Abstract:
The invention relates to a method and an apparatus for performing an assay on chemical, biochemical, or biological fluids. The apparatus includes an assay chamber for fluid reactions and a centrifugal force activated-valve for controlling fluid movement through the assay chamber. An active surface can be positioned in the assay chamber to react with fluids passed through the assay chamber. The active surface may contain biomolecular probes. The biomolecular probes can be DNA, DNA fragments, RNA, RNA fragments, reagents, protein, protein fragments, lipids, and lipid fragments. The apparatus is particularly useful when multiple reactions, dilutions, or washing steps are required to determine a final answer. The centrifugal force activated-valve provides positive control over fluid in the assay chamber and allows for repeated use of the same chamber for multiple reaction steps. The apparatus can be disposed of after an assay to eliminate potential contamination from reuse of the same apparatus. Fluids passed through the assay chamber may be recovered for subsequent analysis, processing, or reactions.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application is a continuation application of application Ser. No. 09/695,008 filed Oct. 23, 2000, which is a divisional of application Ser. No. 09/133,102 filed Aug. 12, 1998 (now U.S. Pat. No. 6,162,400). Priority is claimed under 35 U.S.C. 120 from both of the foregoing applications, which are incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    The present invention relates generally to analytical devices for performing assays and more particularly to an analytical device that includes a centrifugal force actuated valve to control fluid and a method of utilizing the analytical device to perform an assay.  
           [0003]    There are many analytical methods that require one or more reactions or analytical steps to determine a final answer. Examples are heterogeneous immunochemistry reactions, hybridization of DNA to DNA, and hybridization of DNA to RNA. In order to determine the concentration or presence of an analyte, such analytical methods require multiple, serial reactions either with or without washing steps or a single reaction with a washing step. Other assays, such as clinical chemistry assays, often require precise dilutions prior to mixing with other chemicals.  
           [0004]    It would be desirable to reduce the cost of conducting the reactions in an assay by automating the reaction steps rather than using expensive manual labor to perform the steps. Additionally, it would be advantageous to use centrifugal force in the automation process to minimize variations due to surface tension and capillary action and to move and control fluid.  
           [0005]    Centrifugally driven analytical devices have employed several methods for controlling fluid movement, such as differential flow, stop junctions, siphons, and complex, two-axis mechanisms.  
           [0006]    Differential flow allows fluid to enter a chamber quickly but exit slowly. As the fluid enters and exits the chamber there is a finite residence time in which most of the fluid can be manipulated. However, the disadvantage of differential flow is that the entire volume of fluid is never completely controlled.  
           [0007]    Stop junctions employ the pressure created by a capillary to stand off fluid flow until the centrifugal force generated by rotation overcomes capillary back pressure. Stop junctions are sensitive to the exact geometry and surface properties of the junction and to fluid properties of the sample.  
           [0008]    Siphons allow fluid movement into a chamber under the action of centrifugal force and prevent the fluid from exiting the chamber until the siphon is primed or the siphon level is high enough. The disadvantage with siphons is that some of the fluid is lost in the entrance of the siphon and that great care must be taken to prevent the siphon from priming prematurely or from losing prime prematurely.  
           [0009]    Two-axis mechanisms provide better fluid control, but at substantial instrumentation cost, complexity, and size. Two-axis mechanisms are typically mounted on a large turntable. The turntable has two positions, each position having a local center of rotation.  
           [0010]    U.S. Pat. No. 5,171,533 to Fine et al. discloses single use centrifugal valves for performing a biological assay. Sealant materials are used to provide a counteracting force to the centrifugal force of rotation. The sealants are designed to yield at predetermined levels of centrifugal force. The valves in the Fine et al. patent require that the fluid move through different chambers.  
           [0011]    From the foregoing it will be apparent that there is a need for a simple, easy to automate, and economical means for performing an assay or a reaction. Further, it will be apparent that a disposable analytical device is desirable for preventing contamination resulting from repeated use of the same analytical device.  
