General

Techniques for separation and purification are essential in biomedical research, disease diagnostics and drug development. In recent years substantial progress has been made in developing separation methods through magnetic particles. Using these magnetic particles offer unique possibilities for the simple, effective and rapid concentration of purified specific proteins, cells, and other bio-molecules without exposing them to harmful chemical and physical treatment.

Magnetic separation technology offers a distinct advantage by subjecting the analytic to very little mechanical stress (compared to the centrifuge or chromatography membrane). In general MS methods are non-laborious, inexpensive and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization.

Magnetic beads are now widely used for the isolation and detection of bio-molecules in life sciences, microbiology, immunology, molecular biology, drug discovery, research routine laboratories and in diagnostics.

3 Generations in Magnetic Separation

1. First Generation:

A magnet is activated outside the well plate while liquids are pumped out of the well plate. Coated particles stay in the well plate. In general, all first generation separator contact vessels contain magnetic particles suspensions- attracting them to the inner surface of the vessel.

Disadvantage:

• The separation is inefficient since the magnets are not in direct contact with particles due to the thickness of the plate (the vessel side thickness).
• There is an inefficient contact between the magnet and vessel.
• Three steps are needed to collect the particles out of the well:

1. magnetic separation
2. liquid separation
3. re-suspension with fresh liquid, and pumping out the particle suspension

• During the “liquid separation step” a portion of the magnetic particles is pumped out together with the liquid.

2. Second Generation:

Magnetic Rods protected by plastic sleeves or tips: These rods or pins move into the liquid suspension that contains the particles and are then pulled out from a well plate; they can then be transferred to another plate.

Disadvantage:

• All 24 / 96 magnets are moving together in and out from Well Plate, thereby allowing no flexibility in processing.

• All current tips that cover the magnet during the separation are very thick and do not efficiently collect the particles.

3. Third Generation BM CMS:

Combinatorial independent operation of each Magnet in a well:

Bio-Magnetics’ technology allows the user to select and move any combination (or single) of desired magnetic pins into the wells. The user can then pick up and transfer any selected group (or individual) samples chosen from the matrix. Processing then continues with only those selected samples.

Additionally, BM has a patented Tip that has an ultra thin membrane to protect the magnets from adhering to the magnetic particles. Because of its thinness the magnetic field is exceptionally strong. Finally, the distance between the magnet (within the Microtip) and the sample can be controlled to allow a minimum gap between the two-allowing a further magnetic attraction.

The elegance of Bio Magnetics’ solution to magnetic separation is that the robotics can function within a system of exchangeable heads and stations that allow the processing through magnetic pins (encapsulated in special patented Bio-Magnetics’ Microtip). The resulting flexibility makes it possible to meet individual requirements for standard as well as special applications. This state of the art new technology and processing platform is based upon modular stations of separation, Microtip loading, patented FF technology and special plate movement. Elements can be sold as parts to robotic machines already in the workplace or complete customized robotic units that can be sold in new machines-together with user-friendly software.

Why use BM's CMS  Combinatorial  Magnetic Separation?

Classical methods of DNA and RNA isolation are based on column or precipitation methods. These techniques require centrifugation or vacuum steps and often have lengthy processing times as well as volume limitations (complicating their integration into automated high throughput processes). Additionally, the automated processing of these methods may cause cross contamination of the purified nucleic acids making this method useless for many diagnostic purposes. With BM's technology, and its unique ultra thin patented Microtip, sample preparation using DNA blood isolation magnetic reagents are carried out quickly. No centrifugation steps are employed. Quick and pure separation is accomplished with a highly magnetized field that the magnetic pin creates, together with BM’s patented 30 micron Tip membrane. This, along with the new generation of magnetic particles, allows isolation from both very small (e.g. 1 µl) and very large (e.g. 20 ml) sample volumes. Our technology can achieve extraordinarily high retention of particles while older technologies are less efficient. Furthermore, the low unspecific binding properties of the particles matrix ensure the isolation of highly pure nucleic acids from crude samples. The isolated products can be used directly in a variety of downstream applications. While screening the existing technologies, the following obstacles of automated nucleic acid separation have been identified and solved by BM technology or magnetic bead separation

• The working volumes are now between 1 µl and 2500µl.
• The costs of disposable tips decrease the costs of a separation.
• The disposable tip eliminate the cross-contamination of the separation.The method is not limited to the special form of reaction vessels.
• The gap between the tip (with the magnet inside) can be minimized and held constant, special washing methods unique to CMS.

Another problem that was solved with magnetic bead-based separation technologies was the previous insufficient, and/ or ineffective, re-suspension of the magnetic bead pellets to enable sufficient washing and elution steps (which results in higher yields and purity of the separated product compared to other purification methods).

4. The Flip-Flop process

The Flip-Flop (FF) is defined as a stream of magnetic particles in a static liquid suspension moving from one magnet to another:

When one magnet is close to the suspension particles the magnetic particles move towards this, creating an aggregate (button) near the magnetic pole. The magnet is now removed and a second magnet, directly opposite it, is activated; as a result, particles detach from the aggregate (button) and move towards the magnetic pole. The magnetic field creates a stream (flow) of particles moving to the second magnet- while the first aggregate (button) becomes smaller and smaller until all the magnetic beads are moved. This process can be activated as many times as necessary within a given time period. The user can regulate the system and insure that the gap between the Microtip (magnet inside) and the beads is at an absolute minimum.

During this FF process the liquid remains static and, as a result, the FF process becomes a very efficient method of washing of the particles with a maximum of the particle’s surface in contact with the liquid. Furthermore, if the particles have bound DNA, there is less risk for the DNA to become damaged.

If the liquid contains a chemical reagent that binds to the particles, then the binding reaction may be accelerated during an FF process due to a faster interaction of the reagent and particles. Therefore, if the reaction is a detection step, it may be accelerated by the FF. 

Important note
BM's FF is a stream of magnetic particles moving up and down a tube or a well via a tip protected magnetic rod, with another magnet(s) beneath the well(s). These upper & lower magnets move simultaneously up and down creating fast streams of particles.  Please note the attached short movie, in slow motion, demonstrating the movement of the magnetic particles between the two magnets.

Applications:

The applications for BM’s technology are divided into three time frames: 

present, near future and long term applications as detailed down below.

BM’s CMS technology fits perfectly in major present applications.

Present application

• DNA/RNA

• Cell separation

• Diagnostics

There are a few dozens existing magnetic kits for all three above parameters, but especially for DNA /RNA purification and detection, cell separation of white blood cells and bacteria. The magnetic particles (MP) are added to samples such as whole blood or tissue digest. MP’s are supplied from various manufacturers (Agencourt, Dynal, Merck and others). Many of the major diagnostic manufacturers are currently developing, or have developed, products utilizing magnetic microspheres for viral diagnostics, enzyme immunoassay, protein and nucleic acid purification, cholesterol assay, genetic testing, bacterial diagnostics, blood cell diagnostics, and cancer diagnostics.  

Applications for magnetic bead separation technology

The major applications are three:

1. DNA/RNA separation and purification: