JP2011524016A - Systems and methods for hybridization slide processing - Google Patents

Abstract

Disclosed is a system 300 for substantially automated hybridization of a plurality of microarray slides. The system includes a container 310 that is disposed in the cleaning disk on a support shaft 318 for receiving a cleaning disk 312 having an open top end and a plurality of microarray slide substrates 362. And an upper clamp rotor 340 disposed on the support shaft above the lower carrier rotor for receiving a plurality of disposable chamber assemblies 240. The system further includes lowering the upper clamp rotor to engage the lower carrier rotor to attach the plurality of chamber assemblies to the plurality of slide substrates thereby forming a plurality of sealed reaction chambers 244. And removing the plurality of chamber assemblies from the plurality of slide substrates, thereby lifting the plurality of reaction chambers from a sealed state by raising and removing the upper clamp rotor from the lower carrier reader. As designed.
[Selection] Figure 1

Description

  The field to which this invention belongs generally relates to the processing of hybridization slides for the analysis of immobilized DNA samples.

BACKGROUND OF THE INVENTION AND RELATED TECHNIQUES Hybridization slide processing and analysis, such as fluorescent in situ hybridization (FISH), is a well-known technique for detecting the presence of specific nucleic acids in a given sample. This technique generally involves immobilizing a known nucleic acid sequence probe on a glass slide and then introducing sample media into the probe to determine if the sample contains any complementary nucleic acid sequences. A fluorescent indicator can be attached to the sample medium so that the hybridized sample can be examined or analyzed later using a fluorescence microscope or similar slide reader. If a matching sequence is found, the fluorescent indicator appears to confirm the match.

  Hybridization slides are often used for the analysis of DNA samples, but may be used for diagnostic testing of other types of samples. Probe positions in the microarray are formed from a variety of large biomolecules such as DNA, RNA, and proteins, small molecules such as drugs, cofactors, signaling molecules, peptides or oligonucleotides. Typically, a known reactant is immobilized on a substrate, an unknown liquid sample is exposed to an immobilized reactant, the reaction product is examined, and the sample is characterized, but one or more unknown samples are deposited on the substrate. It is also possible to fix them on top and expose them to a liquid containing one or more known reactants.

  Processing of hybridization slides for later analysis typically requires a significant number of processing steps, including a reaction chamber around a portion of the slide that contains an array of immobilized reactant probes. Forming, filling the reaction chamber with a mobile reactant sample in solution, hybridizing the sample with the probe during the incubation step, and without damaging the hybridized reactant sample at the end of the incubation period , Washing and removing unhybridized liquid sample from the microarray slide. While attempts have been made to mechanize one or more of the above steps, to date automation of the entire hybridization process has yielded mixed results with respect to the quality of the exposed microarray slides, or is extremely expensive. is there. A number of the steps still require a number of manual operations to ensure that high quality hybridized microarrays will be available for later analysis.

  Each processing step also requires complex and specialized processing equipment and methods. For example, it is often desirable that reactions performed on a microarray consume only a minimal amount of hybridization sample solution because only a limited sample is available. However, if only a small amount of hybridization solution is diffused over the area of the microarray, the liquid layer will be very thin and if not mixed, the sample solution will be depleted locally on the spot where the specific sequence binds the sequence. There is a possibility that it will be in the state. As the target sample is depleted, the reaction kinetics are slow and only a small signal is produced. This is a major problem with small arrays. It may be particularly desirable for hybridization to be performed with mixing in a small volume reaction chamber. The small volume allows for a high concentration of reactants with limited feed, and mixing maintains the initial reaction rate and therefore produces a larger amount of reaction product.

SUMMARY OF THE INVENTION In the invention embodied and broadly described herein, the present invention includes a hybridization unit for providing a hybridization reaction chamber on a microarray slide. The hybridization unit includes a microarray slide substrate having a reaction region carrying an immobilized reaction product. The slide substrate is substantially rectangular and may have two exposed parallel edges for connection to the carrier fixture of the processing device. A disposable chamber assembly or “mixer” is removably attached to the slide substrate to form a sealed small volume reaction chamber that encloses the reaction region. The chamber assembly or mixer is made of a plastic or polymer material and may be disposable. The hybridization unit further includes coupling means for attaching the disposable chamber assembly to the clamp fixture of the processing device, whereby the disposable chamber assembly is detached from the slide substrate by separating the clamp fixture from the carrier fixture, thereby A sealed reaction chamber is opened.

  The disposable chamber assembly may further include a flexible base layer having a top surface and a bottom surface, the bottom surface forming the top surface of the reaction chamber, and the weakly adhesive gasket seal extending downward from the bottom surface of the base layer, The connection means includes a strongly adhesive top patch extending from the top surface of the base layer for connection to a clamp fixture of the processing device.

  The disposable chamber assembly should be designed with a boundary extending beyond the two parallel edges of the slide substrate to allow attachment of the disposable chamber assembly to the upper clamp fixture of the processing device. You can also. The slide substrate and disposable chamber assembly is further designed to expose the other two parallel edges of the slide substrate for attaching the slide substrate to the lower carrier fixture of the processing device.

  The disposable chamber assembly or mixer may further include a manifold coupled to the exposed surface of the disposable chamber assembly having fill holes and vents aligned with the fill port and vent of the disposable chamber assembly.

  In the invention embodied and broadly described herein, the invention further includes a system for substantially automated hybridization of a plurality of microarray slides. The system receives a basin-type container having an open top end, a lower carrier rotor disposed on a support shaft within the basin-type container for receiving a plurality of microarray slide substrates, and a plurality of disposable chamber assemblies or mixers. And an upper clamp rotor disposed on the support shaft and above the lower carrier rotor. The system is designed such that a plurality of chamber assemblies are attached to a plurality of slide substrates by lowering the clamp rotor to engage the carrier rotor, thereby forming a plurality of sealed reaction chambers. The system is further designed to lift the upper clamp rotor and remove it from the lower carrier rotor, thereby removing the multiple chamber assemblies from the multiple slide substrates, thereby opening the multiple reaction chambers from the sealed state. Is done.

  The present invention also includes the step of inserting a plurality of microarray slides into a processing device, wherein each of the microarray slides has a reaction area covered by a small volume reaction chamber assembly or mixer Including a method of processing. The method continues with filling the reaction chamber with a small amount of hybridization solution to hybridize each reaction region of the microarray slide. The method includes exposing a hybridized reaction region by removing the reaction chamber assembly from each of the microarray slides, washing the microarray slide with a wash bath shared bath, removing the wash solution from the microarray slide, and the microarray slide. Further comprising removing from the processing device.

  The present invention also includes a method for in situ processing of microarray slides for analysis of immobilized samples. The method includes obtaining a slide substrate having a reaction region carrying an immobilized sample and placing the slide substrate in a processing device for automated in situ processing. The in situ processing includes forming a small volume reaction chamber that encloses the reaction region by attaching a disposable chamber assembly or mixer to the slide substrate, filling the reaction chamber with a hybridization solution for reacting with the immobilized sample, During the incubation, the reaction chamber is sealed to prevent contamination, the small volume reaction chamber is opened by removing the mixer from the slide substrate to expose the reaction area, and a large volume of washing solution is flowed through the reaction area. The method further includes the step of removing the hybridization solution and the step of removing the washing solution from the slide substrate.

  Other aspects of the methods of the invention include increasing the reaction with the immobilized sample on the microarray slide by agitating the hybridization solution in the reaction chamber, and a plurality of filler and vent holes in the mixer / manifold assembly. Sealing the reaction chamber by removably inserting the valve pin.

  The invention also includes a post-treatment method for hybridized slides that have been washed away with a wash solution to remove the hybridization solution. The method includes reconnecting a disposable chamber assembly or mixer to a slide substrate to re-form a small volume reaction chamber that encloses the hybridized reaction region, and nucleic acid denaturation on the hybridized and washed microarray slide. And performing various fluidics steps such as recovery.

  In another embodiment of the present invention, instead of removing the mixer from the slide substrate and flushing the reaction area with a large volume of wash solution, the elution buffer is slowly pumped into the reaction chamber to wash the reaction area and the original hybridization solution. Replace the sample, extrude it and collect with a suitable collection device located under the slide substrate. The reaction chamber is then resealed and reheated for a secondary processing step, after which additional elution buffer is pumped through the reaction chamber and the reaction is flushed into another collection device for additional analysis.

BRIEF DESCRIPTION OF THE DRAWINGS Features and advantages of the present invention will become apparent from the following detailed description. The detailed description is to be construed in conjunction with the accompanying drawings, which together illustrate the features of the invention. It is understood that these drawings depict only typical embodiments of the invention and are therefore not to be construed as limiting its scope. Further, it will be readily appreciated that the components of the present invention can be arranged and designed in a wide variety of different configurations, as generally described and illustrated in the drawings herein. However, the accompanying drawings will be used to describe and explain the present invention with additional specificity and detail.