         SUMMARY OF THE INVENTION  
         [0012]    The invention provides an analytical device incorporating a centrifugal force-activated valve for controlling reactions and for moving fluid within the analytical device. The centrifugal force-activated valve allows for several fluids to be passed through the analytical device and allows the analytical device to be used for multiple serial reactions. The centrifugal force-activated valve provides simple and reliable control over fluid movement and can be tuned to open or close over a wide range of rotational speeds.  
           [0013]    An analytical device embodying the invention is economical, easy to use, injection moldable from a variety of plastics, readily adaptable to automation, capable of repeated reaction, mixing, and washing steps, disposable to prevent contamination, and may be transparent to allow an assay by optical detection means.  
           [0014]    In a preferred embodiment of an analytical device according to the present invention, a housing enclosure adapted for rotation about an axis includes an assay chamber, a fluid discharge port in fluid communication with the assay chamber, means for introducing fluid into the assay chamber, and a centrifugal force-activated valve in fluid communication with the fluid discharge port. Preferably, the fluid introducing means is self sealing. Fluid entering the valve through the fluid discharge port may exit the valve through a fluid drain. The fluid drain is in fluid communication with the valve and an exterior portion of the housing enclosure. Chemical, biological, or biochemical fluids may be introduced into the assay chamber.  
           [0015]    Additionally, an active surface can be positioned on an interior surface of the assay chamber. The active surface may contain biomolecular probes. The biomolecular probes can be DNA, DNA fragments, RNA, RNA fragments, reagents, protein, protein fragments, lipids, and lipid fragments. Additionally, the active surface can be disposed on a structure positioned on an interior surface of the assay chamber. For example, the structure can be in the shape of balls or beads. The structure can be attached to an interior surface of the assay chamber or can be loosely contained in the assay chamber. Typically, the active surface is arranged in an array pattern positioned on a surface of the assay chamber.  
           [0016]    An analytical substrate can be positioned in an opening in the housing enclosure. The active surface can be disposed on an interior surface of the analytical substrate and in opposing relation to the assay chamber.  
           [0017]    The analytical substrate can be made transparent to optical detection means external to the housing enclosure. A transparent analytical substrate allows for an assay of the active surface by scanning the active surface through the analytical substrate using the optical detection means.  
           [0018]    The analytical device may also include a reservoir chamber disposed radially outward from the centrifugal valve and in fluid communication with the valve. Fluid exiting the centrifugal valve is stored in the reservoir chamber. Optionally, fluid contained in the reservoir chamber can be extracted through fluid extracting means in fluid communication with the reservoir chamber. Preferably, the fluid extraction means is self sealing.  
           [0019]    In another embodiment, the housing enclosure includes a portion defining a valve chamber for containing the centrifugal force-activated valve. A fluid discharge port is positioned at a radially inward end of the valve chamber and is in fluid communication with the valve chamber and the assay chamber. A valve seat is disposed in the valve chamber and is disposed radially outward from the fluid discharge port. The valve seat is in fluid communication with the fluid discharge port and the valve chamber. An actuator is movably disposed in the valve chamber and is biased to a stationary position relative to the valve seat by a counter acting force provided by bias means positioned in the valve chamber and disposed radially outward from the actuator. The actuator moves radially outward from the stationary position to an actuated position when the housing enclosure is rotated and the centrifugal force acting on the actuator exceeds the counteracting force of the bias means. When the centrifugal force drops below the counteracting force, the actuator moves radially inward from the actuated position to the stationary position.  
           [0020]    A method according to the present invention includes injecting a sample into the assay chamber through the fluid introducing means. The analytical device is then spun at a first rotational speed lower than that required to actuate the centrifugal force-activated valve. The first rotational speed needs to be sufficient to distribute the sample across the active surface. The sample is allowed to react with the active surface. At the conclusion of the reaction period, the analytical device is spun at a second rotational speed to actuate the centrifugal force-activated valve and empty the sample from the assay chamber. Spinning at the second rotational speed is stopped when the assay chamber is empty. Wash fluids are injected into the assay chamber and the analytical device is spun at the first rotational speed to distribute the wash fluids. The analytical device is then spun at the second rotational speed to empty the wash fluids. The washing step can be repeated as necessary to effect complete washing of the assay chamber and the active surface. After the washing step, the active surface can be scanned to detect the presence of an analyte.  
