Maier M, Fritz H, Gerster M, Schewitz J, Bayer E
Research Center for Nucleic Acid and Peptide Chemisty, University of Tübingen, Germany.
Anal Chem. 1998 Jun 1;70(11):2197-204. doi: 10.1021/ac980097w.
Based on the application of cationic polystyrene nanoparticles, a novel method for solid-phase extraction of phosphorothioate oligonucleotides from human plasma has been developed. A high binding affinity, which is required for an effective isolation out of complex mixtures, is mediated by hydrophobic and multiple electrostatic interactions between the oligonucleotides and the nanoparticles. The principle of the method is based on a pH-controlled adsorption/desorption mechanism. Analysis of the extracted samples was performed by capillary gel electrophoresis. Extraction conditions were optimized, providing the isolation of oligonucleotides (> or = 10 nucleotide units) in high yields and purity even at concentrations in the low-nanomolar range (down to 5 nM). The low salt contamination of the samples allows their direct analysis by electrospray mass spectrometry. The combined linearity and accuracy of the assay together with absolute recovery rates in the range of 60-90% indicate that the developed solid-phase extraction method is generally applicable to quantitation of oligonucleotides in human plasma. Further improvement was achieved with an optimized carrier system of 2-fold enlarged particles which reduces the time consumption of the extraction procedure to approximately 30 min.
基于阳离子聚苯乙烯纳米颗粒的应用，已开发出一种从人血浆中固相萃取硫代磷酸酯寡核苷酸的新方法。寡核苷酸与纳米颗粒之间的疏水和多重静电相互作用介导了有效从复杂混合物中分离所需的高结合亲和力。该方法的原理基于pH控制的吸附/解吸机制。通过毛细管凝胶电泳对提取的样品进行分析。优化了提取条件，即使在低纳摩尔范围（低至5 nM）的浓度下，也能以高产量和纯度分离寡核苷酸（≥10个核苷酸单元）。样品的低盐污染使其能够通过电喷雾质谱直接分析。该测定方法的线性和准确性以及60-90%范围内的绝对回收率表明，所开发的固相萃取方法通常适用于人血浆中寡核苷酸的定量分析。通过优化的载体系统，即颗粒尺寸增大2倍，进一步改进了该方法，将提取过程的时间消耗减少到约30分钟。
