Abstract:
A centrifuge includes a centrifuge rotor that rotates to provide supergravity conditions and tilt rotors that provide for sample agitation under the supergravity conditions. The centrifuge rotor is integral with a hollow centrifuge drive shaft. A tilt-drive shaft extends through the hollow and rotates coaxially of the centrifuge rotor. The tilt-drive shaft has a pinion that engages the tilt rotors, which thus are made to rotate about tilt axes that are spaced from and parallel to the centrifuge axis. A centrifuge motor is mechanically coupled to the centrifuge drive shaft to rotate the centrifuge rotor. A tilt motor is mechanically coupled to the tilt drive shaft for imparting a rocking motion of the tilt rotors (and thus of mounted sample reaction cells) relative to the centrifuge rotor. Both motors are controlled by a servo that receives orientation information from orientation encoders associated with the motors; the tilt motor is phase locked to the centrifuge motor to ensure precise relative motion control. The centrifuge motor is typically rotated to achieve a centrifugal force at the tilt rotors of about 1000 G. The tilt motor is controlled differentially relative to the centrifuge motor so that the tilt rotors rotate controllably, at, above and/or below the centrifuge rotation rate to achieve the desired rotation relative to the centrifuge. This centrifuge provides a robust and precise method of controlling fluid motion within a sample container.

Description:
This is a continuation-in-part application of U.S. patent application Ser. No. 09/514,975, filed Feb. 29, 2000. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates generally to chemical instrumentation and, more particularly, to centrifuges. A major objective of the present invention is to provide an improved centrifuge with tilt (i.e., rotation relative to gravitational or centrifugal force) control relative to the centrifugal force generated by the centrifuge. 
     The standard of living in modern societies has been greatly enhanced by advances in chemical, biological, and medical sciences. These fields all involve the separation of samples into constituent components that may then be processed to aid in their identification and/or quantification. The centrifuge is an important instance of instrumentation used to separate s ample components . 
     In addition, as described below, centrifuges that can control the tilt of a sample container relative to the centrifugal force can be used for pouring, mixing, filtering, and facilitating chemical reactions. Furthermore, tilting can be used to control liquid movement among multiple processing chambers of a sample container so that a series of processes can be implemented without manual intervention. Thus, a centrifuge with tilt control can automate sample processing conventionally performed manually by chemists. 
     A simple centrifuge has a centrifuge rotor that is spun, e.g., by a motor. Typically, a liquid-sample container spins with the rotor. The spinning sample components are subjected to a centrifugal force (F=mω 2 r) proportional to their mass, their distance from the centrifuge spin axis, and the square of the spin rate. The effect of the centrifugal force is much like the effect of gravity-liquid components are separated according to their relative densities. However, unlike gravity, the centrifugal force is readily controlled, e.g., by controlling the spin rate. Thus, a centrifuge can generate centrifugal forces orders of magnitude greater than gravity at the earth&#39;s surface. Generally, the “supergravity” conditions of a centrifuge are much more effective than gravity in separating sample components. 
     As suggested above, the supergravity conditions offered by centrifuges have uses other than component separation. Often the quantities of a sample available for analysis or processing are quite small. In the context of small sample volumes and the corresponding small capacities of the carrying the samples, surface tension limits liquid movement. The surface tension can make it difficult to flow a liquid from point to point as required for a series of processing steps or to mix a liquid as may be required to promote a reaction. The following three references disclose various approaches to tilting a sample container on a centrifuge relative to the centrifugal force. In each case, the tilting is used to control the movement of sample from chamber to chamber in a multi-chamber sample container to facilitate a series of reactions. 
     U.S. Pat. No. 5,089,417 to Wogoman discloses a centrifuge in which a holder for a sample container snaps from a first tilt orientation to a second tilt orientation when the centrifuge exceeds a predetermined rotation rate. Similarly, the first tilt orientation is resumed when the centrifuge spin rate falls below the threshold rate. Thus by increasing and decreasing the centrifuge spin rate, sample movement between reaction chambers of the sample container can be controlled. However, this approach provides little flexibility in selecting the centrifuge spin rate or tilt angles relative to the centrifugal force. It would be preferable to control the centrifuge rotation and the tilt actions independently. 
