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Superbugs, or bacteria that are resistant to currently available antibiotic treatments, are of growing concern to human health worldwide. Many of these superbugs are present in hospitals, and are  frequently colonizing surgical sites and causing life-threatening infections and sepsis. The presence of these bacteria in blood is commonly detected by traditional culture methods that require one to two days. There is a need for a faster method to identify the presence of the bacteria and to take measures to prevent the spread of infection  as rapidly as possible. One proposed technology is to use immunomagnetic separation with conjugated fluorescent probes to selectively bind bacteria and quickly visualize their presence in whole blood samples. Recently, fluorescent magnetic multifunctional carbon dots have been coupled with superbug-specific antibodies for the identification of the drug-resistant superbugs Staphylococcus aureus (MRSA) and Salmonella enterica serotype typhimurium definitive phage type 104 (DT 104) present in whole blood samples.

Magnetic DNA or RNA purification relies on the superparamagnetic property of micro- or nano-scale particles. These particles are most often made of iron oxide, with magnetite (Fe3O4) more commonly used than maghemite (Fe2O3). Superparamagnetic particles are not innately magnetic, but they become magnetized when influenced by a magnetic field. So, if the magnetic field is zero, then the particles are not magnetic at all, but when a magnetic field is applied the particles become magnetized.

The traditional solid-phase support system is a static column. These columns are made of silica matrices or anion-exchange resins. They are porous and allow the solution to flow through them. These non-magnetic solid-phase columns require centrifugation to force the solution through. More recently, magnetic particles are being used as mobile solid-phase support systems for capture and purification of DNA and RNA. These magnetic particles are added to solution and are free to move around during the DNA or RNA adsorption period. They are then retrieved by magnetic separation. No centrifugation is needed for magnetic-particle nucleic acid purification.

Our understanding of genetic material has substantially increased since Friederich Miescher first extracted DNA in 1869. He discovered that a material exists within cells that precipitates out of acidic solution and dissolves into alkaline solution. He called it nuclein because it seemed to be located within the nucleus. It took until 1953 for the structure of DNA to be elucidated. It was during this time that procedures to isolate DNA began to emerge. Later, during the 1960's and 70's scientists were furiously untangling the cellular environment, and the discovery of RNA with its various forms and functions further refined DNA purification procedures. It was no longer enough to simply separate DNA from protein and salt impurities; it became necessary to remove contaminating RNA as well. Concurrently, scientists became interested in purifying messenger RNA (mRNA). Soon it became essential to purify not only DNA (genomic or plasmid), but also RNA in its various forms.

Nucleic acid separation can be fickle. DNA is fragile, and RNA even more so. Many commercial kits are designed to streamline the process, but they may not result in high yield or high purity DNA or RNA every time. Every laboratory is different; working habits vary, and experimental goals are not identical. It is tempting to rely on a single kit for routine isolation of genetic material because it is familiar, your lab may have published previous work with an established protocol, or you might not have the luxury or freedom to try something new. However, if you are experiencing consistently low RNA or DNA yield or purity then you may be able to justify taking time to gain a deeper understanding of the process, familiarizing yourself with the tools available, and possibly reworking your strategy.

Biomagnetic separation is a versatile and widely used tool in both industrial and small laboratory settings. It is used for the isolation of target drug molecules in the pharmaceutical realm, for the enrichment of enzymes in industry, and for in-vitro diagnostics in medicine. It is especially useful in the small-scale research environment for inexpensive target cell enrichment, protein isolation, or nucleic acid capture. In the early days of biomagnetic separation it was thought that the process was only reliable for small volumes. However, the development of modern biomagnetic separation racks has made it possible to scale up the process to large volumes and to enable process validation and consistency between batches.

The ability to obtain an enriched population of small molecules, cells, proteins, nucleic acids, or contaminant-free solutions is important for all applications: from small laboratory research up to the large-scale production of pharmaceutical products. Filtration systems are available in all shapes, sizes, and materials for diverse situations. The need to meet regulatory guidelines for purity and consistency of pharmaceutical productsdemands a well-designed enrichment plan and filtration system.

Part 2: Structure/Protection, Functionalization, and Application

Immunoprecipitation

Immunoprecipitation (ip) is a technique for capturing specific proteins via antibody-antigen affinity from a complex solution. A co-ip, instead of identifying individual proteins, is designed to identify protein complexes. The phrase “pulling down” protein is commonly used to explain the process, but this idea is somewhat dated now that magnetic nanoparticles have begun to replace traditional centrifuge-based methods. The protein capture efficiency can be measured by ip input ito SDS page and western blot analysis.