Abstract:
An apparatus for washing magnetic particles including a well plate for receiving an array tube holder having x rows of tubes, each tube containing magnetic particles, the well plate having (x+1) rows of wells for enabling the array tube holder to be shifted from a first position to a second position. The apparatus also includes ((x/2)+1) rows of magnets asymmetrically positioned within the well plate adjacent to the rows of wells. Placing the array tube holder in the first position causes magnetic fields produced by each magnet to attract the magnetic particles in the x rows of tubes to one side of each tube, and shifting the array tube holder to the second position reverses the magnetic fields, causing the magnetic particles to stream across to the other side of each tube. The array tube holder may be a 96 tube array tube holder (8 rows by 12 columns) in which case, the well plate includes 108 wells (9 rows by 12 columns) for simultaneously washing 96 tubes.

Description:
The application is a divisional of Application Ser. No. 09/377,700, filed Aug. 20, 1999, now abandoned, which claims priority to U.S. Provisional Patent Application No. 60/097,487, filed Aug. 21, 1998. 
    
    
     BACKGROUND AND SUMMARY OF THE INVENTION 
     The present invention relates generally to life sciences applications, and more particularly to an apparatus for efficient washing of magnetic particles in molecular biology/immunology applications. One such application is cDNA molecule isolation. 
     One approach for cDNA clone isolation requires the use of streptavidin-coated magnetic particles (approximately 1 μm in size) to capture biotinylated-oligo cDNA hybrids, which are subsequently made double stranded and transformed into a cell such as  E. coli . A time consuming portion of this approach is the cDNA Capture procedure. During the cDNA Capture procedure, extensive washing of the streptavidin-coated magnetic particles are performed after the capture of the cDNA molecules. See generally The GeneTrapper™ cDNA Positive Selection System Instruction Manual, Life Technologies Inc., which is incorporated herein by reference in its entirety. 
     Conventionally, the cDNA Capture procedure uses six 1.7 ml tubes and a six-position magnet to attract the magnetic particles to one side of each of the 1.7 ml tubes. During the cDNA Capture procedure, the nucleic acid containing mixture (comprising a cDNA library hybridized to biotinylated-oligonucleotides) is mixed with the streptavidin-coated paramagnetic beads by gently pipetting. The suspension is incubated for 30 minutes at room temperature. During this process, the suspension is gently mixed every two to three minutes by finger tapping or vortexing at the lowest speed for ten seconds to re-suspend the beads. The tubes are then inserted into the magnet for two minutes and the supernatant is removed and discarded. Finally, the beads are extensively washed. 
     The extensive washing procedure consists of adding 100 μl of wash buffer to the beads, re-suspending the beads by finger tapping or gently vortexing at the lowest speed, and re-inserting the tubes into the magnet for two minutes. The supernatant is removed and discarded, and this process is repeated. 
     An additional 100 μl of wash buffer is added to the beads. The beads are resuspended by pipetting. The solution is then transferred to clean tubes and the clean tubes are inserted into the magnet for five minutes. The supernatant is removed and discarded and 100 μl of wash buffer is immediately added to each tube. Each tube is then finger tapped or vortexed and the tubes are again inserted into the magnet for five minutes. After the five minute incubation, the supernatant is removed and discarded, and 20 μl of 1× elution buffer is added to the beads and mixed by pipetting. The beads are incubated for five minutes at room temperature. During the incubation, the beads are finger tapped for ten seconds every minute. The tubes are then inserted into the magnet for an additional five minutes. The supernatant (which now contains the captured cDNA molecules) is transferred and saved in fresh tubes. 
     Throughout the above defined processes, the tubes are inserted into the magnet and, after a 2-5 minute wait period, the supernatant is removed from the tube and a solution (TE buffer, wash buffer, or elution buffer) is added to the tube by pipetting. Each 1.7 ml tube is then handled separately in order to re-suspend the paramagnetic beads after they have been pulled to one side of the tube by the magnet. This process requires picking up each tube individually and agitating the tube by finger tapping or gently vortexing the tube at the lowest speed to avoid splashing beads to the sides of the tube. The tube is then re-inserted into the magnet for an additional 2-5 minutes and the supernatant is then removed. 