FIG. 1A shows a top view of a disposable reaction chamber assembly for forming a sealed reaction chamber for a reaction zone carrying an immobilized reactant, in accordance with an exemplary embodiment of the present invention. FIG. 1B shows a side cross-sectional view of the disposable reaction chamber assembly of FIG. 1A along section line AA. FIG. 1C shows the plurality of disposable reaction chamber assemblies of FIG. 1A installed on a strip. FIG. 2A shows a side cross-sectional view of another exemplary embodiment of a disposable reaction chamber assembly. FIG. 2B shows a side cross-sectional view of another exemplary embodiment of a disposable reaction chamber assembly. FIG. 3A shows a side cross-sectional view of another exemplary embodiment of a disposable reaction chamber assembly. FIG. 3B shows a side cross-sectional view of another exemplary embodiment of a disposable reaction chamber assembly. FIG. 4 illustrates a method for forming a sealed reaction chamber on a hybridization slide and then filling the chamber with a small amount of hybe solution according to an exemplary embodiment of the present invention. FIG. 5 illustrates a method for applying a small amount of hybe solution and then forming a sealed reaction chamber on a hybridization slide, according to another exemplary embodiment of the present invention. FIG. 6 illustrates a system for semi-automated hybridization of multiple hybridization slides according to an exemplary embodiment of the present invention. FIG. 7 illustrates a method of attaching a plurality of hybridization slides to the hybridization system of FIG. FIG. 8 illustrates a method of applying a small amount of hybe solution to form a sealed reaction chamber on a plurality of hybridization slides mounted in the hybridization system of FIG. FIG. 9 illustrates a method for assembling the hybridization system of FIG. 6 prior to performing a hybridization protocol. FIGS. 10A-10D together illustrate a method of processing multiple hybridization slides in accordance with the exemplary embodiment of the present invention with the hybridization system of FIG. FIG. 11A shows a top view of a hybridization unit of an exemplary embodiment of the present invention. FIG. 11B shows a cross-sectional side view of the hybridization unit of FIG. 1 along section line BB. FIG. 12 shows a perspective view of a system for automated hybridization of multiple hybridization slides in accordance with another exemplary embodiment of the present invention. FIG. 13 shows a top view of the hybridization system of FIG. FIG. 14 shows a cross-sectional side view of the hybridization system of FIG. FIG. 15 shows an exploded view of the hybridization system of FIG. FIG. 16A shows a cross-sectional side view of the rotor in the engaged position. FIG. 16B shows a sectional end view of the rotor in the engaged position. FIG. 17A shows a side cross-sectional view of the rotor after lifting the upper rotor and separating the mixer and slide substrate. FIG. 17B shows a cross-sectional end view of the rotor after lifting the upper rotor and separating the mixer and slide substrate. FIG. 18A shows a side cross-sectional view of the rotor in the cleaning position. FIG. 18B shows a cross-sectional end view of the rotor in the cleaning position. FIG. 19 shows a perspective view of an automated hybridization system of another exemplary embodiment of the present invention. FIG. 20 shows an exploded view of the hybridization system of FIG. FIG. 21 shows an exploded perspective view of a hybridization system of yet another exemplary embodiment of the present invention. FIG. 22 shows a detailed view of one aspect of the hybridization system of FIG. FIG. 23 illustrates a method of attaching a plurality of hybridization units to the hybridization system of FIG. 6 according to another exemplary embodiment of the present invention. FIG. 24 illustrates a method of assembling the hybridization system of FIG. 23 prior to performing the hybridization protocol. FIGS. 25A-25D together illustrate a method of processing multiple hybridization slides with the hybridization system of FIGS. FIG. 26 illustrates a hybridization step of a method for mixing a plurality of hybridization slides attached to a hybridization system using gravity-induced bubble mixing according to another exemplary embodiment of the present invention. Show.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used herein to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Modifications and further modifications of the features of the invention set forth herein, as well as additional applications of the principles of the invention set forth herein, and those of ordinary skill in the relevant arts who receive this disclosure Anything obvious is to be considered within the scope of the present invention.

  The inventors have recognized that it is beneficial to follow the hybridization step of the hybridization process with a washing step in which the volume is much higher than the volume used for hybridization. Also, in certain circumstances, the inventors have recognized that it is beneficial to perform a large volume wash step prior to the hybridization process and to clean and prepare the hybridization slide prior to hybridization. Accordingly, FIGS. 1-10 illustrate various exemplary embodiments of semi-automated hybridization slide processing systems and methods, which include a large volume pre-hybridization wash step, a small volume hybridization step and a large volume. A post-hybridization wash step may be included. Alternatively, FIGS. 11-22 illustrate various exemplary embodiments of a substantially automated hybridization slide processing system and method that includes a small volume hybridization step and a large volume post-hybridization wash step. May include. FIG. 23 illustrates an exemplary hybridization system and method for mixing a plurality of small volume hybridization slides using gravity-induced bubble mixing, the hybridization step being semi-automated or substantially Can be included in an automated slide processing system.

  The hybridization system of the present invention may include various exemplary embodiments of a hybridization unit having a disposable reaction chamber assembly 30, some of which are shown in more detail in FIGS. Referring briefly to FIGS. 4 and 5, the hybridization unit 10 comprises a substantially rectangular glass slide substrate 20 having a sample or reaction region 22 carrying an immobilized reaction 24 such as an immobilized DNA sample and a tissue section. May be included. The reaction region may be covered by a disposable reaction chamber assembly 30 that is removably attached to the slide substrate 20 to form a small volume reaction chamber 26 that encloses the reaction region 22; During processing, a hybridization (“hybe”) solution can be carried. Together, the rectangular glass slide substrates 20 attached to the chamber assembly 30 can form the hybridization unit 10.

  Returning to reference to FIGS. 1A-1B, in one aspect, the chamber assembly 30 may include a flexible base layer 40 having a seal gasket 42 extending downwardly from the bottom surface of the base layer, the bottom surface of the base layer being a small volume reaction. The top surface of the chamber 26 is formed, and the thickness of the seal gasket itself forms the side walls of the reaction chamber. As shown, the gasket seal 42 may be annular or ring-shaped; however, other shapes are envisioned, including ellipses, polygons, or other irregular shapes, and are within the scope of the present invention. It is regarded. The seal gasket 42 can be a removable adhesive gasket, an elastomeric seal (eg, an O-ring or gasket (including silicone gasket)) or a hermetically sealed to hold and support the hybe solution during the filling and incubation phases of the process. Any other sealing mechanism available in the art that is sufficient to form a reaction chamber. The base layer 40 may have a rectangular shape with rounded ends, and the size and position of the rounded ends may be combined with the seal gasket 42 to facilitate connection to the slide substrate. As shown in the figure, a typical chamber assembly 30 includes the introduction of hybridization or hybe solution to one end of the reaction chamber 26 and the enclosed air from the other end when it is filled with hybe solution. Alternatively, a filling port 56 and a ventilation port 58 for allowing gas to flow may be included.

  The square end of the flexible base layer 40 may extend laterally away from the reaction chamber 26 to form a tab with an upper adhesive patch 44 extending upward from the top surface of the base layer. be able to. The chamber assembly 30 may further include a top pressure gasket 46 that also extends upwardly from the top surface of the base layer 40, which top pressure gasket may be substantially aligned with a seal gasket 42 that extends from the bottom surface. it can. As will be discussed in more detail below, the pressure gasket 46 is provided with an upper clamp fixture facing downwards at the interface between the seal gasket 42 and the slide substrate to further seal the reaction chamber during the hybridization protocol. Or it can be operated to transfer the sealing pressure provided by the rotor uniformly.

  An upper release liner strip 52 may be placed on top of the adhesive patch 44 and the annular pressure ring 46, which, along with the lower release liner strip 50, protects and seals the chamber assembly from external contamination during storage and shipping. Can function. A single release liner strip 50 can be installed in a plurality of disposable reaction chamber assemblies 30 after manufacture to facilitate storage, transport and use (see FIG. 3).

  The base layer 40 remains flexible enough to be bent and peeled away from the slide substrate while retaining its shape, supporting the handling of the chamber assembly, and providing an impervious top surface for the reaction chamber 26. It can be formed as a sufficiently rigid semi-flexible structure. In one aspect, the base layer 40 may be a flexible plastic laminate having a primer on the bottom surface and the seal gasket 42 may be an adhesive gasket. The primer surface may be oriented in the direction that forms the inner surface of the reaction chamber and is also the surface to which the adhesive gasket adheres. The adhesive gasket can adhere and bond strongly to the primer surface than the slide, allowing the disposable chamber assembly to be removed cleanly from the slide. These materials are preferred for use in devices used to perform hybridization at about 37 ° C., with deviations up to about 90 ° C., and reactions performed under other conditions are more It is understood that it is desirable. Accordingly, other plastic or polymeric materials having physical and chemical properties selected for specific reaction conditions may be used in the construction of the device. All materials are compatible with any chemical or reactant with which they come into contact, do not degrade by such contact, and do not interfere with the chemical reaction performed within the device.

  The adhesive gasket of the present invention can be formed on the bottom surface of the base layer 40, can create a hermetic seal around the reaction chamber 26, and has sufficient compressibility to create the desired chamber volume. be able to. It is important that when the chamber assembly is removed from the slide substrate, the seal gasket remains adhered to the base layer, not the slide. The reason is that if gasket material remains on the slide, it may interfere with the slide leader. Therefore, the adhesive gasket must preferentially adhere to the base layer primer rather than the slide substrate. A variety of removable and repositionable adhesive materials may be used, including but not limited to acrylic, urethane, silicone, and rubber adhesive materials. Such materials are elastic and undergo plastic deformation and / or elastic deformation. The adhesive gasket may be formed from a commercially available adhesive film or may be applied by spray coating, silk screen, pad printing or other printing methods that produce a suitable finish and thickness.

  Additional embodiments of the disposable chamber assemblies 32-34 are shown in further detail in FIGS. For example, an additional strip 48 of silicone sheet material can be placed under the tab portion of the base layer, and the thickness of the top adhesive patch 44 has been reduced to maintain the base layer in a generally horizontal position over its entire length. Apart from that, the disposable chamber assembly 32 may be very similar to the chamber assembly 30. The chamber assembly 34 also has an upper adhesive gasket that allows the upper pressure gasket 46 to be removed and the upper release liner 52 to lay flush against the upper surface of the base layer 40 that overlies the reaction chamber 26. Similar to previous chamber assembly embodiments, except that the thickness of the adhesive patch 44 is reduced. Chamber assemblies 30, 32 and 34 may or may not include fill and vents.

  As described above, the seal gasket 42 used in embodiments 30, 32 and 34 may have a different adhesion than the adhesive patch 44. The seal gasket 42 is released from the slide substrate when the substrate is lifted at an appropriate time (eg, after hybridization), opening the reaction chamber and exposing the hybridized contents to the adjacent environment. It may be mildly adhesive or non-adhesive. In contrast, the adhesive patch 44 may be highly adhesive to provide a connection between the tab portion of the base layer and the upper clamp rotor or fixture. As described below, when the upper clamp rotor is lifted, the tab portion of the base layer is pulled upward, and the chamber assembly including the seal gasket 42 is then peeled away from the slide substrate.

