Abstract:
A system for holding a slide. The system includes a housing having a side wall and a top. The top includes a recess surrounded by an outer rim. The system also includes an inlet port in communication with the recess and an elevating mechanism capable of receiving the slide and for raising the slide toward the top of the housing to engage the slide with the outer rim to form an analytical cavity. Together these elements form an analytical cavity in which the assay may be performed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Serial No. 60/347,040, filed Jan. 9, 2002, and provisional patent application Serial No. 60/381,196 filed May 17, 2002. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    This invention relates to an improved cassette for holding and applying reagents to a slide that bears samples to be assayed, and methods for use of the cassette.  
         SUMMARY OF THE INVENTION  
         [0003]    An embodiment of the present invention relates to a system for holding a slide. The system includes a housing having a side wall and a top. The top includes a recess surrounded by an outer rim. The system also includes an inlet port in communication with the recess and an elevating mechanism capable of receiving the slide and for raising the slide toward the top of the housing to engage the slide with the outer rim to form an analytical cavity. Together these elements form an analytical cavity in which the assay may be performed.  
         BACKGROUND  
         [0004]    Processing of biological samples on glass slides has a long history. Compared to the relatively simple dyes and stains of previous years, many newer techniques for analysis are significantly more complex and the reagents considerably more expensive. Immunoassays, hybridization assays, and in situ nucleic acid amplification assays are particularly demanding in terms of reagent expense, need for accurate timing, and need for precise temperature control. These are particularly demanding because the reagents should be applied in a precisely controlled thickness. Further, some of these assays involve heating of the slide and reagents to produce enzymatic reactions, yet the reagents must not evaporate during the procedure. In addition, it is desirable to have the assays performed automatically to whatever extent is possible, both to save cost and for increased reliability and precision. 
       
    
    
     DESCRIPTION OF THE FIGURES  
       [0005]    For the present invention to be understood clearly and readily practiced, the present invention will be described in conjunction with the following figures, wherein:  
         [0006]    [0006]FIG. 1 is an exploded diagram that illustrates a cassette according to an embodiment of the present invention;  
         [0007]    [0007]FIG. 2 is a plan view of a cassette according to an embodiment of the present invention;  
         [0008]    [0008]FIG. 3 shows a cross sectional view of a cassette according to an embodiment of the present invention;  
         [0009]    [0009]FIG. 4 is an exploded diagram of a check valve according to an embodiment of the present invention;  
         [0010]    [0010]FIG. 5 is an isometric diagram of an elevator plate for heating or cooling a slide directly below the array according to an embodiment of the present invention; and  
         [0011]    [0011]FIG. 6 is a flow diagram that illustrates an exemplary method for using a cassette in accordance with the present invention.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0012]    An embodiment of the present invention relates to an improved cassette for holding and applying reagents to a microscope slide that bears samples to be assayed. Generally, the cassette may be configured as a small, box-like housing having top, side, and end walls and an opening into which a slide carrying a specimen to be investigated is inserted. The term “analytical cavity,” as used herein, refers to a sealed, shallow space formed between the top, side, and end walls. The term “assay” refers to either assay reagents or fluid applied to a slide that may carry analytical reagents in spots or zones that capture or react with elements of a specimen. For example, an array of nucleic acid spots can either be individual samples to be assayed with a probe or combination of probes. As another example, the array can be used to detect the presence of certain sequences in the solution in the cavity, and thus the solution is the sample. Either type of assay is accessible with the cassette of the invention. Finally, the term “analyte” refers to any material that is subject to analysis including any biological material comprising, for example, one or more of a nucleic acid, a protein including a peptide, a carbohydrate, a lipid or metabolite or other small biological molecule or biological structure such as an organelle, a cell or a tissue.  
         [0013]    The cassette may be designed, for example, to hold the slide in a manner that creates a shallow, sealed analytical cavity over the slide surface to facilitate application and aspiration of a sequence of liquid solutions to a specimen on the slide. In that regard, the cassette may be adapted for applications involving, for example, DNA microarray hybridization, immunohistochemical staining, or any technique or procedure that involves interaction of a thin film of fluid with a lamina. The cassette may also be useful in the automated handling and processing of samples during chemical, clinical, biochemical, or molecular biological analysis or in the creation of analytical structures including, for example, a gel for electrophoresis.  
