Quantitation of Phosphorothioate Oligonucleotides in Human Blood Plasma Using a Nanoparticle-Based Method for Solid-Phase Extraction

Abstract

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.

Analysis of peptides and proteins affinity-bound to iron oxide nanoparticles by MALDI MS

“…This method allows the MALDI MS detection of low concentration of proteins in complex matrix solution. Other types of nanoparticles have also been used to capture the analytes of interest, such as phosphopeptides [30,31], antigen [32][33][34], oligonucleotides [35,36], aminothiol [37], protein [38,39], and bacteria [40,41]. Among the nanoparticles used, carbon nanotube [38], Nile red-adsorbed gold [37], and TiO 2 -coated magnetic nanoparticles [30] were not only employed as affinity probes but also as matrices.…”

Quantitative Analysis of Modified Antisense Oligonucleotides in Biological Fluids Using Cationic Nanoparticles for Solid-Phase Extraction

“…In previous studies it was shown that these conditions are most favorable for the formation of nanoparticle-oligonucleotide conjugates, as the presence of denatured proteins resulted in poor recovery of ONs (14). In order to evaluate the suitability of the developed extraction method for the quantitation of modified antisense ONs in biological fluids, the linearity, accuracy, and reproducibility of the assay were investigated.…”

“…In a previously published work a novel method for solid-phase extraction of phosphorothioate oligonucleotides (PTOs) from human plasma has been developed (14). In contrast to other techniques using strong anion exchange resins (15)(16)(17), this method is based on the application of cationic polystyrene nanoparticles which are capable of reversibly binding negatively charged oligonucleotides with high affinity (18).…”

“…In order to exclude contamination of the samples with residual particles, the supernatant was placed in another 1.5-ml tube and centrifuged again. Subsequently, the samples were dried by rotoevaporation or lyophilization and stored at Ϫ20°C until analysis (14).…”

Nonionic surfactant‐capped gold nanoparticles for selective enrichment of aminothiols prior to CE with UV absorption detection

“…The quantification of various analytes, which range from small molecules to macromolecules, including amino acids and peptides, has been successfully achieved by CE [20][21][22]. However, to date, only cationic polystyrene NPs have been employed for the extraction of oligonucleotides from human plasma, which is followed by separation using CE [23,24].…”