           [0021]    Other aspects and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the invention. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0022]    [0022]FIG. 1 is a cross section of an analytical device according to the present invention.  
         [0023]    [0023]FIG. 2 a  is a cross section of a centrifugal force-activated valve with a valve chamber cap according to the present invention.  
         [0024]    [0024]FIG. 2 b  is a cross section of a centrifugal force-activated valve with a valve chamber cap having fluid extraction means according to the present invention.  
         [0025]    [0025]FIG. 2 c  is a cross section of a centrifugal force-activated valve with a valve chamber cap having a fluid drain according to the present invention.  
         [0026]    [0026]FIG. 3 a  is a cross section of a normally closed centrifugal force-activated valve in the stationary position according to the present invention.  
         [0027]    [0027]FIG. 3 b  is a cross section of a normally closed centrifugal force-activated valve in the actuated position according to the present invention.  
         [0028]    [0028]FIG. 4 a  is a cross section of a normally open centrifugal force-activated valve in the stationary position according to the present invention.  
         [0029]    [0029]FIG. 4 b  is a cross section of a normally open centrifugal force-activated valve in the actuated position according to the present invention.  
         [0030]    [0030]FIG. 5 a  is a cross section of a valve seat with compliant material according to the present invention.  
         [0031]    [0031]FIG. 5 b  is a cross section of an actuator with elastomeric material according to the present invention.  
         [0032]    [0032]FIG. 6 is a cross section of an analytical device having a substrate disposed in the housing enclosure according to the present invention. 
     
    
     DETAILED DESCRIPTION  
       [0033]    In the following detailed description and in the several figures of the drawings, like elements are identified with like reference numerals.  
         [0034]    As shown in the drawings for purposes of illustration, the invention is embodied in an analytical device having an assay chamber for fluid reactions and at least one centrifugal force-activated valve in fluid communication with the assay chamber. The valve provides positive control of fluid contained in the assay chamber and allows for multiple, fluid reaction steps, mixing steps, and washing steps to occur in the assay chamber. The analytical device of the present invention is easy to use, can be manufactured at low cost, is adaptable to automation, provides predictable fluid control that can be tuned over a wide range of rotational speeds, and can be disposed of after use to prevent contamination from repeated use of the same analytical device. Previous apparatus for controlling reactions have incorporated complex fluid control methods, such as, siphons, stop junctions, two-axis rotation, differential flow, and single use centrifugal valves. The analytical device of the present invention provides positive fluid control with less complexity and greater flexibility than the previous apparatus.  
         [0035]    Referring to FIG. 1, there is provided in accordance with the present invention an analytical device generally designated as  1 . The analytical device includes a housing enclosure  3  defining an assay chamber  5 , and a centrifugal force-activated valve  7   a  disposed radially outward of the assay chamber  5 . The housing enclosure  3  is adapted for rotation  6  about a rotational axis  4 . Fluid is communicated from the assay chamber  5  to the valve  7   a , as shown by arrow  19 , thru a fluid discharge port  9  positioned intermediate to the assay chamber  5  and the valve  7   a . An active surface  13  can be positioned on an interior surface of the assay chamber  5 . Fluid is introduced into the assay chamber  5 , as shown by arrow  17 , using fluid introducing means  11 . Fluid introducing means  11  is in fluid communication with the assay chamber  5 . Fluid introducing means  11  can be an opening positioned in the housing enclosure  3  near the rotational axis  4 . Preferably, fluid introducing means  11  is self sealing, for example, a diaphragm or a septum. If the reaction is fast or the volumes of fluid large enough that evaporation is not an issue, the fluid introducing means  11  need not be sealed. Rotation  6  of the housing enclosure  3  generates a radially outward flow of fluid contained in the assay chamber  5 , thereby urging the fluid into contact with the fluid discharge port  9 . Additionally, it is desirable to position the active surface  13  at a radially outward portion of the assay chamber  5  to allow fluid to react with the active surface  13  when the housing enclosure  3  is rotated and the fluid is urged radially outward. When the centrifugal force from rotation  6  of the housing enclosure exceeds the force necessary to actuate the valve  7   a , the valve opens, and fluid enters the valve  7   a  through the fluid discharge port  9 . In a typical application, chemical, biochemical, or biological fluids are reacted in the assay chamber.  