         [0014]    [0014]FIG. 1 is an exploded diagram that illustrates the basic components of a cassette  5  according to an embodiment of the present invention. As shown, cassette  5  comprises a small box-like housing  12  that includes, in large part, rectangularly arranged side walls  14  and end walls  16 , a slide  10 , a transparent top wall  18  (also called “lens  18 ”), a rectangular gasket  24 , an elevator plate  30 , an elevating mechanism shown generally as  32 , an input port  42 , and an output port  44 . One of the end walls  16  may have an opening  20  through which slide  10  carrying a specimen may be inserted. Opposite end wall  16  may include a similar opening  39  through which slide  10  may be manipulated. In operation, elevating mechanism  32  raises slide  10  upwardly toward top wall  18  of housing  12  and into sealed engagement with peripheral gasket  24 , thus defining a sealed, shallow analytical cavity  29  (shown in FIG. 3) between top wall  18  and slide  10 , which may be approximately 0.001 to 0.002 inches deep. The shallow depth of the analytical cavity may be designed to ensure that the liquid in the chamber will be maintained at a uniform thickness and that gas bubbles will not form unless intended as a byproduct of the chemical reaction.  
         [0015]    Lens  18  may be attached to housing  12 , either during manufacture or subsequently, by permanently bond or other attachment means including, for example, gluing, welding, ultrasonic welding, stamping, crimping, press fitting, solvent bonding, brazing, affixation with fasteners, snap fitting, and similar methods as known in the art. According to another embodiment, the lens may be formed as part of the frame during its manufacture.  
         [0016]    Lens  18  may comprise any material compatible with the assay to be performed in the cavity such as a plastic, metal, ceramic, fibrous composite, or any combinations thereof. Lens  18  may be coated to protect the assay from the underlying material. To maintain the surface of the lens that forms the cavity in a planar fashion, lens  18  may be formed by methods that minimize the residual strain or stress in the lens material, such as sequential-compression injection molding.  
         [0017]    Elevating mechanism  32  includes a substantially sinusoidal-shaped leaf spring  34  and a slide release  36  (also referred to as a “sliding spring lock  36 ”). Spring  34  is located between pressure plate  30  and slide release  36 . Slide release  36  has an upper surface with a complementary shape to that of leaf spring  34 . Slide release  36  may be moved into and out of housing  12  through opening  39  in end wall  16  of housing  12 . Contours of leaf spring  34  and slide release  36  are formed so that when slide release  36  is pulled outwardly, an alignment interface exists between slide release  36  and spring  34 , allowing spring  34  and elevator plate  30  to drop to a lowered position within substantially sinusoidal shape of slide release  36  and release slide  10 . In the lowered position, cassette  5  is receptive to insertion or removal of slide  10  through aperture  20 . When slide release  36  is pushed back into housing  12 , a non-alignment interface exists between the release  36  and spring  34 , causing elevator plate  30  to move upwardly to press slide  10  against gasket  24  and a ledge or rim  28 , shown in FIG. 3, formed about the perimeter of the lens  18 . Slide release  36  includes a wedge-shaped finger  38  that is engageable with an aperture on a U-shaped stirrup  40  that extends downwardly from the pressure plate  30 . Stirrup  40  extends through a slot  43  in spring  34 . When slide release  36  is withdrawn, finger  38  engages stirrup  40 , to pull elevator plate  30  downwardly to avoid any possibility of the elevator plate becoming stuck.  
         [0018]    According to one embodiment, lens  18  may have significant transmittance in at least one wavelength band or region of the spectrum or may otherwise be compatible with measurement of a property of the cassette or an analyte by any desired method, so that the assay can be observed, read or controlled without opening the cassette. According to another embodiment, the cassette may incorporate a viewing hole through housing  12  or alternatively through spring  34 , elevator plate  30 , and slide release  36 , or through all of these, that will permit observation of slide  10 . Any analytically useful means of observation is potentially useable with the cassette. Electromagnetic radiation of any wavelength may be used for such observation, including (but not limited to) infrared, visible and ultraviolet light.  