     Independent control of centrifuge spin rate and tilt action is disclosed in U.S. Pat. No. 4,814,282 to Holen et al. Similar to Wogomon, tilt of a sample container is used to transfer liquid from one chamber to another under the influence of centrifugal force. A tilt drive assembly, including motor and drive chain, is attached to the centrifuge rotor so that it rotates therewith. Power is delivered to the tilt-drive motor via slip rings, which tend to wear out as they are not generally designed to operate at centrifuge speeds. In this approach, any sensors used to track tilt would also rotate at high speeds, further complicating operation. In addition, centrifuge forces are applied to the tilt motor and drive train. For example, a 1-pound motor must withstand 1000-pound forces in a readily achievable 1000 G supergravity field. Thus, there are a number of robustness issues that can only be addressed with additional complexity and expense. U.S. Pat. No. 4,776,832 to Martin et al. avoids the need for physical connections to drive a tilt rotor by using inductive motors. The inductive motors include induction rotors that are physically coupled to holders, e.g., for reaction cells, and stationary stators, which are located beneath the centrifuge rotor (wheel). The stators induce eddy currents in the induction rotors, causing them to rotate. No physical connection is required between the stators and the induction rotors, eliminating the need to deliver power through slip rings. On the other hand, the non-physical coupling of drive and induction rotor does not ensure precise and flexible control of sample-container orientation relative to the supergravity field. 
     Related U.S. patent application Ser. No. 09/514,975, which is incorporated by reference herein in its entirety, teaches that agitating a sample container under centrifuge-induced supergravity conditions can overcome surface-tension to achieve rapid and thorough mixing. Such mixing can be invaluable in promoting many types of reactions, e.g., array hybridization. 
     Unfortunately, none of the three patents previously mentioned disclose centrifuges well adapted for this purpose. Wogomon lacks independent control over centrifuge spin rates and tilt angles. Holen does not provide sufficiently robust and precise control over tilt angle in view of the high speeds the tilt motor and any associated sensors must spin at. Martin lacks prescise control over tilt angle in view of the lack of a mechanical connnection between the tilt stators and the tilt rotors. What is needed is an economical and robust centrifuge that provides for independent and precise control of the tilt angle of a sample container relative to a spinning centrifuge rotor. 
     SUMMARY OF THE INVENTION 
     The present invention provides a coaxial-drive centrifuge with tilt control for manipulating liquid samples under supergravity conditions. The coaxial-drive arrangement allows a purely mechanical coupling between a stationary tilt motor to a tilt rotor that rotates relative to a rotating centrifuge rotor. The coaxial drive elements can be aligned with the axis of rotation for the centrifuge rotor. Appropriate gearing between the coaxial tilt-drive element, e.g., a shaft, and the tilt rotor allows the tilt axis to be displaced from the centrifuge axis. A sample container can be attached to the tilt rotor so that the angle of the container relative to the centrifugal force can be controlled to promote mixing or movement of the liquid contents of the container. 
     The invention provides for precise motion control. The centrifuge and tilt motors can be servo controlled. (Alternatively, high-speed stepper motors can be used.) The tilt motor is preferably phase-locked to the centrifuge motor. The desired positive and negative tilts relative to the centrifugal force can be added to the centrifuge servo angle to provide appropriate differential-drive commands to the tilt motor. 
     In a preferred realization of the invention, the tilt axis is parallel to and displaced from the centrifuge axis. Alternatively, the invention provides for any orientation of the tilt axis relative to the centrifuge axis, although, for most applications, the tilt axis is more parallel to the centrifuge axis than orthogonal to it. 