         [0036]    The centrifugal force-activated valve  7   a , includes a valve chamber  23 , a valve seat  25  positioned radially outward of the fluid discharge port  9 , an actuator  27  movably positioned in the valve chamber  23 , and bias means  29  positioned in the valve chamber  23  and radially outward of the actuator  27 . The bias means  29  provides a counteracting force to urge the actuator  27  into a stationary position relative to the valve seat  25 . As will be further described below with respects to FIGS. 3 a ,  3   b ,  4   a , and  4   b , the valve seat may be positioned radially inward or radially outward of the actuator. Additionally, a fluid drain  15  can be positioned in the valve chamber  23 . The fluid drain  15  allows fluid entering the valve chamber  23  as shown by arrow  19 , to exit the valve  7   a  through the exterior of the housing enclosure  3 , as shown by arrow  21 .  
         [0037]    In a typical embodiment of the present invention, housing enclosure  3  can be made from almost any structural material. Obvious choices include metals, ceramics, plastics, fused silica, and glasses. The housing enclosure  3  can be machined from metal, for example, steel, aluminum, and copper. The housing enclosure  3  can also be machined or injection molded from plastic. Stainless steel is a preferred metal since it won&#39;t generally react with the chemical analysis occurring in the analytical device  1 . More preferred, especially for a disposable analytical device, is injection molded plastics. Possible choices for injection molded plastics include acrylic (polymethyl methacrylate), polyethylene, polypropylene, polystyrene, ABS, polycarbonate, and the like. Selection of the material for the housing enclosure  3  is dependent on the specific requirements for the assay. For example, polypropylene is desirable because of its higher temperature limits and inertness; however, the inertness of polypropylene make it difficult to label and difficult to glue in subsequent manufacturing assembly steps. Typically, the valve chamber  23  and the valve seat  25  are made of the same material as the housing enclosure  3 .  
         [0038]    The actuator  27  can also be made of the same materials as the housing enclosure  3 . Preferably, the actuator  27  is made from a dense, inert material, such as, a stainless steel ball. Stainless steel balls are easily obtained and the material is dense and inert. The shape and material properties of the actuator  27  should be compatible with motion of the actuator  27  in the valve chamber  23 . Actuator  27  should move back and forth in the valve chamber  23  with minimal friction, and should be properly shaped and sized to prevent binding in the valve chamber  23 . Suitable shapes for actuator  27  include a ball, a machined shape, and a molded shape.  
         [0039]    Referring to FIG. 1, bias means  29  provides a counteracting force to urge the actuator  27  to a stationary position relative to the valve seat  25 . Rotation  6  of the housing enclosure  3  generates centrifugal forces that urge the actuator  27  to move radially outward in opposition to the counteracting force of bias means  29 . The magnitude of the centrifugal force acting on actuator  27  is proportional to the product of the mass of the actuator  27  and the square of the rotational velocity of the housing enclosure  3  divided by the radial distance of the actuator  27  from the rotational axis  4 . The counteracting force of bias means  29  can be provided by a compression spring, a compressible material, a coil spring, a leaf spring, or a diaphragm. For purposes of illustration, FIG. 1 shows a coil spring. The centrifugal force-activated valve of the present invention can be tuned to actuate at a predetermined rotational speed by selecting the mass of the actuator  27  and the counteracting force of bias means  29 .  