         [0019]    According to another embodiment, cassette  5  may incorporate probe windows (not shown) through, for example, spring  34  and elevator plate  30  that allow the temperature of slide  10  to be measured either directly with a probe, indirectly with an infrared sensor or similar device, or other means for measuring or otherwise inferring the characteristics of the assay. Other probes of analytical cavity  29  and/or slide  10  include, without limitation, ultrasonic and other pressure waves, techniques such as fluorescence, fluorescence polarization, phosphorescence, thermoluminescence, emission or absorption of ionizing radiation, conductivity, magnetic effects, electrostatic effects, and other suitable methods for probing analytical cavity  29 .  
         [0020]    Valved inlet and outlet ports  42  and  44 , which may be secured to lens  18 , enable selected fluids to be admitted into and aspirated from analytical cavity  29  while minimizing evaporation of a liquid, even when cassette  5  is heated such as during an incubation period. At least one port is equipped with a one-way or check valve that may prevent bubble entrapment during exchange of reagents. Input port  42  includes, among other things, an elastic outer seal  46  that forms an airtight engagement with a cannula or similar device used to inject liquid into cassette  5 . Although cassette  5  includes ports  42  and  44  on lens  18 , those of ordinary skill in the art will appreciate that ports  42  and  44  may be placed in any convenient location that communicates with the interior of analytical cavity  29 . Ports  42  and  44  are described in greater detail below in connection with FIG. 4.  
         [0021]    Another embodiment of the slide release, similar in many respects to slide release  36 , incorporates a ramp-like feature so that when the slide release is pulled away from housing  12 , the slide release may contact the bottom of elevator plate  30  and pull elevator plate  30  downward. Pushing the slide release into housing  12  reverses this process and again forces elevator plate  30  upward, sealing analytical cavity  29  if slide  10  is present.  
         [0022]    Although cassette  5  is equipped with spring  34 , those of ordinary skill in the art will appreciate that other lift means may be suitable, including a wedge, clamp, cam, lever, or piston (or similar device driven by hydraulic or pneumatic force or electricity). Those of ordinary skill will also appreciate that the compressive force could be applied by one lift means and retained by others, such as a strip of adhesive or a pin-type interlock.  
         [0023]    Gasket  24  can be made of any suitable material that maintains a desired degree of resilience at the temperatures and pressures of the assay, which may range from about −20 to about 100 degrees C., and generally less than about 1 bar above ambient pressure. According to an embodiment, a groove  26  is formed during the initial molding of lens  18  by insert molding (see FIG. 2). Outlet holes running through lens  18  to the bottom of groove  26  serve both as exit pathways during molding and, after the gasket material is cooled, as retainers of gasket  24 . FIG. 1 illustrates these holes around the periphery of lens  18 .  
         [0024]    Cassette  5  may comprise any suitable material including, for example, plastic, metal, or any combination thereof. Metal may be used when, for example, heat is to be conducted. Any of the standard fabrication techniques may be used to make the parts, including cutting, stamping, casting, machining, press-forming, molding, and injection molding.  
         [0025]    [0025]FIG. 2 illustrates the underside of lens  18  according to an embodiment of the present invention. As shown, the underside of lens  18  includes a slightly recessed area  22 , groove  26  formed at the margin of recessed area  22 , an input channel  58 , an output dam  60 , and an output channel  62 . Input port  42  is located to the left in FIG. 2. Groove  26  is designed to receive elastic rectangular gasket  24 , either permanently or removably, so that the lower edge of gasket  24  projects downwardly beyond ledge  28  (see FIG. 3). The device may be designed so that when slide  10  is urged upwardly toward lens  18 , the peripheral margin of the upper surface of slide  10  will engage ledge  28  and, in doing so, will engage and compress gasket  24 , effecting a seal between the slide  10  and the gasket  24 . Engagement of slide  10  with ledge  28  defines, with precision, the spacing between the upper surface of slide  10  and the underside of the lens  18  and, therefore, the depth of analytical cavity  29 . The underside of the lens  18  is configured to reduce the incidence of bubbles during fluid injection.  
