Fabrication of micron-sized boronate-decorated polyethyleneimine-grafted magnetic agarose beads for specific enrichment of ribonucleic acid.

Exploiting high-performance magnetic beads for specific enrichment of ribonucleic acid (RNA) has important significance in the biomedical research field. Herein, a simple strategy was proposed for fabricating boronate-decorated polyethyleneimine-grafted magnetic agarose beads (BPMAB), which can selectively isolate cis-diol-containing substances through boronate affinity. The size of the basic magnetic agarose beads was controlled through the emulsification of the water-in-oil emulsion with a high-speed shear machine, which enhanced the specific surface area of BPMAB. Subsequently, to modify more boronic acid ligands, branched PEI with excellent hydrophilicity and numerous reaction sites was grafted. 2,4-Difluoro-3-formylphenyl boronic acid (2,4-DFPBA) was covalently immobilized for selectively capturing cis-diol-containing substances under physiological condition (pH 7.4). The BPMAB with a diameter range from 1.86 µm to 11.60 µm possessed clearly spherical structure, and excellent magnetic responsiveness and suspension ability in aqueous solution. ß-Nicotinamide adenine dinucleotide (ß-NAD), a short-chain cis-diol carrying agent, was selected as a target molecule for evaluating the adsorption property of BPMAB and the maximum adsorption capacity of BPMAB for ß-NAD could reach 205.11 mg g-1. In addition, the BPMAB as adsorbent was used to selectively enrich RNA from mammalian cells. The maximum adsorption capacity of BPMAB for RNA was 140.50 mg g-1. Under optimized conditions, the BPMAB-based MSPE successfully enriched the high-quality total RNA with 28S to 18S ribosomal RNA ratios ranging from 2.06 to 2.16. According to the PCR analysis of GADPH gene, the extracted total RNA was successfully reverse transcribed into cDNA. Therefore, we believe that the BPMAB-based MSPE could be applicable for the specific enrichment of RNA from complex biological systems.

High-efficient separation of deoxyribonucleic acid from pathogenic bacteria by hedgehog-inspired magnetic nanoparticles microextraction.

Efficient separation of deoxyribonucleic acid (DNA) through magnetic nanoparticles (MN) is a widely used biotechnology. Hedgehog-inspired MNs (HMN) possess a high-surface-area due to the distinct burr-like structure of hedgehog, but there is no report about the usage of HMN for DNA extraction. Herein, to improve the selection of MN and illustrate the performance of HMN for DNA separation, HMN and silica-coated Fe3O4 nanoparticles (Fe3O4@SiO2) were fabricated and compared for the high-efficient separation of pathogenic bacteria of DNA. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are typical Gram-negative and Gram-positive bacteria and are selected as model pathogenic bacteria. To enhance the extraction efficiency of two kinds of MNs, various parameters, including pretreatment, lysis, binding and elution conditions, have been optimized in detail. In most separation experiments, the DNA yield of HMN was higher than that of Fe3O4@SiO2. Therefore, a HMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated and used to detect pathogenic bacteria in real samples. Interestingly, the HMN-based MSPE combined qPCR strategy exhibited high sensitivity with a limit of detection of 2.0 × 101 CFU mL-1 for E. coli and 4.0 × 101 CFU mL-1 for S. aureus in orange juice, and 2.8 × 102 CFU mL-1 for E. coli and 1.1 × 102 CFU mL-1 for S. aureus in milk, respectively. The performance of the proposed strategy was significantly better than that of commercial kit. This work could prove that the novel HMN could be applicable for the efficient separation of DNA from complex biological samples.