     This process, which is repeated a number of times throughout the procedure for cDNA molecule isolation, is time consuming and can take up to approximately 45 minutes for each tube. Also, the process of finger tapping permits inconsistencies and variability between tubes. 
     An efficient means for simultaneously washing magnetic particles in a plurality of tubes, without finger tapping or vortexing, that renders consistent test results is needed. 
     The present invention satisfies the above mentioned needs by providing an apparatus that can efficiently wash multiple tubes, preferably 96 tubes, simultaneously within 5 minutes without having to individually finger tap or vortex the tubes. As will be evident, the present invention also provides high throughput operation as well as automation for any application which utilizes magnetic particles, particularly magnetic or paramagnetic beads. 
     The present invention is an apparatus for washing magnetic or paramagnetic particles, such as might be useful in life sciences applications, including molecular biology/immunology applications. The present invention includes a well plate which preferably has a substantially flat upper surface, a substantially flat lower surface, and a plurality of wells uniformly placed in rows and columns. Each of the wells has an opening in the upper surface of the well plate that preferably tapers conically to a lower end. The upper surface of the well plate preferably has a recessed area extending around the perimeter and through the first and last rows of the plurality of wells for receiving an array tube holder, preferably a 96 array tube holder. According to a preferred embodiment, the array tube holder can be placed in one of two positions, i.e. a first position and a second position. The array tube holder holds tubes, preferably 0.2 ml tubes, containing a solution comprising magnetic or paramagnetic particles. The lower surface of the well plate preferably includes an asymmetrical arrangement of a plurality of slots positioned in rows and columns. The first row of slots is preferably placed between an edge of the well plate and the first row of wells. Subsequent rows of slots are placed between the remaining rows of wells. 
     The present invention further comprises a plurality of magnets inserted into each slot in the lower surface of the well plate. Each magnet is positioned adjacent to the lower end of a well for attracting the magnetic particles in the tube toward one side of the tube. In each adjacent row of tubes, the magnetic particles are attracted to the opposite side of the tubes. A base plate is preferably attached to the lower surface of the well plate for securing the magnets. 
     The present invention may be used to wash or move the magnetic particles by attracting the magnetic particles to one side of the tube by placing the array tube holder in either the first position or the second position and pipetting a washing solution into each tube. The array tube holder is then shifted to the other of the two positions to reverse the magnetic field. Alternatively, the magnetic field can be reversed by changing the position of the tubes, such as by rotating the tubes, preferably by rotating the tubes 180°. The magnetic field can also be reversed by moving the magnets, such as by shifting the magnets between the rows of wells or by changing the position of the magnets, preferably by flipping the magnets, more preferably by flipping the magnets 180°. The reversal of the magnetic field causes the magnetic particles to stream across the tube in the washing solution, preferably in a widest part of the tube, in response to the magnetic attraction of the magnet on the other side of the tube, thereby efficiently washing the magnetic beads in a short time, preferably about five seconds. The supernatant is removed and discarded and the process of reversing the magnetic field and washing the beads is preferably repeated, more preferably, the process is repeated about three times. 
     Further features and advantages of the invention, as well as the structure and operation of the invention, are described in detail below with reference to the accompanying drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is an exploded view of a preferred embodiment of the present invention, indicating the insertion of the magnets into the slots on the lower surface and the receipt of a 96 array tube holder with tubes onto the upper surface. 
     FIG. 2 is a perspective view of the preferred embodiment of the present invention. 
     FIG. 3 is a top view of the upper surface of the preferred embodiment of the present invention. 
     FIG. 4 is a bottom view of the lower surface of the preferred embodiment of the present invention illustrating magnet slots  410 . 
     FIG. 5 is a top view of the upper surface including a projected-view of magnet slots  410  from the lower surface of the preferred embodiment of the present invention. 
     FIG. 6 is a cross-sectional view of the preferred embodiment of the present invention taken across line A—A of FIG.  5 . 