         [0026]    Input channel  58  may be cut or molded into the inner surface of lens  18  to control the flow of liquid into analytical cavity  29 . Liquid enters the cavity through input port  42 . It first fills channel  58 . As more liquid is admitted, the liquid begins to flow along and towards the opposite end of analytical cavity  29  as a uniform wave front. The wave front advances through the cavity, until it meets output dam  60 , a curved narrow ridge that protrudes slightly from inner surface  22  of lens  18 . Dam  60  forces the flow of the liquid towards the sides and corners of the cavity. Bubbles that otherwise might be trapped in the corners of the cavity are swept into output channel  62 . Output channel  62  is tapered at the corners to facilitate the movement of liquid and bubbles to output port  44 .  
         [0027]    [0027]FIG. 3 is a longitudinal section view of cassette  5  according to an embodiment of the present invention. Elevator plate  30  may be moved between a lower position, in which slide  10  can be placed on its upper surface when slide  10  is inserted through insert opening  20 , and an elevated position in which slide  10  is pressed upwardly into engagement with the ledge  28  and into sealed relation with gasket  24 . Elevator plate  30  is raised and lowered by elevating mechanism  32 .  
         [0028]    [0028]FIG. 4 illustrates the components of an exemplary valve assembly  48  that may be integrated into each port  42  and  44  according to an embodiment of the present invention. Valve  48  includes an input section  50 , an output section  52 , and an elastic septum  54 . Input and output sections  50  and  52  are made of a rigid material, having little resilience at the moderate forces used in the device. The septum is fabricated from an elastic material. Septum  54  is captured and sealed about its peripheral margin between input and output sections  50  and  52  and is stretched over a convex surface of input section  50  during assembly. An inner face of output section  52  is recessed to enable septum  54  to bow sufficiently to unblock an inlet passage  53  and enable flow through a plurality of circumferential holes  51  in septum  54  and outlet passage  55  of output section  52 . Reverse flow of liquid or gas (to prevent evaporation) is blocked by the central portion of septum  54  that is biased, by its inherent elasticity, against inlet passage  53 . Forward flow through valve  48  begins when a sufficient pressure differential is developed across valve  48  to cause the center of septum  54  to bow away from inlet passage  53 . The pressure differential may result from positive pressure of the fluid being emitted to the inlet side of valve  48  or suction applied to the outlet side. The elastic characteristic of the material of septum  54  as well as the thickness and the degree to which septum  54  is stretched determine the cracking pressure of valve  48 .  
         [0029]    The pressure differential may be, for example, between 2 and 5 psi. The upper desirable pressure limit will be determined by the degree to which slide  10  bends under the pressure or vacuum. The lower limit is indicative, to some extent, of quality of the seal because a higher backpressure can diminish reagent loss during incubation.  
         [0030]    Valve  48  may be directly secured to lens  18  to minimize dead volume, and therefore reagent waste. According to one embodiment, positioning output section  52  very close to septum  54  may minimize dead volume. Additionally, to prevent blockages, the inner surface of output section  52  may include shallow grooves that radiate from the outlet hole, which carries fluid from the holes in septum  54  to the outlet hole. Valves  48  may be oriented to allow a flow of liquid through analytical cavity  29  from inlet to outlet so that, when an initial or a replacement reagent passes through analytical cavity  29 , significant mixing will not occur.  
         [0031]    According to an embodiment, outlet port  44  of cassette  5  may be arranged to require a greater cracking pressure than that of inlet port  42  in order to present a continuous back pressure opposing the injection of liquid into the cavity. The back pressure prevents capillary action that might otherwise result between the closely spaced surfaces of lens  18  and slide  10 . If, during the liquid injection process, capillary action were permitted to draw liquid through the analytical gap, bubbles might develop within the liquid layer. The increased cracking pressure of outlet port  44  may be the result of the selection of septum thickness or material or may be provided by connecting output port  44  to an external flow restriction device.  