         [0040]    In a preferred embodiment of the present invention, valve seat  25  is integral to the valve chamber  23  and is made of the same material as the housing enclosure  3 . Valve seat  25  operates to prevent fluid communication between the fluid discharge port  9  and the valve chamber  23  when the actuator  27  is forcefully engaged with the valve seat  25 . Centrifugal force-activated valve  7   a  operates as a normally closed valve when the valve seat  25  is positioned radially inward of the actuator  27 . For a normally open valve, valve seat  25  is positioned radially outward of the actuator  27 .  
         [0041]    Referring to FIGS. 3 a  and  3   b , there is provided an illustration of a normally closed centrifugal force-activated valve  7  according to the present invention. FIG. 3 a  shows actuator  27  biased into a stationary position by bias means  29 . In the stationary position, actuator  27  is forcefully engaged with the valve seat  25  and fluid communication between the fluid discharge port  9  and the valve chamber  23  is prevented, as shown, by arrow  53 . Actuator  27 , moves radially outward from the stationary position to an actuated position when the housing enclosure  3  is rotated and the centrifugal force acting on actuator  27  exceeds the counteracting force of bias means  29 . In the actuated position, as shown in FIG. 3 b , actuator  27  is disengaged from valve seat  25  allowing fluid communication between the fluid discharge port  9  and the valve chamber  23 , as shown by arrow  55 .  
         [0042]    Referring to FIGS. 4 a  and  4   b , there is provided an illustration of a normally open centrifugal force-activated valve  7  according to the present invention. FIG. 4 a  shows actuator  97  biased into a stationary position by bias means  99 . In the stationary position, actuator  97  is forcefully disengaged from the valve seat  95  allowing fluid communication between the fluid discharge port  9  and the valve chamber  93 , as shown, by arrow  55 . Actuator  97  moves radially outward from the stationary position to an actuated position when the housing enclosure  3  is rotated and the centrifugal force acting on actuator  97  exceeds the counteracting force of bias means  99 . In the actuated position, as shown in FIG. 4 b , actuator  97  is forcefully engaged with the valve seat  95  preventing fluid communication between the fluid discharge port  9  and the valve chamber  93 , as shown by arrow  53 . Optionally, it may be desirable to position actuator stops  57  in valve chamber  93  to prevent bias means  99  from urging the actuator  97  into a fluid discharge port  9  blocking position when the actuator is in the stationary position. The actuator stops  57  are disposed radially inward of the actuator  97 .  
         [0043]    In another embodiment of the present invention, a compliant material  59  is conformally disposed on the valve seat  58 , as illustrated in FIG. 5 a . The compliant material  59  forms a tight seal between the actuator  56  and the valve seat  58  when the actuator  56  is forcefully engaged with the valve seat  58 . Alternatively, the valve seat  58  may be a compliant material positioned in the valve chamber, for example, an o-ring or a gasket.  
         [0044]    Referring to FIG. 5 b , in an alternative embodiment of the present invention, an elastomeric material  61  is disposed on the actuator  60 . The elastomeric material  61  forms a tight seal between the actuator  60  and the valve seat  62  when the actuator  60  is forcefully engaged against the valve seat  62 , preventing fluid communication between the fluid discharge port  9  and the valve chamber  23 .  
         [0045]    In another embodiment of the present invention, the valve chamber  23  includes a valve chamber cap  41  disposed in valve chamber opening  43 , as shown in FIG. 2 a . The valve chamber opening  43  is disposed radially outward of the valve chamber  23  and is in fluid communication with the valve chamber  23 . The valve chamber cap  41  has an outer surface  45 , and an inner surface  47 . The inner surface  47  is in fluid communication with the valve chamber  23 . The valve chamber cap  41  allows for the insertion of the actuator  27 , the bias means  29 , and optionally the valve seat  25  into the valve chamber  23 . The valve chamber cap  41  can be permanently attached to the valve chamber opening  43 , by gluing, welding, fasteners, or the like. The valve chamber cap  41  and the valve chamber opening  43  can be adapted to permit the valve chamber cap  41  to be removably attached to the valve chamber opening  43 , for example, using threads, bayonet mount, friction fit, or an o-ring.  
