Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems

摘要

Monodisperse-porous silica microspheres 5.5 mu m in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, beta-nicotinamide adenine dinucleotide (beta-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and beta-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and beta-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 mu m in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and beta-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and beta-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.