     In the case of parallel axes and a planar sample container, the plane of the container can be orthogonal to the tilt axis or generally orthogonal to the centrifugal force. The later orientation works best for agitating a sample by tilting back and forth so as to promote mixing. The former orientation works best for controlling the flow of a sample through a maze of chambers in the sample container. While sample motion control is provided in the prior art, the present invention allows much greater flexibility in the design of the maze since tilt angle is precisely controllable. In either case, the desired motion is achieved using a simple and robust mechanical linkage for both centrifuge and agitation motions. These and other features and advantages of the invention are apparent from the description below with reference to the following drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic sectional view of an array hybridization system in accordance with the present invention. In FIG. 1, course diagonal hatching indicates a centrifuge housing, negative-sloping fine hatching indicates components associated with the centrifuge rotor, while positive-sloping fine hatching indicates components associated with the agitation rotors. 
     FIG. 2 is a schematic plan view of the array hybridization system of FIG. 1 with reaction cells. 
     FIGS. 3A-3C show three orientations of a reaction cell of FIG. 2 being agitated in accordance with the present invention by the system of FIGS. 1 and 2. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In accordance with the present invention, a centrifuge AP 1  for array hybridization of a liquid sample in reactions cells  100  is shown in FIG.  1 . Centrifuge AP 1  includes a housing  11 , a centrifuge rotor  13 , a centrifuge drive system  15 , agitation rotors  17 , a tilt drive system  19 , a computer  21 , and a servo controller  23 . Tilt rotors  17  are rotatably mounted in centrifuge rotor  13  via ball bearings  25 . Thus, while centrifuge rotor  13  rotates about its “centrifuge” axis  25 , tilt rotors  17  rotate about their respective “tilt” axes  27 , which are parallel to and displaced from centrifuge axis  25 . 
     Housing  11  includes a washer-shaped base  31 , a cylindrical sidewall  33 , and a rotor shield  35 . These housing components are indicated by course diagonal hatching in FIG.  1 . Screws  37  attach sidewall  33  to base  31 , while screws  39  attach shield  35  to sidewall  33 . 
     Centrifuge drive system  15  includes a centrifuge motor  41 , a centrifuge-motor pulley  43 , a geared centrifuge-drive belt  45 , a centrifuge-shaft pulley  47 , and a hollow centrifuge shaft  49 , the latter being integral with centrifuge rotor  13 . Centrifuge-motor pulley  43  is rigidly coupled to a centrifuge-motor shaft  51  of centrifuge motor  41 , while centrifuge-shaft pulley  47  is rigidly coupled to centrifuge shaft  49 . Note that centrifuge shaft  49  is rotatably coupled to housing  11  via ball bearings  53 . 
     Centrifuge-drive belt  45  couples pulleys  43  and  47  so that motor  41  can drive centrifuge shaft  49 , and thus, centrifuge rotor  13 . Pulleys  43  and  47  and belt  45  all have gear teeth to ensure a known relationship between motor orientation and rotor orientation. Pulleys  43  and  47  both have 100 teeth so centrifuge rotor  13  rotates on a 1:1 basis with motor shaft  41 . 
     Tilt-drive system  19  includes a tilt-drive motor  61 , a tilt-motor pulley  63 , a tilt-drive belt  65 , a tilt-drive-shaft pulley  67 , a tilt-drive haft  69 , and a tilt-drive pinion  71 . Pinion  71  is monolithic with tilt-drive shaft  69 . Tilt-drive shaft  69  extends along centrifuge axis  25  hrough a hollow in centrifuge drive shaft  49 . 
     Tilt-motor pulley  64  is rigidly mounted on a tilt-motor shaft  73  of tilt motor  61 . Geared tilt-drive belt  65  provides a drive link between pulleys  63  and  67 , which both have gears. The gear ratio of pulley  63  to pulley  67  is 2:1. Tilt-drive shaft pulley  67  is rigidly coupled to tilt-drive shaft  69  so the rotation rate of pinion  71  is the rotation rate of tilt-drive shaft pulley  67 . Pinion  71  drives tilt rotor  17  at 1:2 so that tilt motor  61  drives tilt rotor 1:1. Alternatively other gear ratios can be used, for example a higher motor-to-rotor gear ratio can be used to achieve higher torque for faster acceleration when changing tilt directions. 