         [0046]    One advantage of the removable valve chamber cap  41  is that it allows for application specific selection of a bias means and an actuator. For example, prior to performing an assay using the analytical device  1 , a user can select the mass of actuator  27  and the counteracting force of bias means  29  to actuate the centrifugal force-activated valve  7  at a predetermined rotational speed.  
         [0047]    In another embodiment of the present invention, as illustrated in FIG. 2 b , a valve chamber cap  42  includes fluid extraction means  49  contained in the valve chamber cap  42 . Preferably, the fluid extraction means  49  is self sealing, for example, a diaphragm or a septum. The fluid extraction means  49  is in fluid communication with the inner surface  47  and outer surface  45  of the valve chamber cap  42 . In some assays, it may be desirable to analyze fluids that have been passed through the assay chamber  5 . To extract fluid from the valve chamber  23 , a device such as a syringe is inserted through the fluid extraction means  49  to withdraw fluid remaining in the valve chamber  23 . Optionally, a radially outward portion of the valve chamber  23  can be sized to capture a specific volume of fluid to be extracted from the valve chamber  23 .  
         [0048]    [0048]FIG. 2 c  illustrates another embodiment of the present invention. A fluid drain  51  is disposed in a valve chamber cap  44 . Fluid drain  51  is in fluid communication with the inner surface  47  and the outer surface  45  of the valve chamber cap  44 . Fluid entering the valve chamber  23  exits the valve chamber  23  through the fluid drain  51 . Fluid drain  51  in valve chamber cap  44  may either supplement or replace fluid drain  15  in the valve chamber  23 .  
         [0049]    In an alternative embodiment of the present invention, as illustrated in FIG. 1, housing enclosure  3  has a reservoir plate  31  having a recessed portion defining a reservoir chamber  33  positioned radially outward of the assay chamber  5  and disposed below the valve chamber  23 . Reservoir chamber  33  is in fluid communication with fluid drain  15 . Fluid exiting the valve chamber  23  through the fluid drain  15  enters the reservoir chamber  33 , as shown by arrow  21 . Reservoir chamber  33  can be sized to accommodate any volume, preferably about 3 milliliters. As the volume of the reservoir chamber  33  is decreased the distance between the reservoir chamber  33  and the fluid drain  15  may also decrease. If the distance is such that fluid entry into the reservoir chamber is restricted, a fluid channel  16  may be included to provide a clear and unobstructed path for fluid exiting the fluid drain  15  to enter the reservoir chamber  33 . Fluid channel  16  is disposed below the fluid drain  15  and is in fluid communication with the fluid drain  15  and the reservoir chamber  33 . Fluid channel  16  has a first channel wall  18  disposed opposite to and substantially parallel with a second channel wall  20 . Fluid channel  16  is substantially wider than fluid drain  15  and is substantially symmetrical with fluid drain  15 .  
         [0050]    It may be desirable to recover fluids used in an assay for subsequent analysis or reactions. Reservoir plate  31  may include fluid extraction means  35  in fluid communication with the reservoir chamber  33  and the exterior of the reservoir plate  31 . Preferably, the fluid extraction means  35  is self sealing, for example, a diaphragm or a septum. Typically, a syringe or the like is inserted through the fluid extraction means  35  and fluid is withdrawn from the reservoir chamber  33 .  
         [0051]    [0051]FIG. 1 illustrates an alternative embodiment of the present invention. Air or gas  39  displaced by fluid  21  entering the reservoir chamber  33  from centrifugal force-activated valve  7   a  is vented to the exterior of housing enclosure  3  through a vapor vent  37  of centrifugal force-activated valve  7   b . Vapor vent  37  is in fluid communication with the valve chamber  73  of centrifugal force-activated valve  7   b . Rotation  6  of the housing enclosure  3  above the rotational speed necessary to actuate valve  7   b  from the stationary position to the actuated position, forces the actuator  77  to disengage from the valve seat  75 . Air or gas  39  enters the valve chamber  73  through the fluid drain  78  and exits the valve chamber  73  through the valve seat  75  and enters the vapor vent  37 . Centrifugal force-activated valve  7   a  is in fluid communication with the assay chamber  5 . Fluid  21  entering fluid drain  15  of valve  7   a , displaces air or gas  39  in the reservoir chamber  33 . Valve  7   b  is not in fluid communication with the assay chamber  5 . However, the reservoir chamber  33  is common to both valve  7   a  and valve  7   b ; therefore, fluid entering the reservoir chamber  33  from centrifugal force-activated valve  7   a  displaces air or gas  39  through centrifugal force-activated valve  7   b.    