  Referring now to the disposable chamber assembly embodiments 36 and 38 shown in FIGS. 3A and 3B, the seal gasket can be replaced with a dome-shaped chamber 60 (or “contact lens”), which is a high chamber. During hybridization, it may be formed from molding silicon or similar material that allows moderate distortion of the lower lip 62 under pressure to seal the reaction chamber 26 against the slide substrate. The dome chamber 60 is attached to the base layer 40 with a strong adhesive ring 64 and a circular 66 lower adhesive patch if the lower lip 62 of the dome chamber 60 provides a sealing surface between the chamber and the slide substrate. Can do. Further, in the dome chamber embodiment, the strong adhesive patch upper portion 68 may also be annular or circular and can be placed directly on the reaction chamber 26, with the dome chamber 60 lifted directly upward to The pressure seal formed between the lip and the slide substrate can be released.

  The placement of a representative disposable reaction chamber 30 on the sample or reaction region 22 and the application of hybe solution is shown in FIGS. As can be seen, the reaction region 22 carrying the immobilized reactant 24 extends only to a small portion of the slide substrate 20, and the disposable chamber assembly 30 can be sized accordingly. Although it is expected that only one reaction region will often be formed on each slide substrate, it is possible to use multiple reaction regions and multiple disposable chamber assemblies 30 on a single slide.

  The disposable chamber assembly 30 can be supplied on the release liner strip 50. In the method shown in FIG. 4, the chamber assembly 30 may first be removed from the release liner and placed on the reaction zone 22. The chamber assembly is “brayed” with a hard object, or pressed into the slide substrate 20 to ensure that the gasket seal 42 adheres well to the surface of the slide substrate, the upper release liner strip 52 is removed and the fill port 56 and vent The mouth 58 and the strong adhesive patch 44 may be exposed. Hybridization (“hybe”) or probe solution is then introduced into the small volume reaction chamber 26 with a pipette inserted into the fill port 56 formed in the base layer 40 at one end of the chamber assembly, while formed at the opposite end. The sealed air or gas can be discharged through the vent 58 formed in the air. In one aspect, the fill and vents of the reaction chamber 26 can be sealed when the upper fixture or rotor is lowered onto the top surface of the base layer. Alternatively, the filling and venting ports of the reaction chamber 26 are sealed with a small patch of tape placed on top of the base layer prior to hybridization.

  The method shown in FIG. 5 differs from the method shown in FIG. 4 in that a hybe (or probe) solution is applied to the reaction region 22 before the disposable chamber assembly 30 is placed on the slide substrate 22. In this embodiment, the fill and vent are not formed in the base layer because there are concerns about sealing the reaction chamber 26. The reason is that the formation of the reaction chamber is completed by connecting the chamber assembly 30 to the slide substrate 20. However, additional care is required to ensure placement of the chamber assembly around the apply portion of the hybe solution and in a manner that minimizes the amount of air or gas trapped within the reaction chamber 26.

  FIG. 6 shows a processing device 104 for semi-automated hybridization of a plurality of hybridization slides. The processing device 104 may include a basin-type container 110 having an open top end. The basin-type container may include an internal basin designed to hold a large amount of washing solution for washing the hybridization slide after completion of the hybridization process. The basin-type container is circular as shown, or any other shape or configuration capable of temporarily holding or carrying a sufficient amount of wash solution to immerse or sink a plurality of hybridization slides The range can include, but is not limited to, 0.1 to 3.0 liters of cleaning solution.

  The basin-type container 110 can be fluidly attached to a controllable pump / valve unit 120 that includes an ethanol or similar liquid container or bottle for prehybridization washing, and a post-high. Selection from a variety of liquid sources 122, such as a wash buffer solution container for hybridization washing, is possible. The liquid source 122 can be stored at room temperature or maintained at a predetermined temperature, such as in a hot water bath. The basin-type container 110 also includes internal piping such as valves (not shown) and pipes or tubes 126 for filling and / or discharging cleaning liquid from the basin, and exhaust for controlled release of steam or gas. A mouth 128 may be included.

  As shown in FIG. 7, the processing device 104 can include a slide carrier disposed within a ladle-type container 110, which slide carrier includes a plurality of hybridization slide substrates 20 having one or more reaction regions 22. Designed to receive. The slide carrier may be a lower carrier rotor 130 supported on a support shaft 118 and designed to be driven by a spin motor (not shown), which spin motor is held in the basin 112 and therein. Allows the lower carrier rotor (including the received slide substrate) to rotate relative to the washed liquid. The lower carrier rotor 130 can also be designed for vertical movement up and down the axis of rotation. The lower carrier rotor 130 and the basin-type container 110 together immerse the lower carrier rotor in a bath of cleaning liquid contained in the basin 112, then remove the lower carrier rotor from the bath and centrifuge to leave all residue. It can be designed to remove the cleaning liquid.

  The slide substrate 20 can be inserted into equally spaced carrier rotor “windows” 132 formed in the lower carrier rotor 130. The carrier rotor window 132 may have a slot or groove formed in the inner surface for receiving and gripping the side and outer edges of the slide substrate. The slot opens towards the center part of the carrier rotor, closes at the outer end, and the slide substrate is installed from the center of the rotor, so that the high speed centrifugation of the lower carrier rotor 130 during the rotation, in particular to remove residual cleaning liquid. During this time, the slide may be reliably prevented from sliding off the window 132.

  The processing device 104 extends upwardly from the floor of the basin 112 and typically includes a slide carrier window 132 when the slide carrier is lowered to the bottom of the basin-type container 110 during the hybridization reaction or incubation time. A slide heating pad 124 may be included that enters the bottom. The heating pad 124 is aligned with the bottom surface of the slide substrate 20 or is pressed against the slide substrate 20 to provide heat to the hybe solution, which further excites the suspended reactants and moves. And increase the efficiency of the reaction. In one aspect of the invention, the slide heating pad heats the slide substrate 20 to a temperature of about 95 ° C. during the hybridization protocol to denature the sample and probe solution, and then prolonged heating and incubation at a lower temperature. Thus, hybridization can be completed.

  A pre-hybridization wash procedure can be performed using the processing device 104 designed as in FIG. For example, one or more slide substrates 20 having a sample or reaction region 22 carrying an immobilized reactant 24 are placed in a lower carrier rotor 130 and the lower carrier rotor is placed on a support shaft 118 in a ladle-type container 110. Can be put. The top opening of the basin-type container can be closed with a cleaning cover (not shown), and the basin 112 is filled with a pre-hybridization cleaning solution such as ethanol or a similar cleanser at a constant concentration, so that the slide substrate and the reaction area 22 are separated. Purify together and prepare sample for hybridization. In one aspect, the lower rotor may be placed at the bottom relative to the base of the container such that the slide carrier window is captured by a slide heating pad 124 extending upward from the floor of the basin 112. Alternatively, the lower rotor may be raised and rotated in the prehybe cleaning solution. In any position, the slide heating pad can be driven to heat the ethanol solution directly or through the slide substrate. During the prehybridization protocol, the basin is alternately discharged and refilled with a cleaning solution of varying composition, after which the lower carrier rotor 130 is removed from the bath and centrifuged at high speed to remove any residual from the rotor or slide substrate. The cleaning liquid can be removed.

For example, an exemplary embodiment of the prehybridization wash procedure is the following protocol:
Incubate the slide for 15 minutes at 37 ° C. in 2 × SSC / 0.5% lgepal, pH 7.0;
Incubate the slide for 1 minute in 70% ethanol;
Incubate the slide for 1 minute in 85% ethanol;
Incubate the slide for 1 minute in 100% ethanol; and • Centrifuge at room temperature to dry.

  One method of filling the reaction chamber 26 with hybe or probe solution after completion of the prehybridization wash procedure is shown in FIG. In this method, after the hybe solution is applied to the reaction region 22, the reaction chamber assembly 30 is connected to the slide substrate 20 to form the assembled hybridization unit 10 (see also FIG. 5). However, as described above, after the reaction chamber assembly 30 is connected to the slide substrate 20 to form the hybridization unit 10, the reaction chamber can be filled through the filling port (see FIG. 4). Regardless of the procedure used to fill the reaction chamber with the hybridization solution, the processing device 104 may form one exemplary embodiment 100 of the present invention with the hybridization unit 10.

  As shown in FIG. 9, once the hybridization unit 10 has been formed and filled with hybe or probe solution, an upper “clamp” rotor 140 is placed on the lower carrier rotor and the installed slide substrate to provide a top surface of the base layer. The reaction chamber can be further sealed. When the disposable chamber assemblies 30, 32, and 34 described in FIGS. 1A-2B are used, the clamp rotor 140 may be pressed against the upper pressure gasket or directly against the substrate to remove the chamber assembly from the slide substrate after hybridization. The top surface of the reaction chamber is formed along a strong adhesive patch formed on the end tab of the chamber assembly. Alternatively, if a disposable chamber assembly 36 or 38 is used (FIGS. 3A-3B), the clamp rotor may be pressed directly onto the strong adhesive patch.

  The upper clamp rotor 140 shown in FIG. 9 can be designed to be supported on a support shaft 118 above the lower carrier rotor 130 and immersed and rotated with the lower carrier rotor in a bath of cleaning liquid. After the chamber assembly 30 is connected to the slide substrate 20 to form the hybridization unit 10 and then the hybridization unit is installed in the lower carrier rotor, the upper rotor 140 and the lower rotor 130 are meshed together to further define the reaction chamber. Seal and allow the upper adhesive patch to contact the clamp rotor. The adhesive patch is highly adhesive to provide a connection between the base layer of the chamber assembly and the upper clamp rotor even after various heating and soaking steps performed during hybridization and post-hybridization protocols. It's okay. Therefore, due to the strong adhesion, when the upper and lower rotors are later removed, the chamber assembly 30 is detached from the slide substrate 20, the sealed state of the reaction chamber is released, and the hybridized contents are adjacent. Can be sure to be exposed to the environment.