     Centrifuge motor  41  and tilt motor  61  are controlled by servo controller  23 . To this end, each motor  41 ,  61 , includes a respective orientation encoder  75 ,  77 . Each motor has 1000 marks indicating 1000 evenly spaced orientations. Each encoder detects these marks optically and provides information necessary for servo controller to track the orientations of each motor  41 ,  61 , and thus of respective rotors  13 , 17 . 
     Servo controller  23 , manufactured by Galil Corporation in Mountain View, California, typically commands centrifuge motor  41  to rotate at a constant speed in a single direction. For example, a rotation rate of 3000 rpm can be used to achieve 1004 G supergravity given a 10 centimeter spacing (using the formula F=mω 2 R) between centrifuge axis  25  and tilt axis  27 . To ensure the rotation rate is maintained, servo controller  23  compares actual orientation with expected orientation over time. Deviations are compensated for as in conventional servo operation. 
     Note that, if the tilt rotor rotation rate matches the centrifuge rotation rate, the orientation of a reaction cell  100  relative to the centrifuge rotor (and thus the direction of supergravity) does not change. If a stirring motion is required, the desired stirring rate can be added or subtracted from the centrifuge rotation rate to obtain the desired tilt rotation rate. This operation is indicated by the tilt TLT and centrifuge CNT signals being summed by a summing amplifier  79 . Such a stirring motion can be used with reaction cells lying flat on the tilt rotor (perpendicular to tilt axes  27 ). The drive signals from servo control  23  are amplified by Copley amplifiers  81  and  83 . 
     In general, for reaction cells oriented along tilt axes  27 , as shown in FIG. 1 with reaction cells  100 , a rocking motion is desired for agitation. In this case, a preferred motion would be to rotate to a desired tilt relative to the centrifuge radius (down in the direction of supergravity), optionally hold that orientation for a time, then rotate in the opposite direction to the opposite tilt, optionally hold that orientation for a time, and iterate. 
     To achieve the desired rocking motion, computer  21  determines the desired plus and minus differential orientations for tilt motor  61  relative to the orientation of centrifuge motor. These serve as stops between tilts. Computer  21  then programs servo controller  23  accordingly. To tilt in one direction, tilt rotors  17  must rotate (relative to the inertial frame) faster than centrifugal motor  13 . To tilt in the opposite direction, tilt rotors must rotate slower than centrifugal motor  13 . 
     For example, centrifuge motor  41  and thus centrifuge rotor can rotate at 3000 rpm. The desired centrifuge rotor rotation motion and tilt rotor rocking motions are indicated by arrows  91  and  93 , respectively, in FIG.  2 . The agitation amplitude is selected to be about ±6° to effect full “sloshing” of the sample liquid. The agitation rate depends on the sample liquid and the centrifugal force. A typical value would be a 5 Hz agitation, which would yield ten replenishments per second. These values are programmed into servo  23  by computer  21  using a high-level programming language used by Galil servo-controller  23 . 
     Encoders  75  and  77  are  250  slot A quad B encoders that resolve 360° into 1000 orientations. Thus, sixteen encoder counts roughly corresponds to 6°. Thus, a command sequence to servo controller  23  can take the form of the following commands processed at the rate of ten per second: “advance the tilt motor sixteen counts relative to the centrifuge motor, reverse for thirty-two counts, forward again for thirty-two counts, reverse again for thirty-two counts, and so on, ending with a sixteen-count return to center. 
     Other embodiments employ other gear ratios. In such cases, the tilt motor rate is still determined from the sum of the centrifuge rotor spin rate and the desired tilt rate. In this case, however, the sum is multiplied by the tilt motor:rotor gear ratio. Also, the centrifuge rotor spin rate is the quotient of the centrifuge motor spin rate divided by the centrifuge motor:rotor gear ratio. These relationships determine how a servo is to control the tilt motor and centrifuge motors. These relationships assume that the tilt and centrifuge motors and encoders are equivalent. If they differ, the drive commands will have to take these differences into account as is known in the art. 