         [0052]    In a most preferred embodiment, the housing enclosure  3  has a portion defining an opening  63 , as shown in FIG. 6. The opening  63  is positioned over the assay chamber. An analytical substrate  65  having an inner surface  67  and an outer surface  68  is disposed in the opening  63 . The inner surface  67  is positioned above and in opposing relation to the assay chamber  5  and is in fluid communication with the assay chamber  5 . An active surface  13  may be positioned on the inner surface  67  of the analytical substrate  65 . Additionally, the analytical substrate  65  can be made transparent to optical detection means  69 . The optical detection means  69  is external to the analytical device  1 . The optical detection means  69 , scans through the analytical substrate  65  to assay the active surface  13 , as shown by arrow  71 . Suitable materials for the analytical substrate  65  include glass, plastics, fused silica, and quartz. The analytical substrate  65  can be secured in the opening  63  using glue, adhesives, a weld, fasteners, or the like. Preferably, the active surface  13  is positioned at a radially outward portion of the inner surface  67 , so that fluid directed radially outward by rotation  6  of the housing enclosure  3  will be urged into contact with the active surface  13 .  
         [0053]    In alternative preferred embodiments, the active surface  13  includes biochemical, chemical, or biological moieties. The active surface  13  can be disposed on the surface of a structure attached to the inner surface  67  of the analytical substrate  65 , for example, balls, beads, or other structure that can be fixedly attached to the inner surface  67 . The active surface  13  may comprise an array or other orderly structure of biomolecular probes. The biomolecular probes can be DNA, DNA fragments RNA, RNA fragments, reagents, protein, protein fragments, lipids, and lipid fragments. A dye, label, tag, or reagent may be attached or dried onto to the active surface. Alternatively, fluid introduced into the assay chamber  5  may contain a dye, label, tag, or reagent. Preferably, the active surface  13  is disposed at a radially outward position on the assay chamber  5  or the analytical substrate  65 . Rotation  6  of the analytical device  1 , generates a radially outward flow of the fluid, bringing the fluid into contact with the active surface  13 .  
         [0054]    Additionally, the active surface  13  can be disposed on the surface of a structure that is not attached to the analytical substrate  65  or to the interior surface of the assay chamber  5 . For example, the active surface  13  can be disposed on balls or beads loosely contained in the assay chamber  5 . Further, after the conclusion of a reaction, the balls or beads can be extracted from fluid remaining in the valve chamber  23  via the fluid extraction means  49  in valve chamber cap  42  as illustrated in FIG. 2 b  or from fluid captured in the reservoir chamber  33  via the fluid extraction means  35  in the reservoir plate  31  as illustrated in FIG. 1.  
         [0055]    The best mode for making the analytical device  1  is to injection mold the housing enclosure  3 , preferably from plastic. The bias means  29  is a coil spring and the actuator  27  is a stainless steel ball. Preferentially, an o-ring is positioned on the valve seat  25  between the actuator  27  and the valve seat  25 , to supply compliance. The coil spring holds the stainless steel ball against the o-ring of the valve seat  25 .  