  10A-10D show the upper clamp rotor and lower carrier at various positions relative to the floor of the ladle vessel 110 and the slide heater 124 during various stages of the prehybridization, hybridization, and posthybridization cycles. A side and end cross-sectional view of the rotor are shown. As shown in FIG. 10A, the lower carrier rotor 130 is open to the bottom, which can occur after the hybridization unit 10 is formed by the connection of the disposable chamber assembly 30 and the slide substrate 20 after the prehybridization cleaning protocol. You may place it first in the place you put it. After formation of the hybridization unit 10, both the upper clamp rotor and the lower carrier rotor can occur during the hybridization protocol, in a closed, bottom position (FIG. 10B), which can occur at the start of the post hybe wash protocol. Closed, lifted and rotating position (FIG. 10C) and placed in an open, lifted and rotating position (FIG. 10D) that can occur during later stages of the post hybe washing protocol You can do it.

  For example, after the prehybridization protocol is completed as described above, the lower carrier rotor 130 is lowered and fitted around the slide heating pad 124, as shown in FIG. 10A, to dispose the disposable chamber assembly 30 with the installed slide. Connected around the 20 reaction zones, the reaction chamber 26 may be filled with hybe (or probe) solution. The upper clamp rotor 140 is then installed and / or lowered until it contacts and over the chamber assembly 30 as described in FIG. 10B to engage the adhesive patch and / or pressure gasket to hybridize the hybridization chamber 126. May be completed. Once this configuration is reached, the sample can be hybridized according to the hybridization protocol.

For example, an exemplary embodiment of the hybridization protocol described in FIGS. 4 and 10B includes the following steps:
Connecting the disposable chamber assembly onto the reaction zone;
Filling the reaction chamber with probe solution;
Denaturing the sample and probe for 5-10 minutes at 75 ° C .; and • Incubating overnight at 37 ° C.

Similarly, an exemplary embodiment of the hybridization protocol described in FIGS. 5 and 10B includes the following steps:
Applying a probe solution onto the reaction area;
Connecting the disposable chamber assembly onto the reaction zone;
Denaturing the sample and probe for 5-10 minutes at 75 ° C .; and • Incubating overnight at 37 ° C.

  After the hybridization protocol is complete, the wash plate 112 may be filled with wash buffer, the rotors 130, 140 may be lifted together, and slowly rotated while immersed in the buffer solution 106, as described in FIG. 10C. 10D, the upper clamp rotor 140 is then moved away from the lower carrier rotor 130, the chamber assembly 30 is adhered to the bottom surface of the upper rotor, completely removed from the slide substrate 20, and the top surface of the slide substrate 20 is cleaned. May be left exposed for. Sinking the slide substrate and slowly rotating it to break the gasket seal ensures that the reaction area is protected from contamination of airborne particles. Also, a certain amount of fluid sheer is applied immediately to the reaction area to ensure that the hybe solution is pushed out quickly, reducing the risk of cross-contamination with the hybe solution used on adjacent slides.

  Once the rotor is separated as in FIG. 10D, purifying and preparing the hybridized sample for analysis can be completed according to a post-hybridization wash step such as a typical post-hybe protocol. This may include the step of alternately discharging the basin-type container and filling with various washing buffer solutions 106 and rotating the lower carrier submerged in the buffer solution to completely remove the hybe solution. Removal of the lower carrier rotor from the buffer solution can be accomplished by lifting the lower carrier rotor out of the bath or by draining the wash solution from the basin-type container.

  During the post-hybe phase, the upper rotor 140 may be raised above the surface of the buffer solution and centrifuged separately at high speed, which may cause contamination by dropping on the slide substrate during the subsequent drying or washing buffer removal phase. Any remaining cleaning solution may be shaken off. In one aspect, before the cleaning of the lower carrier rotor is complete, the upper clamp rotor along with the connected chamber assembly may be completely removed from the processing device for cleaning and removal of the connected chamber.

For example, an exemplary embodiment of a post-hybridization wash procedure is the following protocol:
Wash the slides in 1 × Post Wash Buffer II (2 × SCC / 0.1% lgepal) for 2 minutes at room temperature;
-Wash slides in 1x Post Wash Buffer I (0.4xSCC / 0.3% lgepal) at 72 ° C (+/- 1 ° C) for 2 minutes without agitation;
• Wash slides in 1x Post Wash Buffer II (2x SCC / 0.1% lgepal) for 1 minute at room temperature without agitation;
-It may include centrifuging and drying at room temperature.

  The hybridization system 100 of the present invention is advantageous over the prior art by providing a reaction step that uses a very small volume reaction chamber, but can perform a large volume wash step both pre- and post-hoc. . As disclosed above, this can be accomplished by temporarily forming a sealed small volume reaction chamber on the surface of the slide, releasing the seal to open the reaction chamber and washing liquid. Can expose slides to large volume washouts or baths. It has been recognized that the benefits of large volume washing cannot be realized by forcing washing liquid through the small volume reaction chamber utilized during the incubation cycle. Further, it was recognized that removing the reaction chamber and exposing the slide to a large volume of wash solution or bath could remove the used hybe solution from the slide more completely and quickly.

  It will be appreciated that a large volume washout or bath of wash solution may be common to each of the plurality of hybridization slides. Moving multiple slide substrates through the same bath of both pre-hybe and post-hybe cleaning liquids provides simultaneous cleaning of multiple slides and efficient and economical use of the cleaning liquid. Furthermore, the use of large volume cleaning can also reduce the chance of cross contamination. This is because a microliter-sized hybe solution sample can be completely washed away and diluted in a much larger liter-sized amount of washing solution.

  While the above description teaches several exemplary embodiments of a semi-automated hybridization slide processing system and method of use thereof, hereinafter, substantially automated hybridization slide processing system will be described in FIGS. Various embodiments of the method and its use are shown, which also includes a small volume hybridization step and a large volume post-hybridization wash step.

  Each exemplary embodiment of a substantially automated hybridization system may include a hybridization unit 210 shown in more detail in FIGS. Hybridization unit 210 may include a substantially rectangular glass slide substrate 220 having a reaction region 224 carrying an immobilized reaction 226, such as an immobilized DNA sample. The reaction region can be covered by a disposable chamber assembly, or mixer 240, and the disposable chamber assembly or mixer can be removably attached to the slide substrate 220 to form a small volume reaction chamber 244 that encloses the reaction region 224. The mixer may be attached to the slide substrate with a mixer seal 242, which may be a removable adhesive material, an elastomeric seal (eg, an O-ring or gasket (including silicone gasket)) or during the filling and incubation stages of the process. Any other sealing mechanism available in the art to form a sufficiently closed reaction chamber to hold and carry the hybridization solution. If a non-adhesive material or elastomeric seal 242 is used, a small amount of corner adhesive material 258 is placed at the corner of the slide so that the hybridization unit 210 is in the processing device as discussed in more detail below. The mixer 240 can be lightly attached to the slide substrate 220 until it is placed on the slide substrate 220. The reaction chamber 244 can be provided with a filling port 250 and a vent 252 to allow introduction of hybridization fluid and venting of enclosed air or gas.

  Typically, the reaction region 224 carrying the immobilized reactant 226 substantially covers the top surface of the slide substrate 220 and provides a mixer seal 242 around the edge of the microarray slide to delineate the outer periphery of the reaction chamber 244. Can leave room for. A single reaction chamber may cover the entire reaction area on the surface of the slide. However, it is possible to divide the immobilized reactant into different compartments and subdivide the reaction chamber 244 into a plurality of individually sealed subchambers 246, each subchamber extending across the surface of the slide. It is separated from adjacent subchambers by a sealing portion. For example, the exemplary reaction chamber shown in FIGS. 11A-11B is subdivided into eight subchambers 246, each subchamber having its own fill port 250 and vent 252. In other aspects of the invention, the number of sub-chambers may include, but is not limited to, 2, 4, 6 or 12 sub-chambers as needed.

  The height of the reaction chamber (s) 244, 246, defined by the distance between the top of the slide substrate 220 and the bottom of the disposable chamber assembly or mixer 240 (or the thickness of the mixer seal) is about 1/1000 inch (or 25 μm). ), But higher heights are often used. Controlling the height of the reaction chamber to about 1/1000 inch allows the chamber volume, and hence the volume of hybridization solution required, to be limited to about 25 μL or less. However, it is understood that the reaction chamber volume can vary from about 5 μL for the small subchamber 246 to about 100 μL for the large single reaction chamber 244. One skilled in the art can consider that this range provides a small volume of hybridization that contacts a high concentration of sample suspended in the hybridization solution with the immobilized probe on slide 220. Make it possible.

  The disposable chamber assembly or mixer 240 may be made of a multi-layer, flexible polymer material, forming a transparent laminate structure, filling the reaction chambers 244, 246, and hybridization as the current air volume is pushed out of the vent 252. Provides the user with the ability to observe liquid progress. The mixer may include a built-in agitation system such as a bladder (not shown), which can be formed in the top surface portion of the mixer and when inflated causes the ceiling to fall downward into the reaction chamber. Can operate to widen the surface portion. The bladder can be inflated and deflated with air pressure to continue mixing the hybridization solution inside the reaction chamber during the incubation. Air vents 254 and pneumatic lines 256 communicating the bladder and the hybridization system can be formed in one end tab 248 of the mixer, preferably the internal end tab. The mixer's pneumatic agitation system is further detailed in US Pat. No. 7,234,400, “Laminated Microarray Interface Device”, filed Aug. 2, 2002, which reference is incorporated herein in its entirety. It is described in.