     FIG. 7 is a cross-sectional view of the preferred embodiment of the present invention taken across line B—B of FIG.  5 . 
     FIG. 8 is a perspective view of a 96 array tube holder holding tubes. 
     FIG. 9A is a perspective view of the preferred embodiment of the present invention receiving a 96 array tube holder with tubes in a first position for washing the magnetic particles. 
     FIG. 9B is a perspective view of the preferred embodiment of the present invention receiving a 96 array tube holder with tubes in a second position for washing the magnetic particles. 
     FIG. 10A is a cross-sectional view of the preferred embodiment of the present invention taken across line B—B of FIG. 5 with the insertion of a 96 array tube holder with tubes in a first position for washing the magnetic particles. 
     FIG. 10B is a cross-sectional view of the preferred embodiment of the present invention taken across line B—B of FIG. 5 with the insertion of a 96 array tube holder with tubes in a second position for washing the magnetic particles. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is an apparatus for efficiently washing magnetic particles. One feature of a preferred embodiment is the provision of 108 wells. The 108 wells provide the ability to accommodate up to 108 tubes, preferably 0.2-ml tubes. The 108 wells also preferably permit an array of tubes, preferably an array of 96 tubes, to be moved between a first position and a second position. Another feature of this preferred embodiment is the asymmetrical location of five sets of 12 cylindrical rare-earth magnets. This design permits the rapid attraction of magnetic particles to the sides of the tubes such that a manual or robotic pipette tip can be inserted without disrupting the magnetic particle pellets. The geometry of the 108 wells and the asymmetrical location of the rare-earth magnets provide for the reversal of the magnetic field when moving the array of 96 tubes from the first position to the second position or vice versa. As will become apparent to one skilled in the art; the above features allow for the simultaneous washing of the magnetic particles in all 96 tubes of the array. 
     Alternatively, the magnetic field can be reversed by changing the position of the individual tubes, such as by rotating the tubes, preferably by rotating the tubes 180°. The magnetic field can also be reversed by moving the magnets, such as by shifting the magnets between the rows of wells or by changing the position of the magnets, preferably by flipping the magnets, more preferably by flipping the magnets 180°. 
     Although the present invention is described herein as being used to capture biotinylated cDNAs for cDNA molecule isolation, it will become apparent to one skilled in the art that the present invention can be used in any life sciences application, including molecular biology/immunology applications in which magnetic particles are used. Such applications include for example clinical diagnostic testing, i.e. with antibodies to sort cells, molecular biology, immunology and/or enzymology. 
     FIG. 1 is an exploded view of a preferred embodiment of the present invention. The apparatus  100  is preferably comprised of a well plate  110 , a plurality of rare-earth magnets  120 , and a base plate  130 . The well plate  110  is provided to receive an array of 96 0.2-ml tubes  140  containing a solution of magnetic particles and to house the plurality of rare-earth magnets  120 . Alternatively, the well plate  110  is equipped to receive anywhere from one to 108 0.2-ml tubes  140 . The base plate  130  provides means for securing the plurality of rare-earth magnets  120  housed within the well plate  110 . The base plate  130  is preferably an adhesive material, but can be any non-ferrous material that secures the rare-earth magnets  120  housed within the well plate  110 . 
     FIG. 2 is a perspective view of well plate  110  according to a preferred embodiment. Well plate  110  is preferably comprised of a substantially flat upper surface  210 , a substantially flat lower surface  220  (not directly shown from this view), and  108  wells  230  arranged in a 9×12 array. Five sets of 12 cylindrical rare-earth magnets  120  (not shown) are inserted within well plate  110 . The well plate  110  is preferably composed of a white plastic material, such as acetal plastic. Alternatively, it would be apparent to those skilled in the art that other types of materials or plastic materials can be used for the well plate  110 . Although any magnetic material can be used for the cylindrical rare-earth magnets  120 , neodymium-iron-boron (Nd—Fe—B) is preferred. 