         [0056]    In a preferred embodiment of a method according to the present invention, a fluid sample may be introduced into the assay chamber  5  via fluid introducing means  11 . The analytical device  1  is spun at a first rotational speed to generate a radially outward flow of the sample, whereby the fluid reacts with the active surface  13 . The first rotational speed is below the threshold rotational speed necessary to actuate the centrifugal force-activated valve  7 , from the stationary closed state to the open actuated state. The closed valve  7  prevents the sample from exiting the assay chamber  5  through the valve  7 . Spinning at the first rotational speed continues until a predetermined reaction time has passed or the reaction has reached a desired state of completion. After spinning at the first rotational speed, the analytical device  1  is then spun at a second rotational speed greater than the first rotational speed. The second rotational speed exceeds the threshold rotational speed necessary to actuate the valve to the open actuated state. The centrifugal force-activated valve  7  opens and fluid exits the assay chamber  5  through the valve  7 . The introducing step, the first rotational speed step, and the second rotational speed step may be repeated as necessary to complete the reaction.  
         [0057]    In an alternate embodiment, after spinning the analytical device  1  at the second rotational speed, a wash fluid may be introduced into the assay chamber  5  via fluid introducing means  11 . The analytical device  1  is spun at a first rotational speed to distribute the wash fluid. The wash fluid removes un-reacted sample from the active surface  13  and the assay chamber  5 . The analytical device  1  is then spun at the second rotational speed to remove the wash fluid from the assay chamber  5 . The introducing step, the first rotational speed step, and the second rotational speed step may be repeated as necessary to ensure complete washing of the active surface  13  and the assay chamber  5 .  
         [0058]    In another embodiment, the analytical device  1  is spun at the second rotational speed to remove any sample remaining in the assay chamber  5 . The active surface  13  is assayed by scanning the active surface  13  with optical detection means  69 . The optical detection means are external to the analytical device  1 .  
         [0059]    In the most preferred embodiment of a method according to the present invention, the analytical device  1  includes an analytical substrate  65  having an active surface  13  disposed on a radially outward portion. A fluid sample is introduced into the assay chamber  5  via fluid introducing means  11 . The analytical device  1  is agitated to mix the sample and to generate a radially outward flow of the sample, whereby the sample reacts with the active surface  13 . The agitation can be accomplished, for example, by reversing the direction of rotation  6  of the analytical device  1  multiple times. Mixing continues until a predetermined reaction time has passed or the reaction has reached a desired state of completion. The analytical device  1 , is then spun at the second rotational speed to empty the sample from the assay chamber  5 . The introducing step, the mixing step, and the second rotational speed step may be repeated as necessary to complete the reaction.  
         [0060]    In an alternate embodiment, after spinning the analytical device  1  at the second rotational speed, a wash fluid may be introduced into the assay chamber  5  via fluid introducing means  11 . The analytical device  1  is spun at a first rotational speed to generate a radially outward flow of the wash fluid. The wash fluid removes un-reacted sample from the active surface  13 , the analytical substrate  65 , and the assay chamber  5 . The analytical device  1  is then spun at the second rotational speed to empty the wash fluid from the assay chamber  5 . The introducing step, the first rotational speed step, and the second rotational speed step may be repeated as necessary to ensure complete washing of the active surface  13 , the analytical substrate  65 , and the assay chamber  5 .  
         [0061]    In another embodiment, the housing enclosure  3  has a reservoir plate  31  positioned radially outward of the assay chamber  5  and positioned below the centrifugal force-activated valve  7   a . Reservoir plate  31  includes a reservoir chamber  33 . The analytical device  1  is spun at the second rotational speed to remove fluid from the assay chamber  5 . Centrifugal force-activated valve  7   a , opens and fluid exits the assay chamber, enters the valve, and is stored in the reservoir chamber  33 . Optionally, fluid in the reservoir chamber  33  may be removed via fluid extraction means  35 . A syringe or the like may be used to withdraw fluid from the reservoir chamber for further analysis or other use.  
         [0062]    In another embodiment, the analytical device  1  is spun at the second rotational speed to remove any sample remaining in the assay chamber  5 . The analytical substrate  65  is transparent to optical detection means  69 . The active surface  13  is assayed by scanning the active surface  13  through a transparent portion of the analytical substrate  65  via the optical detection means  69 . The optical detection means are external to the analytical device  1 .  
         [0063]    Although several specific embodiments of the invention have been described and illustrated, the invention is not limited to the specific forms or arrangements of parts so described and illustrated. The invention is only limited by the claims.