  The mixer 240 can be combined with an optional manifold device 270 that can facilitate filling and sealing of the reaction chamber 244 or sub-chamber 246 and reduce the risk of sample cross-contamination. The manifold may include a series of inlet holes and vents 272 aligned with the mixer inlet 250 and vent 252 respectively. The tip of the pipette can be captured and directed to the injection / vent hole to form an injection / vent hole 272 with a funnel-shaped opening 274 for guiding the hybridization solution into the reaction chamber or sub-chamber. After filling and venting, the inlet 250 and vent 252 of the mixer 220 can be connected to a plug-in plug, a sliding sealing bar built into the manifold, or a piercable septum layer built into the mixer itself. The reaction chambers 244, 246 can be closed by various means including layers), etc., to be liquid tight containers that are protected from external contamination during the incubation phase of the hybridization process. Further, the manifold 270, the mixer 240 and the mixer seal 242 can be designed as a mixer / manifold subassembly 280. Both the mixer 240 and the mixer / manifold subassembly can be disposable and designed to be easily attached and removed from the top surface of the slide substrate 220.

  Hybridization unit 210 can also be designed such that the boundary of mixer 240 extends beyond two parallel edges 230 of the slide substrate and the other two parallel edges 232 are exposed. In the exemplary embodiment shown in FIGS. 11A-11B, the mixer further extends along the long axis of the slide substrate (beyond the short edges) and provides two end tabs 248 at both ends 230 of the hybridization unit. To do. At the same time, the disposable chamber assembly or mixer 240 is narrower than the width of the slide substrate 220 and can expose two edges 232 that edge the length of the slide substrate. In another aspect of the invention, the set of parallel edges is exchanged, the mixer flap extends over and beyond the long edge of the slide substrate, and the short edge on one side of the slide substrate is exposed. Can be left.

  As discussed in more detail below, this design allows the mixer 240 to be attached to the upper clamp fixture of the processing device and the slide substrate 220 to be attached to the lower carrier fixture of the processing device. After receiving the mixer and slide substrate, the upper and lower fixtures can be mated together to join the mixer and slide substrate to form reaction chambers 244,246. When the upper and lower fixtures are removed, the mixer is removed from the slide substrate, and the sealed state of the reaction chambers 244, 246 is opened and opened.

  A processing device 304 that, together with the hybridization unit 360, forms an exemplary embodiment 300 of the present invention for substantially automated hybridization of a plurality of microarray slides is generally illustrated in FIGS. The processing device 304 may include a basin container 310 having an open top end. The basin-type container may include a basin 312 designed to hold a large amount of washing solution for washing the microarray slide after completion of the hybridization process. The basin 312 is circular as shown, or has any other shape or configuration capable of temporarily holding or carrying a sufficient amount of cleaning solution to immerse or sink a plurality of microarray slides. The range may include, but is not limited to, 0.1-3.0 liters of cleaning liquid. The basin-type container 310 may further include a side compartment 314 having a recess 316 for holding a cleaning liquid bottle, and internal piping such as valves and pipes for filling and discharging cleaning liquid from the basin.

  The processing device 304 includes a slide carrier disposed within the basin-type container 310 and can be designed to receive a plurality of microarray slide substrates 362. The slide carrier may be a lower carrier rotor 330 that is supported on a support shaft 318 and driven by a spin motor 320, as shown in the figures of the embodiments, the spin motor being in the basin 312 and therein. Allows rotational movement of the lower carrier rotor (including the received slide substrate) relative to the retained cleaning liquid. Further, both the lower carrier rotor 330 and the basin-type container 310 immerse the lower carrier rotor in a bath of cleaning liquid accommodated in the basin 312 and then remove the lower carrier rotor from the bath to remove all remaining cleaning liquid. Can be designed as Removal of the lower carrier rotor from the bath can be accomplished by lifting the lower carrier rotor out of the bath or by draining the cleaning liquid from the basin-type container. In one aspect of the present invention, the lower carrier rotor 330 can be lifted away from the floor of the basin 312 and rotated around the support shaft 318 while discharging the cleaning liquid.

  The processing device 304 further includes a clamp plate disposed on the slide carrier and can be designed to receive a plurality of mixer / manifolds 364 or individual mixers 366. The clamp plate may be an upper clamp rotor 340 supported on the same support shaft 318 and above the lower carrier rotor 330 as shown in FIGS. The support shaft 318 can be separated to allow rotational movement of the upper clamp rotor 340 that is distinct compared to both the ladle vessel 310 and the lower carrier rotor 330. The upper clamp rotor can also be designed for immersion and rotation in the bath of cleaning liquid contained in the basin 312.

  In the rotating embodiment of FIGS. 12-15, the upper clamp rotor 340 and the lower carrier rotor 330 can be designed to mesh one with the other by providing a relative vertical motion between the two disks. When engaged, the plurality of mixers / manifolds 364 pre-accommodated by the upper clamp rotor 340 are coupled to the plurality of slide substrates 362 pre-contained on the lower carrier rotor 330 to provide a plurality of sealed hybridization reaction chambers. A hybridization unit 360 can be formed. When removed, the separate movement between the upper clamp rotor and the lower carrier rotor causes the mixer / manifold 364 to be removed from the slide substrate 362 and the mixer is withdrawn from the slide substrate to form a plurality of reaction chambers. The mixer seal is opened and opened.

  The mixer / manifold 364 attached to the upper clamp rotor 340 can be angularly aligned with the slide substrate 362 attached to the lower carrier rotor 330 before bringing the two rotors together. This can be accomplished by monitoring and controlling the angular position of both rotors until the mixer / manifold and slide substrate are aligned.

  The slide substrate 362 can be inserted into equally spaced carrier rotor “windows” 332 formed in the lower carrier rotor 330. The carrier rotor window 332 is a slot or groove formed on the inner side to receive and grab the exposed edge of the slide substrate that is not covered by the mixer, and bends open during installation, then during rotation, In particular, during the high speed centrifugation of the lower carrier rotor 330, it may have a flexible tab at the end of the slot that snaps closed to prevent the slide substrate 362 from popping out of the window 332.

  The mixer / manifold 364 can be coupled to the upper clamp rotor 340 via an end tab of the mixer 366 that extends longitudinally beyond the edge of the slide substrate (see FIG. 15). The end tabs can be grasped by clips or tabs formed at both ends of a clamp rotor “window” 342 that extends downwardly toward the lower carrier rotor. The clip not only functions as a point of contact with the end tab, but can also act as an interlocking alignment and interlocking mechanism that aligns and secures the two rotors together. Additional embodiments using clips 642 are shown in FIGS. 24 and 25A-25D.

  Returning to the reference to FIG. 15, the upper clamp rotor 340 can be designed to receive both a separate mixer 366 or a mixer / manifold subassembly 368, and the manifold is loaded into the mixer before being loaded onto the clamp rotor. Can be attached to facilitate subsequent filling, venting and sealing of the reaction chamber or subchamber.

  In one aspect of the present invention, the clamp rotor window comprises a flexible “floating lid” 346 secured around the inner edge of the clamp rotor window 344 that spans the gap between the inner edge of the window and the manifold 368. be able to. As the two rotors move apart, the floating lid snuggly fits around the manifold and can be operated to grab the manifold and further secure the mixer / manifold 364 to the clamp plate rotor. When the upper clamp rotor 340 engages the lower carrier rotor 330 with or without the manifold, the floating lid 346 is pressed against the upper surface of the mixer 366 to completely compress the mixer seal and create a liquid-tight reaction chamber. Can function as a spring. Pressing against the upper surface of the mixer using a spring-like floating lid provides greater tolerance when the upper and lower rotors are engaged, and the slide plate 362 may break or break due to excess clamping plate rotors Application of extra force is avoided.

  In another aspect of the invention, the manifold 368 may be permanently connected to the upper clamp rotor 340, and only the mixer 366 may be removed and disposable in each cycle of the hybridization process. In addition, manifolds with universal pattern filling funnels and vents can be designed to adapt the mixer shell to the various subchamber designs available.

  In yet another aspect of the invention, the mixer / manifold 364 or mixer 366 can be attached to the slide substrate 362 prior to placing the slide into the lower carrier rotor 330. After receiving the pre-assembly hybridization unit 360, the clamp rotor 340 can be lowered to engage the carrier rotor and the required pressure can be applied to the top of the mixer 366 to properly seal the reaction chamber. As described above, the clamp rotor is automatically connected to the end tab of the mixer, and then lifting the clamp rotor releases the mixer seal so that the mixer 366 or mixer / manifold 364 is removed from the slide substrate 362. Can do.

  The air line can be formed in the upper clamp rotor or connected to the upper clamp rotor for communication with the mixer air line. The air line can be terminated with an outlet hole having an elastomeric seal aligned with the air port in the mixer. Pressing the upper clamp rotor against the top surface of the mixer to create a liquid-tight reaction chamber between the mixer and the slide substrate creates a hermetic seal between the end of the air line and the air port in the mixer end tab at the same time. .

  An additional aspect of the hybridization system extends upward from the floor of the basin 312, typically when the slide carrier is lowered to the bottom of the basin container 310 during the hybridization reaction or incubation time. A slide heater 324 that enters the bottom of the window 332 may be included. The slide heater 324 is arranged in contact with the bottom surface of the slide substrate 362 or is pressed against the bottom surface of the slide substrate 362 to supply heat to the hybridization solution, and the hybridization solution further supplies the suspended reaction product. It can be excited and moved to increase the efficiency of the reaction. In one embodiment of the present invention, the slide heater can heat the slide substrate 362 to a temperature of about 95 ° C.

  16A and 16B show side and end cross-sectional views of the upper clamp rotor 340 and the lower carrier rotor 330 in a closed position that can occur during the filling and incubation phases of the hybridization process. In this position, the coupled rotor is located at the bottom of the basin-shaped container 310, and the heater 324 projects upward into the carrier rotor window and contacts the bottom of the slide substrate 362. The upper clamp rotor can press the outer edge of the upper surface of the mixer 366 with the floating lid 346 and firmly press the mixer seal against the upper surface of the slide substrate 362 to form a reaction chamber having a liquid tight seal. A manifold 368 can be attached to the interior portion of the top surface of the mixer to align with the inlet and vent. In addition, the air line 348 in the upper clamp rotor can be placed in air communication with the air port of the mixer to allow manipulation of the mixer bladder to agitate and mix the hybridization solution during incubation. .