         [0032]    According to an embodiment, tubing may be connected to valves  48  to supply and receive liquid. For example, a tubing attachment nipple may be included on the inlet or outlet side of valve  48  during fabrication (not shown). Alternatively, the exposed surfaces of valves  48  may be designed so that a robotic connector can mate with them and form a simple connection that is sealed by a slight force of the connector against inlet or outlet ports  42  and  44 .  
         [0033]    [0033]FIG. 5 is an isometric diagram of an elevator plate  500  for heating or cooling a slide directly below the array according to an embodiment of the present invention. Elevator plate  500  is similar to elevator plate  30  in many respects except that elevator plate  500  includes a gas injection port  502 , a gas exhaust port  504 , and a beveled edge  506  that may facilitate insertion of slide  10 . In operation, elevator plate  500  raises slide  10  upwardly toward top wall  18  of housing  12  against peripheral gasket  24  and rim  28 , leaving a gap of approximately 0.010 inches between slide  10  and elevator plate  500 . Gas injection port  502  and gas exhaust port  504  communicate with the gap so that a gas can flow beneath slide  10  for heating or cooling by either forced or natural convection. According to another embodiment, the gap between slide  10  and elevator plate  500  may be designed to accommodate liquids of various temperatures.  
         [0034]    Variations in the height of analytical cavity  29  may create undesirable flow characteristics during the process of introducing liquids into this cavity. Chemical reactions within this cavity may often become diffusion limited, making local variations in the cavity&#39;s height important. An analytical cavity of uniform depth may contribute to uniform flow characteristics through the cavity.  
         [0035]    During incubation, cassette  5  may be heated by direct infrared radiation or by placing a ferrous metal plate at the top of elevator  32  directly below slide  10  and using inductive heating. Alternately, slide  10  may be heated by injecting warm gas through the cavity while monitoring the temperature of slide  10  through a viewing port formed in elevator plate  30 . According to another embodiment, slide  10  may be heated by placing the whole assembly in an oven or other controlled temperature space, such as for long incubations or overnight hybridization. Additionally, liquids in analytical cavity  29  may be mixed or agitated by mechanical, ultrasonic, or other mixing means to improve the quality of the reaction.  
         [0036]    In many assays, liquids must be introduced into the cassette, incubated, removed, and, optionally rinsed. To accomplish this in a manner that is reproducible, the cassette must allow liquids to be applied to the surface of the slide in a uniform manner. In particular, application of liquids should be without the formation of gas bubbles during the application step. However, bubble formation may be a byproduct of chemical reactions between or among samples and reagents. The cassette must allow the liquid to be removed from the surface of the slide and other applications of the same or of a different liquid to be made without violating the above requirements. The removal of liquids from the cassette may be accomplished using a vacuum source, or by pressure applied to the inlet side of the cassette. According to an embodiment, the cassette may be prepared for a second liquid by flushing the first liquid with an inert liquid, such as a saline solution.  
         [0037]    Any suitable method may be used to add liquids to analytical cavity  29  or to remove them. For example, when liquid or gas is injected into the inlet port  42  of cassette  5 , the fluid pushes against septum  54 , thus distorting its shape. This process opens paths for liquid or gas to flow through valve  48 . Applying a vacuum to outlet port  44  produces the same result. As noted above, elastic characteristics of the septum&#39;s material and the thickness of septum  54  determine the overall backpressure of valve  48 . Equivalent considerations apply to other one-way valve designs, such as the one-piece “duckbill” valve and other forms. For example, it is possible to adjust opening force by adjusting spring strength in a “poppit and spring” type of check valve, or by varying the length of the slit or the thickness of the plastic in a duckbill-type valve.  
         [0038]    Cassette  5  of the present invention may be assembled in any suitable manner, such as by placing slide release  36  and spring  34  into housing  12 , and then elevator plate  30 . Independently, gasket  24  may be formed in groove  26 , and valves  42 , and  44  may permanently joined to the lens. Next, lens  18  may be placed into the top of housing  12  and permanently bonded thereto. Finally, cassette  5  may be package and sterilized, if required.  