     In the preferred embodiment shown in FIG. 2, each of the 108 wells  230  has an opening in the upper surface  210  of the well plate that tapers conically toward a lower end to accommodate the array of 96 0.2-ml tubes  140  (not shown). Alternatively, well plate  110  could have wells of a variety of configurations depending on the type of test to be run in the apparatus  100 . One skilled in the art will recognize that other well shapes capable of accommodating the array of 96 0.2-ml tubes  140 , such as circular or rectangular wells, could also be used. The 108 wells  230  are preferably uniformly positioned in the upper surface  210 . 
     The substantially flat upper surface  210  of the well plate  110  according to the preferred embodiment is shown in FIG.  3 . The substantially flat upper surface  210  of the well plate  110  preferably uniformly positions the 108 wells  230  as an array of rows and columns (9 rows by 12 columns). The substantially flat upper surface  210  of the well plate  110  further comprises a recessed area  310  that extends around the perimeter of the well plate  110  and through the first row  320  and last row  330  of the 108 wells  230  for receipt of a 96 array tube holder (tray retainer set) manufactured by, for example, Perkin Elmer Corporation (Catalogue No.403081). Alternative embodiments may contain other forms of recessed areas for accommodating other models of array tube holders or no recessed area at all. 
     The substantially flat lower surface  220  of well plate  110  according to the preferred embodiment is shown in FIG.  4 . The substantially flat lower surface  220  is comprised of a plurality of cylindrical slots  410 . The plurality of cylindrical slots  410  are preferably positioned asymmetrically in an array format of rows and columns. Each of the cylindrical slots  410  is used to house one of the rare earth magnets  120  (not shown). 
     In an alternative embodiment (not shown), the substantially flat lower surface  220  of the well plate  110  is comprised of elongated slots, preferably five elongated slots, each row having one slot. The elongated slots are positioned asymmetrically in an array format of rows, preferably five rows, and one column. Each of the elongated slots is used to house one bar magnet. Each elongated slot and bar magnet extends the approximate length of the well plate  110 . 
     The relative positioning of the plurality of cylindrical slots  410  with respect to each of the 108 wells  230  according to a preferred embodiment will now be described with reference to FIG.  5 . FIG. 5 is a top view of the substantially flat upper surface  210  of well plate  110  with the positions of the plurality of slots  410  from the substantially flat lower surface  220  shown in dotted lines on the upper surface  210 . The first row of twelve (12) cylindrical slots  410  is positioned between the upper edge of the long axis of the well plate  110  and the first row  320  of wells  230 . The remaining four (4) rows of twelve (12) cylindrical slots  410  are positioned along the long axis of the well plate  110  between the remaining 8 rows of wells  230 . Thus, each well  230  is adjacent to one side of cylindrical slot  410 . 
     The five sets of cylindrical rare-earth magnets  120  are inserted in the plurality of cylindrical slots  410 . FIG. 6 represents a cross-sectional view of the well plate  110  taken across line A—A of FIG.  5 . The projection of the plurality of cylindrical slots  410  are indicated by the dotted lines. FIG. 6 also indicates in dotted lines the placement  610  of each cylindrical rare-earth magnet  120  inside each slot  410 . Each cylindrical rare-earth magnet  120  is placed adjacent to a well  230  in order to attract the magnetic particles contained in a tube  140  (not shown) inserted in the well  230  to one side of the wall of the tube  140 . 
     FIG. 7 illustrates a cross-sectional view of the well plate  110  taken across line BB of FIG.  5 . The asymmetrical placement of the cylindrical rare-earth magnets  120  are shown in relation to each well  230 . As shown in FIGS. 10A and 10B, the placement of the cylindrical rare-earth magnets  120  causes the magnetic particles inside of a tube  140  to be attracted to opposite sides of the tube  140  for adjacent rows. 