  17A and 17B show the side of the upper clamp rotor 340 and the lower carrier rotor 330 after the upper clamp rotor is lifted away from the lower carrier rotor and the mixer / manifold 366 and slide substrate 362 separated when the incubation phase is complete. A cross-sectional view and a terminal cross-sectional view are shown. The lifting movement of the upper clamp rotor can open and open the mixer seal that forms the reaction chamber, pull the mixer out of the slide substrate, and leave the top surface of the slide substrate exposed for cleaning. After the basin 312 is filled with the cleaning liquid and the two rotors are completely immersed, the upper rotor can be lifted and released to remove the mixer from the slide substrate. When the slide substrate is submerged and the mixer seal is released, the reaction area on the slide substrate is immediately flushed with cleaning liquid when the reaction chamber is opened, and any reaction chamber contents can be cross-contaminated to adjacent arrays. It is possible to minimize the possibility.

  During the washing process, the basin-type container is alternately discharged and filled with various washing solutions, whereby the hybridization solution can be completely removed. At this stage of the hybridization process, the upper rotor 340 can be lifted out onto the basin container, centrifuged separately at high speed, and then contaminated by dropping or dropping onto the slide substrate during the subsequent drying or washing water removal stage. Any remaining cleaning solution that has the property may be shaken off. In one aspect of the invention, the upper clamp rotor, along with the connected mixer and manifold, may be completely removed from the processing device for cleaning and removal of the mixer / manifold 364 before the cleaning of the lower carrier rotor is complete. .

  In addition, FIGS. 17A and 17B show a clamp rotor clip or tab 344 that extends downward from the clamp rotor and connects to an end tab of the mixer 366 to lift the upper clamp rotor 340 and lift the lower carrier rotor. When separated from 330, the mixer can be pulled out of the slide substrate 362 and operated to disassemble the hybridization unit 360. Also shown is a float lid 346 that, when the two rotors are tied together, is pressed against the top surface of the mixer 366 to fully compress the mixer seal and create a hybridization unit with a liquid tight reaction chamber. Also, the manifold / 368 can be grabbed and the mixer / manifold 364 can be further secured to the clamp plate rotor 340 during the separation of the two disk rotors.

  18A and 18B, the lower carrier rotor 330 is lifted away from the protruding slide heater 324 and partially away from the bottom of the basin 312 so that the disk rotates around its support shaft during the cleaning phase. Thus, it may be possible to create relative movement or flow on and around the slide substrate 362. This can provide faster and more complete cleaning of both the reaction area and the bottom surface of the slide substrate. In addition, the rotational speed can be adjusted to avoid damage to the hybridized immobilized reactant probe.

  In another aspect of the invention, the combined rotors 330, 340 are lifted together and moved away from the projecting slide heater 323, and before the disk is separated, the basin 312 is filled with sufficient cleaning liquid to sink the rotary rotor, Can be rotated together. This ensures that there is some liquid avoidance when removing the mixer 366 or mixer / manifold 364 from the slide substrate 362, quickly flushing out the hybridization solution on the slide and reducing the risk of cross-contamination. This is particularly beneficial for microarray slides having multiple sub-chambers that allow undesired mixing of various hybridization samples if all liquid is not quickly removed when opened. Inducing the flow of cleaning fluid across the surface of the slide through rotation of the rotor disk can minimize the risk of cross-contamination.

  Similar to the semi-automated hybridization slide processing system described above, the substantially automated hybridization system 300 provides a reaction stage using a very small volume reaction chamber and a subsequent large volume wash stage. Is more beneficial than the prior art. As disclosed above, this can be accomplished by temporarily forming a sealed small volume reaction chamber on the surface of the slide, releasing the seal to open the reaction chamber and washing liquid. Can expose slides to large volume washouts or baths. It has been recognized that the benefits of large volume washing cannot be realized by forcing washing liquid through the small volume reaction chamber utilized during the incubation cycle. In addition, it was recognized that the hybridization solution used could be removed more completely and faster from the slide by removing the reaction chamber and exposing the slide to a large volume of wash solution or bath.

  It will further be appreciated that a large volume washout or bath of wash solution may be common to each of the plurality of microarray slides. Moving multiple slide substrates through the same bath of cleaning liquid provides simultaneous cleaning of multiple slides and efficient and economical use of the cleaning liquid. Furthermore, the use of large volume cleaning can also reduce the chance of cross contamination. This is because microliter-sized hybridization liquid samples can be completely washed away and diluted in much larger liter-sized volumes of wash solution.

  In addition, FIGS. 18A and 18B show a slide mounting groove or slot 334 formed in the carrier rotor window 332 to receive and grab the exposed edge of the slide substrate not covered by the mixer. Can be made.

  Returning to the reference to the rotary embodiment shown in FIGS. 12-15, the cleaning stage involves the use of a plurality of cleaning liquids, and alternately discharging and filling the basin 312 in the basin mold 310 during the process. In the meantime, the lower carrier rotor 330 and the connected slide substrate 362 can continue to rotate. After the cleaning phase is completed, all liquid can be drained from the basin-type container, the lower carrier rotor can be centrifuged at a higher rotational speed, and all remaining cleaning liquid can be shaken off by centripetal force. In another aspect of the present invention, the upper clamp rotor 140 located directly above the lower carrier rotor is blown away from the slide surface with all remaining wash solution before they dries and contaminates the hybridized reaction area. A downward nozzle that supplies a jet of nitrogen gas or humidified or ozone-free air can be provided.

  Another exemplary embodiment 400 of the present invention that uses non-rotating components is generally shown in FIGS. The embodiment may include a basin-type container 410 having an open top end. The basin-type container can be designed to hold a sufficient amount of wash solution to immerse or submerge a plurality of microarray slides after completion of the hybridization treatment incubation state. The basin-type container may be rectangular and has a side compartment (not shown) having a recess for holding a cleaning liquid bottle, and internal piping such as valves and pipes for filling and discharging the cleaning liquid from the basin-type container May further be included. The internal tubing can be designed to drain and fill at high speed to reduce the time that the slide is not immersed.

  The processing device can be designed to receive a plurality of microarray slide substrates 414 including a lower carrier plate or fixture 412 disposed within a basin-type container. In the illustrated embodiment, the lower fixture 412 can be formed in the bottom surface of the basin-type container 410. The processing device may include an upper clamp plate or fixture 422 disposed on the lower plate and may be designed to receive a plurality of disposable mixers 426. The upper clamp fixture is common to all mixers or, as shown in the figure, a processing device can be designed with a separate clamp fixture for each mixer.

  In the non-rotating embodiment of FIGS. 19 and 20, the clamp fixture 422 may be coupled to the top cover 420 of the processing device, and the relative motion and engagement between the upper fixture 422 and the lower fixture 412. Can be designed to have a piston-like actuator 424. When engaged, a plurality of mixers 426 having mixer seals 428 previously accommodated by clamp plates are attached to a plurality of slide substrates 414 previously accommodated on a carrier plate 412 to form a plurality of sealed hybridization reaction chambers. Can be made. And when removed, due to the separation movement between the upper clamp fixture and the lower carrier fixture, the multiple mixers are removed from the multiple slide substrates, the mixer seals are pulled out of the slide substrates, and each of the multiple reaction chambers Is opened and opened.

  An upper cover 420 can be attached to the basin-type container 410 and sealed with an outer wash chamber seal 402 to form an outer chamber 404 that completely surrounds and encloses a plurality of hybridization units. Once the cover is secured on the basin, the piston-like actuator 424 is actuated to close the gap between the mixer 426 and the slide substrate 412 to form a separately sealed hybridization reaction chamber and after incubation is complete , Pull out and remove the mixer from the slide substrate.

  Flushing and washing of the hybridized slide substrate after completion of the incubation step can be accomplished by flowing the wash solution through an enclosed outer wash chamber 404 common to all microarray slides installed in the processing device. . The cleaning liquid can be moved or flowed relative to the accommodated slide substrate 412 with a liquid pump or similar device. Cleaning solution removal after the cleaning cycle is completed drains the cleaning solution from the cleaning chamber and supplies a downward jet of nitrogen gas or humidified or ozone-free air onto the slide substrate to remove any remaining cleaning solution. Can be achieved.

  FIG. 21 shows yet another embodiment 500 of the hybridization system of the present invention, in which three disk plates or rotors are used. The lower rotary disk includes a lower carrier rotor 510 and an upper clamp rotor 520, both of which can move up and down and rotate around the support shaft 502. The embodiment shown in FIG. 21 may include a third upper valve disc 550. The valve disc can be designed for axial (up and down) movement and may or may not rotate with the lower disc rotor.

  The upper valve disk 550 can be designed to have a plurality of valve stations 560 designed for interconnection with a plurality of manifold / mixers installed on the lower clamp rotor. The outward or bottom-down projection of each valve station 560 is a set of valve pins 566 that connect a series of injection / vent holes 540 formed in the manifold (holes 272 in the manifold shown in FIG. 11B). And similar to wax 274). The valve pin may be solid and can be formed from a hardened material or a stainless metal material. The valve pin can be used to control both the flow of liquid into and out of the reaction chamber (s) and to seal the reaction chamber during the incubation phase. Can work as a spigot. Although described with a hybridization system embodiment 300 using a rotating processing device, the valve station can also be designed to work with a non-rotating processing device.

  One embodiment of valve station 560 is shown in more detail in FIG. The valve station includes a plurality of plates including a fixed top plate 562 that provides a base of movement, a valve pin actuation plate 564, and a plate 568 that holds an O-ring to guide and maintain the valve pin 566 in the proper position and orientation. A plate may be included. The actuation plate 564 may be biased downward by a spring 574, but its vertical position can be controlled by an air-powered (pneumatic) piston 572 or similar actuation device. An O-ring 570 concentric with each valve pin 566 creates a seal between the valve pin and the waxy opening 542 in the manifold when the valve station 560 is attached to the lower mixer / manifold.