         [0039]    [0039]FIG. 6 illustrates an exemplary method  600  for using cassette  5  in accordance with the present invention. An array of spots containing DNA to be tested (step  602 ) is printed on a glass slide (step  604 ) and dried and baked sufficiently long to ensure adhesion. The slide is inserted into a cassette of the invention in step  606 , and the cassette is closed in step  608 . The cassette is then transported to a workstation and placed in line for processing. In the processing station, an injector and an evacuation line are pressed against the inlet and outlet ports of the cassette in steps  610  and  612 .  
         [0040]    The plate is first washed with a series of buffers to hydrate the sample and partially denature it in step  614 . The evacuator evacuates the cavity for 5 seconds, and then the injector injects enough of the first wash solution to fill the cavity. This step is repeated twice, and then the cassette is incubated with the wash fluid for a preset period. The same procedure is applied with a second buffer, and then a third wash with hybridization buffer, each injected by a different injector connected to a supply of the wash solution.  
         [0041]    Next, the cassette is evacuated in step  616 , and the hybridization buffer containing the labeled probe is injected into the cassette enough to fill the cavity. The cassette is placed in a humidified incubator at 42 deg. C (or a different temperature, depending on the particular hybridization and desired degree of stringency) for 12 to 16 hours. Humidification reduces the driving force for evaporation.  
         [0042]    After hybridization is completed, the cassette is rinsed to be free of the probe, using 3 changes of wash buffer, i.e. with evacuation followed by filling with the more buffer. Then the cassette is cooled, and rinsed similarly with other buffers and evacuated. The cassette is dried by forcing warm dry nitrogen gas through the analytical cavity at a convenient rate, for example about 100 microliters/sec, for a time known to be long enough to dry the slide. The entire cassette, with the nitrogen retained by the backpressure-retaining valves, is placed in a dark place until later analysis is performed. The absence of oxygen and of light may be important to preserve the fluorescent probes typically used in such procedures.  
         [0043]    Typically, the slide will be removed from the cassette in step  618  and placed directly in a standard fluorescence reader capable of reading the particular spot size and array size used in the particular assay. The cassette may be cleaned or discarded in step  620  to prevent any possibility of cross contamination with another assay, before ending process  600  in step  622 .  
         [0044]    Similar procedures can be devised for immunoassays or other assays involving proteins or carbohydrates, or other biological material including cells, tissues and organelles; and for binding assays of any sort, not necessarily biomedical. The ability of the cassette to remain sealed allows anaerobic assays to be conducted easily. Moreover, the ability to rapidly replace reagent solutions is an advantage in many situations, including kinetic analysis. For example, the three-fold exchange rinse described above can be done in substantially less than one second, with appropriate machinery.  
         [0045]    The precision of the thickness and humidity control inherent in these cassettes can also be useful in related assays requiring a support. For example, a thickness of 25 microns is suitable for thin layer electrophoresis, which could be conducted in these cassettes by providing for multiple sample injection ports or by providing samples in a porous material fixed to the slide, and then flowing in an electrophoretic separation medium, either of the gelling or the non-gelling type. Electrodes would be fitted into the lens specifically for this purpose. Voltage would then be applied to these electrodes for the purpose of electrophoretic separations.  
         [0046]    The result and process would be generally similar to results obtained with “capillary” electrophoresis, as the thickness can be made to fall within the same general range. Thin electrophoretic layers below about 250 microns (0.25 mm) in thickness can be difficult to cast. Capillaries in present use are generally in the range of about 40 to about 100 microns. Therefore, as suitable range for cavity thickness in electrophoresis is in the range of about 10 to about 250 microns.  
         [0047]    The foregoing description has been limited to a few specific embodiments of the invention. It will be apparent, however, that variations and modifications can be made to the invention, with the attainment of some or all of the advantages of the invention. Therefore, it is the object of the appended claims to cover all such variations and modifications as come within the true spirit and scope of the invention.