     Reactions cells  100  are similar so the following description of any one is representative. A reaction cell  100  includes a substrate  101  and a cover  103  so as to define a 2 cm×2 cm×0.25 mm interior volume or “reaction chamber”  105 . (In the figures, the thickness of reaction cell  100  is exaggerated for clarity.) During hybridization, this interior volume is partially filed with sample liquid  109 , with the remainder of the cell interior volume being filled with gas  107 , e.g., dry air or nitrogen. A hybridization array  110  with 10,000 probes arranged in a 100×100 array is defined on substrate  101  on the side contacted by sample liquid. Two septa (not shown) in each cover  103  permit fluid to be introduced and removed from reaction cell  100 . 
     In FIG. 3A, reaction cell  100  is shown titled counterclockwise +6° relative to a central centrifugal force vector  115  at the beginning of an agitation cycle. (Agitation angles are exaggerated in FIGS. 3A-3C for clarity.) In this orientation, all sample liquid  109 , other than a thin film, is at the end  117  shown to the left in FIGS. 3A-3C. The surface of sample liquid  109  in the static state represents a constant radius from centrifugal axis  113  (FIG.  1 ). 
     In FIG. 3B, reaction cell  100  has rotated clockwise past a level (orthogonal to a centrifugal force vector  115 ) orientation to a −2° clockwise orientation. In this orientation, some of the liquid has reached the opposite end  119  (to the right in FIGS.  2 A- 2 C). Most of the remaining liquid is still at the clockwise end  117 , while a tapered sheet of sample liquid  109  extends between the ends  117  and  119 . 
     In FIG. 3C, reaction cell  100  has rotated to an extreme clockwise position at −6°. In this position, except for a thin film, sample liquid  109  is at the right end  119  of reaction cell  100 . This completes the first half of an agitation cycle. The second half of the agitation cycle begins with the orientation of FIG.  3 C and ends with the orientation of FIG.  3 A. The return motion provides for highly desirably vertical mixing. 
     The vertical mixing assures that every target molecule spends some time close enough to array  110  for binding to occur. The centrifugal force  115  helps overcome the inertia of the liquid and its non-specific binding forces with the substrate so that a high agitation rate can be maintained. The advantages of the invention can be understood with the following, admittedly approximate, understanding of the hybridization process. 
     When the agitation rate is doubled, each target molecule is likely to be found half as far from a respective probe for half the time. When it is half as far, it is four times as likely to hybridize. However, the interval over which it can hybridize is half as long. Thus, in principle, doubling the agitation rate doubles the hybridization rate. This linear relationship applies until non-specific binding fluid forces prevent sample liquid from completing its motion across the array. The stronger the centrifugal force, the higher the agitation rate can be raised before this limiting consideration applies. Thus, the centrifuge rate can be increased until the forces involved adversely affect specific binding or threaten the integrity of the hybridized or non-hybridized species. 
     In FIG. 1, tilt axes  27  are parallel to the centrifuge axis  25  and the hybridization arrays are generally orthogonal to the centrifugal force. In other embodiments, the sample containers are also generally orthogonal to the centrifugal force, but the tilt axes are not parallel to the centrifuge axis. For example, the tilt axes can be circumferentially (in other words, “tangentially”) oriented relative to the centrifuge axis. 
     Reaction cells  100  of FIG. 1 are oriented so that arrays  110  generally orthogonal to the centrifugal force. Oblique orientations are also provided for. For example, reactions cells can be oriented so that they are more orthogonal to the centrifugal force than along it. However, reaction cells can be oriented both along and orthogonal to the centrifugal force. 
     Particularly with a circumferentially oriented tilt axis, but also in other cases in which the array is orthogonal to the centrifugal force, the substrate can be curved cylindrically, for example, along a radius slightly less than (e.g., 90% of) the distance between the tilt axis and the centrifuge axis. In this case, the centrifugal force is more orthogonal to the array away from the array center and even at the extremes of the tilt motion. This provides a more uniform sample liquid distribution across the array, which in turn allows less sample liquid to be used without risking drying of the array. In addition, the agitation is gentler on the sample. 