  Valve pin 566 interconnects with injection / vent hole 540 in the manifold and can seal the hole during hybridization. The infusion / vent in the manifold can include a funnel-shaped opening 542 for receiving the valve pin 566 and guiding it into the infusion / vent. In one aspect of the invention, the manifold can be divided into an upper manifold 530 and a lower manifold 532, in which an internal liquid path can be formed. For example, the manifold may have a main liquid line 534 that communicates with a plurality of moving liquid lines 536, the moving liquid lines being filled with the inlet / vent 540 at the break between the upper manifold 530 and the lower manifold 532. You can cross. In one aspect of the invention, the valve pin 566 can be partially withdrawn to allow liquid 544 from the main line 534 to flow down through the inlet 540 and into the reaction chamber. Similarly, the valve pin can be partially removed to allow reversible flow of liquid expelled from the vent path through the liquid path and into a suitable collection device (not shown).

  The method of the present invention utilizing the valve disc 550 includes installing a slide substrate in the lower carrier rotor 510, installing a mixer / manifold in the upper clamp rotor 520, and lowering the clamp rotor to engage the carrier rotor. A step of attaching a mixer / manifold to the slide substrate to form a reaction chamber may be included. The reaction chamber can then be filled with hybridization solution through a funnel-shaped opening 542 in the manifold. After filling, the valve disc 550 with the downwardly projecting valve pin 566 can be lowered into the manifold inlet / vent 540 to seal the reaction chamber. Hybridization can then be performed with mixing during an incubation step controlled by compressed air delivered to the bag formed in the mixer.

  After hybridization is complete, the valve disc 550 may be raised and the basin filled with sufficient wash buffer to immerse the lower rotor 510, 520. The lower rotor can be rotated in the wash buffer to move the clamp rotor away from the carrier rotor, creating an immediate liquid flow on the slide substrate when the sealed chamber covering the reaction area is opened and opened. . The cleaning cycle of the slide substrate housed in the lower carrier rotor can be continued as previously described.

  In another aspect of the embodiment 500 of FIGS. 21-22, the method may further include reconnecting the clamp rotor 520 and the carrier rotor 510 to reestablish the reaction chamber after the cleaning cycle is complete. The valve disk 550 can be lowered and the valve pins 566 can be reinserted into the mixer / manifold to perform various fluid steps such as nucleic acid denaturation and recovery on the hybridized and washed microarray slides.

  It will be appreciated that the valve station 560 provides additional flexibility for processing and cleaning of the slide substrate after hybridization. After incubation is complete, for example, the valve pin 566 inserted into the injection / vent 540 is partially opened by the air piston 572, and the elution buffer is slowly pumped into the reaction chamber to wash the reaction region and the hybridization solution. And the hybridization solution can be pushed out through the vent and collected with a suitable collection device located under the vent outlet. The valve pin is then lowered again to seal the reaction chamber, and the slide substrate and mixer (eg, hybridization unit) can be reheated. Upon completion of the second processing step, the valve pin can be reopened and additional heated elution buffer can be pumped through the reaction chamber to force the reacted liquid into another collection device.

  Further, in the case of a non-rotating processing device, after hybridization is completed, the valve pin 566 inserted into the injection hole 540 is partially opened, the clamp fixture is pushed up, the mixer seal is released, and the mixer is removed from the slide substrate. Washing liquid can be pumped into the reaction chamber with sufficient pressure to release. The outer cleaning chamber seal may remain intact so as to maintain a high volume cleaning chamber. After the cleaning sequence is complete, the cleaning solution can be replaced by nitrogen gas or humidified or ozone-free air to remove any remaining cleaning solution from the slide.

  In both rotating and non-rotating embodiments of the processing device, the use of valve pin 566 (see FIGS. 21-22) can provide significant advantages over the prior art. For example, the valve pin and valve station 560 are simple to assemble with minimal moving parts and can reduce the manufacturing cost of the processing device. Using a valve pin to seal a reaction chamber filled for incubation is also cheaper than current conventional sealing methods such as tape application by hand. Solid metallic valve pins can provide a more secure seal when used repeatedly. The soft contact surface of the disposable manifold wax-like opening 542 with respect to the injection hole 540 becomes a wear point and is continuously replaced. Most importantly, however, the valve pin and manifold system can reduce the “dead” volume between the reaction chamber inlets and vents and the tip of the pin valve to a very small amount in the range of 1-3 μL. Thereby saving the amount of hybridization solution required to carry out the test.

  FIGS. 23 and 24 show another exemplary embodiment 600 of the present invention. Similar to the semi-automated hybridization slide processing system and method described above, the disposable chamber assembly 624 is removably attached to the slide substrate 620 to create a sealed reaction chamber 626 that surrounds the reaction region, and the hybridization unit 610 is Can be formed. In this case, the hybridization unit 610 does not require a manifold for automated dispensing of the hybe solution into the reaction chamber, but to manually fill the reaction chamber with a pipette prior to installation into the carrier rotor 630. Of filling holes and vents. However, again, similar to the substantially automated hybridization system described above, the hybridization unit 610 can include one or more reaction chambers 626 formed in the disposable chamber assembly 624, with a short slide substrate 620. Two end tabs 628 may also be included that extend beyond the edges. Each end tab 628 may include a connection hole 622 for flexibly receiving a clip 642 extending downward from the upper clamp rotor 640.

  The lower carrier rotor 630 may include a recess 632 at each end of the carrier window designed to receive the clip 642. For example, after the lower carrier rotor 630 in which the hybridization unit 610 is installed is placed in the basin-type container 604 and the slide substrate 620 is adjacent to the slide heater 606 (FIG. 23), the upper clamp rotor 640 is placed in the basin-type container. The clip 642 extending downward from the upper clamp rotor 640 can be pushed through the connecting hole 622 in the disposable chamber assembly 624 and into a recess 632 formed in the lower carrier rotor (FIG. 24). In one aspect, the clip 642 can be coupled to the recess 632 to better align the two rotors and secure them together. The basin-type container 604 in which the rotors 630 and 640 are installed can be sealed with the cover 608.

  In addition, manipulation of the downwardly extending clip 642 during the hybridization protocol is shown in FIGS. The figure shows the side of the upper clamp rotor and lower carrier rotor at various positions relative to the floor of the ladle vessel 604 and the slide heater 606 during the various stages of the prehybridization, hybridization, and posthybridization cycles. Sectional and end sectional views are shown. As shown in FIG. 25A, the lower carrier rotor 630 may first be placed in an open, bottom position adjacent to the slide heater 606 after the hybridization unit 610 is connected. The upper clamp rotor and lower carrier rotor are then both closed and lifted, which can occur during the hybridization protocol, in a closed, bottom position (FIG. 25B), which can occur at the beginning of the post hybe wash protocol. , Rotated position (FIG. 25C), and may be opened, lifted and placed in the rotated position (FIG. 25D), which may occur during subsequent stages of the post hybe wash protocol.

  For example, after the disposable chamber assembly 624 is connected around the reaction area of the slide substrate 620 and the reaction chamber 626 is filled with a hybe (or probe) solution, the hybridization unit 610 may be installed in the lower carrier rotor 630. . After installation of the hybridization unit, the lower carrier rotor may be placed around the slide heating pad 606 as shown in FIG. 25A. Then, as shown in FIG. 25B, the upper clamp rotor 640 is installed over and / or lowered over the chamber assembly 624 and formed in the holes 622 formed in the tabs of the disposable chamber assembly and in the slide carrier rotor. The clip 642 may be engaged in the recessed portion 632 thus completed to complete the sealing of the hybridization chamber (s) 126. Once this configuration is reached, the sample can be hybridized according to the hybridization protocol.

  After the hybridization protocol is complete, the washer can be filled with wash buffer 602, the rotors 630, 640 can be lifted together, and slowly rotated while immersed in the buffer solution, as described in FIG. 25C. Then, as described in FIG. 25D, the upper clamp rotor 640 is moved away from the lower carrier rotor 630 and the chamber assembly 624 is held on the bottom surface of the upper rotor by the clip 642 and completely removed from the slide substrate 620. The top surface may be left exposed for cleaning. As described above, sinking the slide substrate and rotating it slowly to break the reaction chamber seal ensures that the reaction area is protected from contamination of airborne particles. Also, a certain amount of liquid avoidance is applied immediately to the reaction area to ensure that the hybe solution is pushed out quickly, reducing the risk of cross-contamination with the hybe solution used on adjacent slides.

  FIG. 26 shows yet another exemplary embodiment 700 of the present invention, in which the basin-type container 710 includes a rotating arm 720 that rotates downward prior to the hybridization step. Thus, the upper clamp rotor 740 and the lower carrier rotor 730 can be connected to each other. The rotating arm then lifts the associated rotors 740, 730 together as a unit, rotates the assembly 90 degrees, the support shaft 718 is aligned substantially in a horizontal plane, and the hybridization unit 710 is pivoted in a substantially vertical plane. Can be rotated around. In addition, a small bubble of air or inert gas is introduced into the reaction chamber of each hybridization unit, and the rotation of the associated rotor around the substantially horizontal support shaft 718 during the hybridization / incubation step Mixing by bubbles induced by gravity can occur inside the reaction chamber. After the hybridization step, the rotating arm 720 can return the associated rotors 740, 730 to a horizontal position and allow the wash step to proceed as described above.

  While the bubble mixing embodiment 700 is particularly useful for the semi-automated hybridization system 100 described above and shown in FIGS. 1-10, the bubble mixing embodiment of the present invention is described above. And is also applicable to the substantially automated hybridization system 400 shown in FIGS. 11-22 as a complementary or alternative mixing system to the air agitation system during incubation.

