July 1, 2012 (Vol. 32, No. 13)

For better or worse, the technology that performs biological measurements often takes center stage when new discoveries are reported.

Although the measurement yields the interesting result, in many cases such a result could not have been produced without the careful sample preparation that took place beforehand. Recognizing this fact, a growing number of scientists are becoming interested in using biomagnetic separation products to perform sensitive measurements.

Biological sample preparation may not be the most glamorous aspect of research, but scientists shouldn’t overlook its importance, says Brett R. Richards, technical account manager at Thermo Scientific. “If you don’t have good sample preparation, the rest of the experiments will suffer.”

With the goal of developing tools for improved biological sample prep, scientists at Thermo Scientific have developed superparamagnetic beads that come prefunctionalized with antibodies and other capture probes, or present chemical functionalities for the customized attachment of antibodies, oligos, proteins, or nucleic acids.

The newest line of magnetic bead products from Thermo Scientific, known as Sera-Mag Magnetic SpeedBeads, run as small as 1 micron in diameter and have two layers of magnetite encapsulated with a polymer, carboxylate-modified shell.

This enables them to respond faster to a magnetic field and gives them a bumpy, cauliflower-like outer surface that provides a greater surface area for attachment of capture probes.

Since they are superparamagnetic and highly charged, the beads have no residual magnetism outside of an applied magnetic field, resulting in less aggregation, which allows for more reproducible recovery and cleaner sample prep, Richards says.

Using magnetic beads for biological sample preparation has certain advantages over traditional techniques such as anionic exchange columns, including ease of scalability and the absence of filters and shear forces, which can cause sample loss and degradation, he adds.

An additional benefit of Thermo’s magnetic bead product line is that separations can be accomplished using a basic rare earth magnetic rack. Researchers interested in scaling up and automating the separations process can take advantage of magnetic particle processor instruments such as the Thermo Scientific KingFisher.

In the future, scientists at Thermo Scientific hope to develop even smaller beads that settle out of solution less quickly and will open the way to new applications in microfluidics and point-of-care technologies, Richards says. They are also working on developing tools for performing cell separations, in conjunction with partner companies.

The bumpy, cauliflower-like outer surface of Thermo Scientific’s Sera-Mag beads gives them a higher surface area-to-volume ratio for greater attachment of capture probes.

Biological Sample Prep

Scientists at Sigma Life Science are also working on magnetic beads for biological sample preparation. The beads are also about 1 micron in diameter, which customers tend to favor over larger beads since they stay in suspension longer and have better surface area to volume ratios, says John Dapron, principal scientist at Sigma Life Science.

The beads are made of magnetite and encapsulated with a biocompatible coating, such as polystyrene or agarose. Beads are available preconjugated with capture probes, such as anti-FLAG™ or a quadridentate chelate that binds to poly-histidine-tagged proteins, or functionalized with standard bioconjugate coupling groups for custom applications.

Sigma Life Science’s biomagnetic separations products can be used in a single tube or scaled up to accommodate 96 samples in a plate format that is compatible with liquid-handling platforms.

Scientists at Sigma Life Science are actively working on the development of affinity-enrichment applications on even smaller beads. One of the big pushes for smaller beads, says George Lipscomb, market segment manager at Sigma Life Science, is for biological sample preparation for mass spectrometry analyses of low-abundance proteins.

Many biomarkers are present at such low levels that mass spectrometric analysis is not possible without enrichment, Dapron says.

Also, for diagnostic purposes, scientists need to look at multiple biomolecules in concert, Lipscomb adds. For this reason, smaller beads in conjunction with high-throughput platforms will make sample processing easier and could open the way for scientists to detect multiple low-abundance biomarkers in small volumes of biological samples.

Sigma Life Science’s MISSION ExpressMag Super 96-Well Magnetic Kit allows researchers to perform high-throughput assay optimization for applications such as lentiviral transduction of cell lines.

Kit Application

A new business field of PerkinElmer is the development and production of magnetic beads applied in kits for biological sample preparation. The micrometer-sized beads are made of polyvinyl alcohol and are mainly used in nucleic acid purification processes. Functionalized with chemical linkers, they enable the specific binding of DNA, RNA, and microRNA from a huge variety of different sample materials.

