenced, suggesting that there are many sequences that can bind HIVRT with low picomolar dissociation constants.
