Preparation Of Multifunctional Magnetic Nanoparticles And Application To Enrichment Of Cells And Extraction Of Plasmid DNA

Because plasmid DNA (pDNA) is routinely used as an important genetic engineering vector, the development of a rapid, simple, cost-effective and environmental-friendly bacteria enrichment and pDNA extraction method amenable for automation is of considerable advantage. Conventional pDNA extraction techniques requiring centrifugation, precipitation are not easily adapted to automated systems. In contrast, magnetic nanoparticle extraction methods demonstrate remarkable simplicity owing to the nature of the particles, which can fish bioentities directly out of biological matrices, and received considerable attention since 1990'. However, the present magnetic adsorbents and related protocols are limited in terms of binding capacity. Although considered as an important cell enrichment method, the immunomagnetic separation of cells are limited in terms of sample preparation, cost and shelf life, and especially not suitable for large-scale cell separation purpose. Nevertheless, there are few successful cell enrichment methods based on nonspecific adsorption between cells and magnetic particles. Second, most magnetic solid phase procedures involve two centrifugation steps:the first to harvest cells from liquid culture and the second to pellet denatured genomic DNA/protein complexes after cell disruption and neutralization. These centrifugation procedures are both time-and labor-intensive; moreover, this step was not amenable to the miniaturization and automation required of high-throughput biological sample preparation. Furthermore, the potential for shearing damage to biomacromolecules during extensive centrifugation is unavoidable.In view of above-mentioned reasons, the present study prepared multifunctional carboxylated magnetic nanoparticles (CMNPs) with high binding capacities. With the help of the nanoparticles, a facile method was constructed for plasmid DNA extraction by integration of bacteria capture, lysate clearance and DNA purification. A new idea for enrichment of microorganisms and nucleic acid based on co-agglomeration with magnetic nanoparticles was also proposed. Even without digestion of RNA primarily by RNase, plasmid can be separated selectively from DNA/RNA mixture by carful tune of temperature.1. Iron oxide nanocrystals were prepared by coprecipitating di and trivalent Fe ions in alkaline solution. The as-prepared iron oxide was then coated by ultrasound assisted in situ polymerization method by using Bis[2-(methacryloyloxy) ethyl] phosphate as coupling agent, methacrylic acid as functional monomer and potassium persulfate as initiator. The results indicated iron oxide could act as polymerization center. When the ultrasound power increased from 400w to 600w and then 800W, the related hydrodynamic size was 96.87,55.97 and 49.61 nm, respectively. The spinel crystalline with size 7 nm was indicated by transmission electron microscopy (TEM) and X-ray diffraction (XRD) characterization. The fourier transform infrared spectroscopy (FTIR) study and thermogravimetric analyses (TGA-DTG) confirmed the successful functionalization of carboxyl groups on the surface of magnetic nanocrystals with organic content 7-12%. The saturation magnetization value for coated particles obtained by vibrating sample magnetometer (VSM) measuring was 47 emu/g. The coated particles were sensitive to environmental pH. When flocculated on addition of acid, the particles are easily separated within 30 s, while it generally needs 5 min to reach a recovery of nearly 100%under neutral condition.2. Using "cell capture efficiency" and the "firmness of immobilized cell/CMNPs on the wall" after magnetic separation as index, we found acid, calcium salt and zinc salt were effective CMNPs-E.coli cell coupling agents, of which HCI and CaCI2 were the best choice. The performance of HCI and CaCl2 was studied sufficiently to optimize best conditions. The results indicated that capture of cells with CMNPs increased sharply when pH was below 5.0, then achieving steady value of upward 90%capture. The cells capture efficiency increase with elevating of cell density, more addition of CMNPs amount and elongated separation time. Under optimized condition (0.85 mg of CMNPs, pH 3.5-1.5), typically, more than 90%of E.coli cells could be recovered within 3 min from 1.5 ml of overnight culture with OD6oo higher than 0.2. When CaCl2 was used as binding agent, the cell capture efficiency was controlled by ratio of [ca2+] vs. OD600, and also affected by CMNPs amount and separation time, which fit Logistic model equation perfectly. In this event, Ca2+ might act as salt bridge between CMNPs and E.coli cells.3. A rapid protocol for extraction and purification of high quality plasmid DNA from bacteria with CMNPs was reported using solid phase reversible immobilization method (SPRI). In this case, a more expressive DNA adsorption was achieved at a PEG800>0.15 g/mL and an NaCl concentration>0.5 mol/L. The recoveries of plasmid DNA increase with more addition of CMNPs, while decrease when used too much. The adsorption and elution of plasmid DNA was rapid, with equilibrium time of 150 and 40 s, respectively. Below 70℃, plasmid DNA adsorption was not affected while RNA recoveries decrease dramatically with increase of temperature. Hence, an RNase-free method will work for purification of pDNA by carful modulation of temperature.4. By using pH sensitive multifunctional magnetic nanoparticles, a facile, centrifugation-free and small-scale plasmid DNA extraction method was established by integration of bacteria capture, lysate clearance and DNA purification. This method was simple without involvement of expensive apparatus and hazard agents; the recovered pDNA was mainly composed of supercoli form with A260/A280>1.75 generally. The biological quality of pDNA was validated by restriction enzyme digestion. The quality and quantity of pDNA were comparable with that extracted by QIAGEN Kit, yet, the cost associated with the current method was only 1/5 of the commercial one. The entire procedure took less than 20 min, whereas the other method took at least 30 min.5. To generate adsorption or elution conditions, the present magnetic-particle-based purification method usually employ high concentration chaotropic salts which are damage to nucleic acid, or vicious agents which make particles difficult to disperse or recover. Besides, these agents are expensive and not environmental-friendly, and large volume consumption of these agents inevitably increased the handle volume. For samples with low salt concentration (e.g. PCR product), the present methods suffer one or more of above mentioned problems. In view of these considerations, a new method was constructed for DNA/RNA enrichment by using high biocompatible spermine or spermidine as binding buffer. The results showed neither of the two compaction agent could generate a selectivity for pDNA or RNA. Only when spermidine concentration was 1-2 mmol/L can pDNA adsorb to CMNPs'surface, and that for RNA was 0.5-5.5 mmol/L. When spermine is 1-7 mmol/L,100%of pDNA can be recovered, while RNA was around 90%when spermine was 1.5-5 mmol/L, leaving 10%of small RNA in supernatant. DNA and RNA could completely elute when compaction agents were scripted. The saturation binding capacity was 2μg DNA/μg particle when spermine was used as binding agent, which was higher than other methods ever reported.6. Low pH could also produce driving fore for the binding of nucleic acid to the surface of CMNPs. In solutions with pH higher than 8.0, the adsorption was nearly zero, while in acidic solutions,100%DNA and RNA could be fished out of solution with slight degradation of DNA.