     The reaction cell can also include fastening means effective to press the cover and the substrate together, i.e., to immobilize the cover on the substrate, thereby forming a watertight, temporary seal therebetween. The fastening means ensures stable, effective and secure positioning of the cover over the substrate. Optional gasket means adjacent the surface of the cover may be included to aid in equalizing the pressure provided by the fastening means. The optional gasket may be, for example, placed between the cover and the rigid frame to provide compliance in the system and to even the pressure applied to the cover and the substrate. The apparatus further comprises fluid transfer means that enables introduction of fluid from the exterior of the apparatus to the reaction chamber, and removal therefrom. In a preferred embodiment, the fluid introduction means comprises one or more ports in the cover. 
     It is preferred that the cover be made of plastic and the substrate of glass, plastic, fused silica or silicon, the seal between plastic and either glass, plastic, fused silica or silicon being advantageous for producing the apparatus of the invention. The cover material should be thermally stable, chemically inert, and preferably non-stick. Furthermore, when the apparatus is used in hybridization, the cover should be comprised of a material that is chemically and physically stable under conditions employed in hybridization. In a preferred embodiment, the plastic cover is polypropylene, polyethylene or acrylonitrile-butadiene-styrene (“ABS”). In the most preferred embodiment, the plastic cover is comprised of polypropylene. The cover may be constructed by machining or molding technologies. 
     As noted above, the cover preferably has a lip along its cover bordering a large central recessed portion of the inner face of the cover. Applying pressure to the outer face of the cover directly above the perimeter lip is required to form the tight seal between the cover and the substrate. Any means that presses the lip of the cover securely to the substrate is suitable. Such pressure may be applied evenly by, for example, clamps, a press, or by coverturing the substrate and cover within a two-part rigid frame and compressing the two together to supply an even pressure to the cover and substrate. If desired, the peripheral lip of the cover may be modified to provide for an improved seal; for example, one or more continuous ridges can be incorporated into the lip so that the pressure supplied to the cover is higher at those locations and preferentially causes them to compress. 
     In any of these embodiments, the reaction cell may be re-used, as the peripheral seal is temporary and the fastening means may be removed when desired. Thus, the reaction cell may be readily disassembled after use, cleaned, and re-assembled (with alternate components, such as a different substrate, if desired) so that some or all of the components of the reaction cell may be re-used. 
     This reaction cell interior height may range from about 0.002″ to 0.02″ (50 μm to 500 μm). The dimension of the cover, the peripheral lip, and the reaction area are such that the reaction area is generally in the range of about 4 mm 2  to 500 mm 2 , preferably about 20 mm 2  to 350 mm 2 , and the reaction chamber has a volume in the range of about 0.2 μl to about 312 μl, preferably about 1 μl to 200 μl. 
     Hybridization array  110  has a plurality of molecular probes bound thereto. Preferably, the molecular probes are arranged in a spatially defined and physically addressable manner, i.e., are present in one or more “arrays.” In a most preferred embodiment, the probes are oligonucleotide probes (including cDNA molecules or PCR products), although other biomolecules, e.g., oligopeptides and the like, may serve as probes as well. 
     The present invention has applicability to analytical chemistry and industry fields that rely on its techniques. While centrifuge AP 1  is described in its use for hybridization and other reaction requiring liquid agitation, it can also be used for sequencing reactions by moving a liquid from chamber to chamber in a sample container. In addition, it can be used for sample-component separation. 
     Variations on the disclosed embodiments provide for other orientations of the sample container relative to the centrifuge and/or tilt axis. Alternative embodiments provide for non-parallel relationships between the centrifuge and tilt axes and even for varying the angle between these axes. Where precision is not critical, nonservo motors can be used in applications. On the other hand, fast stepper motors can be used as an alternative to servo-controlled motors where precision is required. These and other variations upon and modifications to the disclosed embodiments are provided for by the present invention, the scope of which is defined by the following claims.