Functionalized cryogel monoliths for fast and selective separation of nucleic acids directly from crude lysate.

The fast and selective separation of nucleic acids has been attractive recently because of their wide number of applications in the biomedical field such as the development of vaccines for infectious diseases, gene therapy, and diagnosis. Traditional approaches of nucleic acids separation are costlier, lengthy, and associated with possible denaturation because of the use of organic solvents in the elution step. Under this perspective, cryogels represent an attractive choice as a monolith stationary phase in column chromatography, which have proven efficient in recent chromatographic studies. Cryogels are the macroporous hydrogels with interconnecting properties between the pores. They allow the easy flow of large biomolecules with minimum mass transfer resistance. They are spongy in nature and possess good mechanical strength. Current article represents different developed functionalized cryogel monoliths for nucleic acids separation, their separation strategies, and challenges associated with further advancement in separation science.

Applications of magnetic materials separation in biological nanomedicine.

As a result of their advantages for superparamagnetic properties, good biocompatibility, and high binding capacity, functionalized magnetic materials became widely popular over the past couple of decades, being applied on large scale in various processes of sample preparation for biomedicine. In this work, we perform an in-depth review on the current progress in the field of magnetic bead separation, discussing in detail the physical basis of this process, various synthesis methods and surface modification strategies. We place special focus of attention as well on the latest applications of magnetic polymer microspheres in cell separation, protein purification, immobilized enzyme, nucleic acid separation, and extraction of bioactive compounds with low molecular weight. Existing problems are highlighted and possible trends of magnetic separation techniques for biomedicine in the future are proposed.

Application of monolithic chromatographic supports in virus research.

Key properties of monolithic chromatographic supports, make them suitable for separation and/or concentration of large biomolecules, especially virus particles and viral genomes. One by one, the studies that have been completed so far, contributed to the knowledge that monolith chromatography has hardly any limitation to be applied in virus research. Viruses of different sizes, possessing icosahedral structure and symmetrical morphology, as well as rod-shaped or filamentous viruses with helical structure, even enveloped ones, all of them could be successfully managed by means of monolith chromatography. Same is true for viral genomes, primarily when being distinct from other nucleic acid forms present in a host cell. This review is exclusively focused on viruses. It describes the application of monolith chromatography to different problematics within the virus research field. The reviewed achievements offer new possibilities and trigger new aspects in virology.

Mediator Monomer Regulated Emulsion Interfacial Polymerization to Synthesize Nanofractal Magnetic Particles for Nucleic Acid Separation.

Polymer-based magnetic particles have been widely used for the separation of biological samples including nucleic acids, proteins, virus, and cells. Existing magnetic particles are almost prepared by coating polymers on magnetic nanoparticles (NPs). However, this strategy usually encounters the problem of poor magnetic NPs loading capacity. Here, a series of nanofractal magnetic particles (nanoFMPs) synthesized by a strategy of mediator monomer regulated emulsion interfacial polymerization is presented, which allows effective magnetic NPs loading and show efficient nucleic acid separation performance. The mediator monomers facilitate the dispersion of magnetic NPs in internal phase to achieve higher loading, and the hydrophilic monomers use electrostatic interactions to form surface nanofractal structures with functional groups. Compared with magnetic particles without nanofractal structure, nanoFMPs exhibit a higher nucleic acid extraction capability. This strategy offers an effective and versatile way for the synthesis of nanoFMPs toward efficient separation in various fields from clinical diagnosis to food safety and environmental monitoring.

A method for high-concentration agarose gel preparation and its application in high-resolution separation of low-molecular-weight nucleic acids and proteins.

Separation of nucleic acids and proteins using gels has always been a crucial part of molecular biology research. For low-molecular-weight nucleic acids and proteins, low- and medium-concentration agarose gels cannot achieve the high resolution as polyacrylamide gels. We found that 6 %-14 % high-concentration agarose gels (HAGs) could be easily dissolved in an autoclave and the vertical gel cast can be effortlessly filled using an easy-made plastic box. Coupled with the improved buffer condition, HAG electrophoresis resulted in a good resolution of DNA and protein bands. With conventional TBE buffer plus 0.2 % NaCl, DNA fragments that differ by 2-5-bp within the 50-200-bp size range can be resolved on 6 %-8 % HAGs. By using TBE without NaCl, DNA fragments that differ by 2-bp or 2-nt within the 10-100-bp size range can be well resolved on >8 % HAGs. Using a buffer system comprising 1 M Tris-Cl for gel preparation, 0.2 M Tris-Cl/0.2 % SDS as upper tank buffer, and 0.2 M Tris-Cl as the lower tank buffer, HAGs achieved good molecular weight separation of total bacterial and plant proteins in the 10-200 kDa range. In conclusion, we developed a method for HAG preparation and electrophoresis of low-molecular-weight nucleic acids and proteins.

Selective precipitation of RNA with linear polyacrylamide.

Selective precipitation of RNA is often used in molecular biology as one of the methods for separation of nucleic acids to obtain samples enriched with DNA or RNA molecules alone or for purification of RNA samples. In the present study a simple and fast approach for selective precipitation of RNA with linear polyacrylamide is proposed for the first time. The method is based on the different predispositions of the DNA and RNA molecules to bind with the polyacrylamide. In this process, the linear polyacrylamide is used as the flocculant, collecting RNA particles to form aggregate, which then precipitated at low alcohol concentration. During and after precipitation the temperature is adjusted to maintain high solubility of DNA and other contaminates at given pH, salt and alcohol concentrations on the one hand, and globular state of polyacrylamide, preventing solubility of the RNA-LPA aggregate, on the other hand. The precipitated RNA can be used directly for RT-qPCR assay. The principal advantage of the present approach is the fast and quantitative precipitation of most RNA species from very dilute solutions. This makes it possible to obtain both almost DNA-free RNA and RNA-free DNA samples in one process.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2007397 .

DNA-Iron Oxide Nanoparticles Conjugates: Functional Magnetic Nanoplatforms in Biomedical Applications.

The use of magnetic nanoparticles (MNPs), such as iron oxide nanoparticles (IONPs), in biomedicine is considered to be a valuable alternative to the more traditional materials due to their chemical stability, cost-effectiveness, surface functionalization, and the possibility to selectively attach and transport targeted species to the desired location under a magnetic field. One of the many main applications of MNPs is DNA separation, which enables genetic material manipulation; consequently, MNPs are used in numerous biotechnological methods, such as gene transfection and molecular recognition systems. In addition, the interaction between the surfaces of MNPs and DNA molecules and the magnetic nature of the resulting composite have facilitated the development of safe and effective gene delivery vectors to treat significant diseases, such as cancer and neurological disorders. Furthermore, the special recognition properties of nucleic acids based on the binding capacity of DNA and the magnetic behavior of the nanoparticles allowing magnetic separation and concentration of analytes have led to the development of biosensors and diagnostic assays; however, both of these applications face important challenges in terms of the improvement of selective nanocarriers and biosensing capacity. In this review, we discuss some aspects of the properties and surface functionalization of MNPs, the interactions between DNA and IONPs, the preparation of DNA nanoplatforms and their biotechnological applications, such as the magnetic separation of DNA, magnetofection, preparation of DNA vaccines, and molecular recognition tools.