Chemical apparatus and process disinfecting – deodorizing – preser – Control element responsive to a sensed operating condition
Reexamination Certificate
1995-07-06
2001-02-13
Le, Long V. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Control element responsive to a sensed operating condition
C422S063000, C422S105000, C436S043000, C436S054000, C436S174000, C436S177000, C436S180000, C436S807000, C210S222000, C210S695000
Reexamination Certificate
active
06187270
ABSTRACT:
The present invention relates to a device and a method for the separation of magnetic microparticles from a suspension of magnetic microparticles in a liquid by means of a magnetic field. The device comprises a pipette, a pump connected to said pipette, a magnet, and a device for moving the pipette toward the magnet and away from it.
The separation of magnetic microparticles is used in particular in immunoassays and in DNA testing. Immunological reactions on the surface of coated, magnetic particles allow a simple separation of bound from free reaction partners as the magnetic particles are retained by the magnetic forces while the remaining reaction solution can be removed.
In prior art, the separation of coated magnetic particles has already been used to carry out immunoassays. European patent application EP-A-0 339 980 describes a device (
FIGS. 10 and 11
) used to separate a suspension of magnetic microparticles by means of a magnetic field. The particles are then separated on a special wire located in the inside of the reaction vessel. In order to release the microparticles from the wire, the latter is exposed to vibrations. A drawback of the device is that it requires the presence of high magnetic fields and the action of an agitator. The instrument described is also difficult to integrate in existing instrument concepts as neither strong magnets, nor shaking devices, nor agitators, nor special wires are present in conventional analyzers.
It was, hence, an object of the invention to provide a device and a method for separating and washing magnetic microparticles which could be easily integrated in existing instrument concepts and require only a small number of additional devices.
This object was accomplished by providing a device for the separation of magnetic microparticles from a suspension of magnetic microparticles in a liquid by means of a magnetic field. The device comprises a pipette, a pump connected to said pipette, a magnet, and a device for moving the pipette toward and away from said magnet, or a device for moving the magnet toward and away from said pipette while the magnetic microparticles are deposited at the inner wall of the pipette.
Numerous immunological assays have been simplified with the introduction of coated magnetic microparticles. In these processes, the magnetic particles assume the function of a solid phase. The use of magnetic microparticles is advantageous when the test to be carried out requires the separation of bound from non-bound reaction partners. Since immunological tests with magnetic microparticles are known in prior art (e.g. U.S. Pat. No. 4,219,335), the chemical and/or immunological aspect thereof is not discussed in further detail at this point.
Magnetic microparticles are understood to be microparticles that are attracted by means of a magnetic field. The magnetic particles can, hence, themselves be magnetized. Preferred materials are those that exhibit only a minor remanence. The material of the microparticles can be a compound material, e.g. a matrix that contains magnetically attractable particles. Magnetically attractable materials are, for example, iron, iron oxide, nickel, cobalt, or chromium oxide. One or several particles of this material can be embedded in a matrix. The matrix can consist of a multitude of materials, e.g. organic or inorganic polymers.
The present invention makes use of coated microparticles. The coating can be a polymer, for example, that carries reactive groups on its surface (U.S. Pat. No. 4,454,234) or is coated with a biologically active component (U.S. Pat. No. 4,177,253, U.S. Pat. No. 3,933,997). Both the manufacture of magnetic microparticles and their coating are known from prior art (U.S. Pat. No. 4,297,337, DE-A-3014036). Microparticles coated with biologically active groups are also commercially available, for example, by Dynal Tech and Rhône-Poulenc. The microparticles marketed by these companies have the advantage of having a relatively low density allowing the use of relatively large microparticles. An increasing density entails an increase in the sedimentation rate of the microparticles which then requires a reduction of the diameter of the microparticles in order to avoid an undesired separation of the suspension through gravity. The beads manufactured by Dynal have a diameter of 2.8 &mgr;m with a density of approximately 1.3. The microparticles manufactured by Rhône-Poulenc have a diameter of 0.5 to 2 &mgr;m with a density of also 1.3. If the beads manufactured by these firms are suspended in aqueous solutions, it takes hours to accomplish a separation through gravity.
These beads can therefore be used in analysis procedures without requiring additional equipment for shaking or generating ultrasound in order to keep the suspension stable.
An apparatus in accordance with the invention comprises a pipette to hold a suspension of magnetic microparticles. Pipettes usually have a cylindrical shape with an opening at each of their front sides. In connection with an apparatus of the invention, it is also possible to use pipettes which have areas with rectangular cross sections. It is essential to the invention that the interior of the pipette can be penetrated by a magnetic field, provided a magnet is set up at the outer wall of the pipette. The pipette material must, hence, be permeable to magnetic field lines with glass and plastic being usually employed as materials. In a preferred manner, the pipette has a cylindrical base onto which a plastic tip is placed. The liquid held in the pipette is located inside the tip where the separation of the magnetic microparticles is accomplished. The use of tips has the advantage that they can be discarded after use. The removal of possibly remaining beads is therefore not required. If possible, the interior of the pipette should not have any recesses or edges as this may interfere with a quantitative release of magnetic microparticles separated in the pipette. In a preferred manner, the cylinders for pipettes have relatively small diameters, approximately in the range of a few millimeters up to 2 cm. Pipettes that have a uniform inner diameter over their longitudinal extension are suitable; it is, however, advantageous to have pipettes with tapered ends.
The inner wall of a pipette should be smooth. It is also possible to use pipettes whose inner walls are provided with a non-adherent coating, for example, a silane coating.
In order to fill the pipettes with suspensions and solutions, one of their openings is connected to a pump. Both the elements to implement the coupling of the pipette to the pump as well as the pump itself are known in prior art on automated analyzers.
Magnets for separating the magnetic microparticles can be electrical magnets as well as permanent magnets. Permanent magnets are preferred as these do not generate heat during operation which could interfere with the analysis. Strong permanent magnets exhibit a magnetic flow that is sufficient to separate magnetic microparticles from a suspension with a thickness of up to a few centimeters. The magnets used preferably have the form of a bar magnet. It is, however, also possible to use magnets with various other shapes. The shape is usually selected such that only one magnetic pole is located in the vicinity of the pipette.
The surface of the magnet located in the vicinity of the pipette should have a cross section that is essentially not smaller than the diameter of the pipette. This magnet surface can be planar; a concave curvature of the surface is, however, preferred so that the magnet matches the surface area of the pipette. In a preferred manner, this concave surface is so small that it covers less than one half of the surface area of the pipette so that a highly inhomogeneous magnetic field is generated in the inside of the pipette.
The magnet can be fixed in its position inside the apparatus or attached to a device for moving it spatially. A fixed positioning of the magnet is preferred in accordance with the invention to simplify the structural design.
Further, a device in ac
Maurer Eberhard
Pappert Gunter
Schmitt Urban
Arent Fox Kintner & Plotkin & Kahn, PLLC
Le Long V.
Roche Diagnostics GmbH
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