  In the foregoing detailed description, the invention has been described with reference to specific exemplary embodiments. However, it will be understood that various modifications and changes may be made without departing from the scope of the present invention as set forth in the claims. The detailed description and accompanying drawings are to be regarded as illustrative rather than restrictive, and it is intended that the invention, as described and shown herein, be any and all such modifications or changes. It shall fall within the range of

  More specifically, exemplary exemplary embodiments of the present invention have been described herein, but the present invention is not limited to these embodiments, and those skilled in the art based on the foregoing detailed description. And any and all embodiments having modifications, omissions, combinations (eg, combinations of aspects across the various embodiments), adaptations and / or changes understood by. Limitations in the claims shall be construed broadly based on the terms used in the claims and shall not be limited to the examples described during the foregoing detailed description or performance of the application, which examples are non-exclusive. It should be interpreted as appropriate. For example, in the present disclosure, the term “preferably” is non-exclusive and shall mean “preferably, but not exclusively”. Any steps recited in any method or process claim may be executed in any order and are not limited to the order recited in the claims. Means-plus-function or step-plus-function limitations are expressly cited with all the following conditions regarding the limitations of a particular claim: (a) "means for" or "steps for"; and (B) Adopted only if the corresponding function is explicitly cited, if present in the limitation. Structures, materials or actions that support the means plus function are explicitly cited in the description herein. Accordingly, the scope of the invention should be determined not by the above description and examples, but only by the claims and their equivalents as permitted by law.

Claims (30)

A unit for providing a reaction chamber on a slide comprising the following components:
A slide substrate having a reaction region and two exposed parallel edges for connection to the carrier fixture of the processing device;
A chamber assembly removably attached to the slide substrate to form a sealed reaction chamber that encloses the reaction region; and coupling means for attaching the chamber assembly to a clamp fixture of a processing device, the clamp attachment The unit, wherein the chamber assembly is removed from the slide substrate by releasing the tool from the carrier fixture, thereby opening the sealed reaction chamber. The following components:
A flexible base layer having a top surface and a bottom surface, wherein the bottom surface forms the top surface of the reaction chamber; and the weakly adhesive gasket extending from the bottom surface of the base layer forming the side wall of the reaction chamber The chamber assembly comprising: a seal; and the coupling means comprising a strongly adhesive top patch extending from an upper surface of the base layer for coupling to a clamp fixture of the processing device. unit. The following components:
The chamber assembly comprising a dome-shaped shell having a flexible annular lip to form a sealed reaction chamber enclosing the reaction region; and an upper surface of the dome for connection to a clamp fixture of the processing device The unit of claim 1, further comprising the connecting means comprising a strongly adhesive top patch extending from the top.   The coupling means comprises a set of chamber assembly boundaries, the boundaries extending beyond two further parallel edges of a slide substrate for attaching the chamber assembly to a clamping fixture of a processing device; The unit according to claim 1.   The unit of claim 4, wherein the set of chamber assembly boundaries extends beyond a substrate edge parallel to the minor axis of the slide substrate.   The unit of claim 4, wherein the set of chamber assembly boundaries extends beyond a substrate edge parallel to a long axis of the slide substrate. A system for multiple microarray slides comprising the following components:
Ladle-type container;
A slide carrier rotor disposed on a support shaft in the basin-type container for receiving at least one slide substrate therein;
A clamp rotor disposed on the support shaft and proximate to the carrier rotor for receiving at least one chamber assembly therein;
The chamber assembly is attached to the slide substrate by engaging the clamp rotor with the carrier rotor, thereby forming at least one sealed reaction chamber, and the clamping rotor is removed from the carrier rotor. A chamber assembly is removed from the slide substrate, thereby releasing the at least one reaction chamber from sealing;
Above system. The disposable chamber assembly comprises the following components:
A flexible base layer having a top surface and a bottom surface, wherein the bottom surface forms a top surface of at least one reaction chamber;
A weakly adhesive gasket seal extending from the bottom surface of the base layer forming a side wall of the at least one reaction chamber; and a strength extending from the top surface of the base layer for connection to a clamp fixture of the processing device The system of claim 7, comprising an adhesive top patch. The chamber assembly comprises the following components:
A dome-shaped shell having a flexible annular lip to form the at least one sealed reaction chamber;
A flexible base layer having a top surface and a bottom surface;
An adhesive bottom patch extending from the bottom surface for connecting the dome-shaped shell to the base layer; and an adhesive property extending from the top surface of the base layer for connection to a clamp fixture of the processing device. The system of claim 7, comprising a top patch.   And at least one manifold coupled to the exposed surface of the at least one disposable shell, wherein the manifold includes at least one fill hole aligned with a fill port and a vent in the disposable shell; and The system of claim 7, having at least one vent.   A valve rotor having at least one valve station with a valve pin disposed on the support shaft proximate to the clamp rotor and projecting outwardly; and meshing the valve rotor with the clamp rotor; The system of claim 10, wherein the valve pin is removably inserted into the at least one fill hole and the at least one vent hole of the at least one manifold. A method for processing multiple slides, the following steps:
Inserting a plurality of slides into a processing device, each of the plurality of slides having a reaction region forming a small volume reaction chamber by being enclosed by a small volume chamber assembly;
Filling the reaction chamber with a small amount of liquid to react with the reaction zone;
Exposing the reaction region by removing the chamber assembly from the plurality of slides;
Washing the plurality of slides with a common bath of washing solution;
Removing the plurality of slides from a shared bath of wash solution.   13. The method of claim 12, wherein the processing device further comprises at least one rotor disk disposed in a basin-type container designed to accommodate a shared bath of cleaning liquid.   14. The step of cleaning the plurality of slides further comprises immersing the at least one rotor disk in a shared bath of the cleaning liquid contained in the basin-type container and rotating there. Method.   15. The step of removing the plurality of slides from a cleaning liquid further comprises separating the at least one rotor disk from the cleaning liquid shared bath and shaking the cleaning liquid by centrifuging the rotor disk. The method described. A method for in-situ processing of slides for analysis of immobilized samples comprising the following steps:
Obtaining a slide substrate having a reaction region carrying an immobilized sample;
Installing the slide substrate in a processing device for automated processing,
The process consists of the following steps:
Attaching a chamber assembly to the slide substrate to form a small volume reaction chamber that encloses the reaction region;
Filling the reaction chamber with a liquid to react with the immobilized sample;
Sealing the reaction chamber during incubation;
Releasing the reaction chamber from a sealed state by removing the chamber assembly from the slide substrate;
Removing the reaction solution by flowing a large volume of washing solution into the reaction region; and removing the washing solution from the slide substrate; and the step of removing the slide substrate from the processing device. , The above method.   The method of claim 16, wherein the small volume reaction chamber holds less than 100 μL of liquid.   The chamber assembly further comprises an attached manifold having at least one fill hole and at least one vent aligned with the fill and vent in the disposable chamber assembly, whereby the reaction with reaction liquid The method of claim 16, wherein the method facilitates filling of the chamber.   19. The method of claim 18, wherein sealing the reaction chamber further comprises removably inserting a plurality of valve pins into the at least one fill hole and the at least one vent hole.   The method according to claim 16, further comprising agitating the reaction solution by alternately expanding and contracting air bags provided in a chamber assembly portion of the reaction chamber.   The method of claim 16, further comprising agitating the reaction solution by introducing bubbles into the reaction chamber and rotating the slide substrate about a substantially horizontal axis.   The method according to claim 16, further comprising improving the reaction between the reaction solution and the immobilized sample by heating the slide substrate.   17. The method of claim 16, wherein the large volume of cleaning liquid comprises at least about 0.1 liter of cleaning liquid.   The method according to claim 16, wherein the step of removing the cleaning liquid uses centrifugal force to centrifuge and drop the cleaning liquid from the slide substrate.   The method of claim 16, wherein removing the cleaning liquid further comprises blowing the cleaning liquid from the slide substrate using a compressed gas stream.   The method of claim 16, further comprising co-processing at least two slide substrates in the processing device, wherein the at least two slide substrates are washed away in a shared volume of cleaning solution. An in situ processing method of at least two slides for immobilized sample analysis comprising the following steps:
Obtaining at least two slide substrates having a reaction region carrying an immobilized sample;
Attaching a chamber assembly to each slide substrate to form a small volume reaction chamber enclosing the reaction region;
Filling the reaction chamber with a reaction solution for reacting with the immobilized sample;
Installing the at least two slide substrates in a processing device for processing,
The process includes the following steps:
Sealing the reaction chamber during incubation;
Increasing the reactivity of the reaction solution by agitating the hybridization solution during the incubation;
Releasing the reaction chamber from a sealed state by removing the chamber assembly from the slide substrate;
Removing the reaction solution from the reaction region by rinsing the at least two slide substrates with a common washing solution; and removing the washing solution from the slide substrate; and the at least two slides The above method comprising the step of removing the substrate from the processing device.   The chamber assembly further comprises an attached manifold having at least one fill hole and at least one vent aligned with the fill and vent in the chamber assembly, whereby the reaction chamber with reaction liquid 28. The method of claim 27, which facilitates filling.   29. The method of claim 28, wherein sealing the reaction chamber further comprises removably inserting a plurality of valve pins into the at least one fill hole and the at least one vent hole. A method for processing multiple slides, the following steps:
Inserting a plurality of slides into a carrier fixture of a processing device, each of the plurality of slides having a reaction region carrying an immobilized reactant;
Washing the plurality of slides in a shared bath of washing fluid according to a protocol;
Removably attaching a plurality of disposable chamber assemblies to the plurality of slides to form a sealed reaction chamber enclosing the reaction region;
Filling the reaction chamber with a small amount of reaction solution to react with the enclosed reaction zone;
Further sealing the reaction chamber during a reaction protocol by attaching a clamp fixture to the chamber assembly;
Lifting the clamp fixture to remove the chamber assembly from the plurality of slides to expose the reaction area;
Washing the plurality of hybridization slides in a shared bath of washing fluid according to a protocol; and removing the plurality of slides from the carrier fixture of the processing device.

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