     FIG. 8 represents a 96 array tube holder  810  with tubes  140  according to a preferred embodiment, such as the 96 tube tray retainer set manufactured by Perkin Elmer Corporation. The 96 array tube holder  810  is a two-piece plastic tube holder. The bottom piece  820  of holder  810  allows for 1 to 96 tubes  140  or up to an 8 by 12 array of tubes  140  to be inserted into holder  810 . The top piece  830  of holder  810  secures tubes  140  by clamping tubes  140  within holder  810 . Holder  810  further comprises brackets  840  surrounding bottom piece  820 . The recessed area  310  of the well plate  110  is designed to accept brackets  840  surrounding bottom piece  820  in either of two positions (described further herein). 
     The well plate  110  9×12 array of wells  230  allows the 8×12 tube holder  810  to be placed in one of two positions. FIGS. 9A and 9B illustrate the insertion of tube holder  810  into apparatus  100  in first and second positions, respectively. In FIG. 9A, holder  810  with tubes  140  is positioned in the first position. In FIG. 9B, holder  810  with tubes  140  is positioned in the second position. Note that the first position and the second position are interchangeable such that FIG. 9B could represent holder  810 -with-tubes  140  positioned in the first position and FIG. 9A could represent holder  810  with tubes  140  positioned in the second position. 
     The ability of the present invention to simultaneously wash the magnetic beads in all of the 96 tubes  140  according to the preferred embodiment is directly correlated to the additional row of wells  230  (i.e., 9 rows in well plate  110 , as compared to 8 rows in array tube holder  810 ), the asymmetrical positioning of slots  420  in which rare-earth magnets  120  are inserted, and the ability to reverse the magnetic field by shifting the 96 tube array tube holder  810  with tubes  140  from the first position to the second position (and vice versa). The operation of the present invention will now be described with reference to FIGS. 10A and 10B. 
     FIG. 10A represents a cross-sectional view of apparatus  100  taken across line B—B of FIG. 5 with tube holder  810  inserted in apparatus  100  in a first position. As shown, apparatus  100  contains an additional row of wells  230  as compared to tube holder  810 . The magnetic particles in solution in each tube  140  are shown as magnetic beads  1010  attracted to the walls of tubes  140 . The magnetic beads  1010  are attracted to the tube walls adjacent to the rare-earth magnets  120  and are held onto the tube  140  walls by the magnetic field. Thus, each adjacent tube  140  has magnetic beads  1010  attracted to opposite sides of the walls of tubes  140 . The attachment of the magnetic beads  1010  onto the sides of the tube  140  walls allows for the insertion of a manual or robotic pipette tip into each tube  140  without disrupting the magnetic beads  1010 . Therefore, supernatant material can be removed and solutions can be added without disturbing the magnetic beads  1010 . 
     After the removal of the supernatant from each of the tubes  140 , a washing solution is added to each of the tubes  140 . The 96 tube array tube holder  810  with tubes  140  is then shifted to a second position. FIG. 10B represents a cross-sectional view of apparatus  100  taken across line B—B of FIG. 5 with tube holder  810  inserted in apparatus  100  in the second position. The rare-earth magnets  120  are now located on the opposite side of each tube  140 . The magnetic field is therefore reversed, and because of the position of the magnets  120  relative to the tubes  140 , the magnetic particles stream across the widest diameter of the washing solution because of the attraction of the magnetic field. The supernatant is removed and more washing solution is added. The process is preferably repeated a plurality of times. For example, the process may be repeated as many times as is necessary to achieve the required amount of washing. 
     With regards to the approach for cDNA molecule isolation, the use of the present invention reduces the washing process from approximately 45 minutes for six 1.7 ml tubes to approximately 5 minutes for 96 0.2 ml tubes. The need for individual finger tapping or vortexing of the tubes containing the paramagnetic beads is eliminated. During the extensive washing process of the cDNA Capture procedure, the time in which the tubes are placed in apparatus  100  is reduced from 2 minutes to 5 seconds. According to a preferred embodiment, the extensive washing process is repeated for a total of three (3) times. Where the cDNA Capture procedure requires 5 minute stays in the magnet, the present invention reduces the length of time in apparatus  100  to about one (1) minute. The amount of washing solution needed to wash the magnetic particles is also reduced with apparatus  100 . 
     While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.