PerkinElmer magnetic beads and kits are optimized for use with different instruments including chemagic MSM I and chemagic Prepito. These instruments perform magnetic separation in their standalone version or can be integrated with standard liquid-handling systems, says Stan Klein, director of sales, molecular diagnostics at PerkinElmer.

To ensure successful integration, PerkinElmer sends company representatives to help get the instrument hooked up and running within the lab’s existing environment and ensure that it meets the customer’s individual needs.

The growing trend in the field is “to get away from a machine that does a general job to one you can put a sample in and walk away,” Klein says.

Standardization and automation are becoming increasingly important with the growth of biorepositories in hospitals and universities, Klein adds. More and more researchers access biorepositories to obtain DNA or RNA for large screening projects in order to identify disease-related mutations, making it one of the biggest growth industries with a strong need for nucleic acid extraction tools.

For this reason, scientists at repositories routinely process numerous DNA and RNA samples for long-term storage, says Alex Lopez, senior field applications scientist at PerkinElmer. With so many samples to manipulate, it becomes even more crucial to use high-throughput automated technologies that track samples through each step and ensure greater consistency and better results than manual sample handling, Klein adds.

The technology of the chemagic MSM I can be used with customizable kits that make it suitable for samples including blood, tissue, saliva, plant, and food, and can handle sample volumes ranging from 10 microliters to 10 milliliters.

PerkinElmer says that its chemagic Magnetic Separation Module I enables researchers to perform automated, high-throughput DNA or RNA isolation in a platform that can be combined with standard liquid-handling units.

Really Small Beads

At Miltenyi Biotec, scientists are also heeding the call for high-throughput biological sample preparation and automation, with a technology that enables both biomolecule purification and cell sorting.

At about 50 nm in diameter, Miltenyi Biotec’s beads are much smaller than most magnetic beads for biomagnetic separations applications. Beads are applied to the biological sample in suspension while the magnetic separation is performed by placing the columns in a magnet. Following a series of wash steps, researchers can elute the desired biomolecules or cells off the column by displacing the column from the magnetic field.

For cell-sorting applications, smaller beads have an advantage over large beads by exerting faster binding kinetics and not precipitating out of solution. Moreover, they carry a smaller magnetic moment on the cell, allowing an almost quantitative magnetic labeling according to the number of surface receptors similar to that of a single antibody.

They show less cross-linking on the cell surface, and sorted cells are directly compatible with flow cytometry without the need of bead detachment, says Andreas Bosio, head of R&D, stem cells, neuroscience and molecular biology at Miltenyi Biotec.

The beads are nontoxic and degradable, composed of an iron oxide core with a dextran coating that enables functionalization with antibodies for the capture of specific cell types. Customers can run columns manually or purchase an instrument such as the MultiMACS or the AutoMACS for higher throughput and automation.

One interesting feature of Miltenyi Biotec’s instruments is the ability to connect directly to a flow cytometer for highly sensitive analysis of enriched cells, a combination that has been integrated in the MACSQuant. Also, the company offers a comprehensive portfolio of cell-separation products that enable the sorting of all types of cells, including blood cells and tissue-derived cells.

Tissue-derived cells, such as cancer cells in a tissue biopsy, present a unique set of separation challenges due to the cells being associated with the tissue, Bosio says. The firm’s latest instrument for tissue dissociation, known as the gentleMACS Octo Dissociator, performs both mechanical and enzymatic dissociation simultaneously and allows the user to select from a series of programs or to develop customized protocols for unique applications.

Of particular interest to researchers in clinical settings is a fully integrated instrument known as the CliniMACS Prodigy, which was introduced this year. The CliniMACS Prodigy carries samples through from separation to cell culture, containing all the necessary features for many applications in clinical cell therapy, which has been a hot research area recently, Bosio notes.

Although flow-assisted cell sorting has been the go-to technology for cell sorting for several decades, magnetic cell sorting (MACS) has begun making strides in the field in recent years. It’s just a matter of time, Bosio says, before MACS becomes even more well-known, due to its simplicity, reproducibility, and potential for use with more complex cell samples.

According to Miltenyi Biotec, its latest instrument, the CliniMACS Prodigy, provides researchers with a fully integrated, customizable cell-processing workflow for separating almost any cell type.

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