Abstract:
Methods and apparatus for automated sample analysis are provided in which a plurality actuators are involved in moving a samples from one compartment to another, and appropriate reactants are combined with the sample in one or more of the compartments. The actuators are preferably contained in a device that also has a detector, data reduction capabilities, and a printer. Contemplated signal detectors include a photomultiplier tube, a photodiode, and a charge-coupled device. Steps contemplated to be performed automatically include aliquoting the sample, diluting the sample, contacting at least a portion of the sample with a reagent having a substantially selective binding towards the analyte. Contemplated reactants include sense and antisense nucleic acids, antibodies and antigens, solid-phases such as paramagnetic beads, reagents, other substrates, and wash solutions.

Description:
This application claims the benefit of U.S. provisional application number 60/095193, filed Aug. 3, 1998, and is a continuation in part of U.S. utility application number 08/905261, filed Aug. 1, 1997, which are both incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The field of the invention is diagnostics. 
     BACKGROUND OF THE INVENTION 
     In recent decades, advances in modem chemistry and more sophisticated instrumentation have led to a plethora of clinical tests. However, the equipment and the trained personnel needed to perform such tests led also to an increase in costs. To cut down on the costs related to clinical diagnosis, many physicians frequently outsource testing of blood and other specimens to centralized or specialized laboratories. Outsourcing clinical diagnostics, however, often increases the time between acquiring a sample and obtaining a test result. A delay in obtaining a test result is especially disadvantageous when time is a critical factor in differential diagnosis, for example, in the treatment of heart attacks, poisoning or strokes. Furthermore, a delay in obtaining test results adds to the overall cost. 
     The time span between acquiring a sample and obtaining a test result is not only of paramount importance in clinical diagnosis, but also in a variety of other fields. Such fields are, for example, environmental chemistry to detect a source of pollution, military field tests to detect poisonous gases, or criminological investigation to find traces of chemical markers. Time constraints, as well as the requirement to perform diagnostic tests at the place of sample collection led to the development of compact, self-contained test systems. Such self-contained test systems may be categorized into two different classes. 
     The first class may be characterized as qualitative test systems. Many qualitative test systems provide all required reagents, and a sample can be analyzed without further need of instrumentation. In U.S. Pat. No. 3,726,645 to Kaezmarek et al., U.S. Pat. No. 3,713,779 to Sirago et al., and U.S. Pat. No. 3,689,224 to Agnew et al., for example, small, flat hand-held test kits are described, in which a liquid or gaseous sample reacts with reagents provided by the test kit. A color change of an indicator reveals the presence of analyte. In these test kits, manual application of pressure is usually used to move and mix reagents; and the sample. In other test systems, for example in U.S. Pat. No. 4,806,316 to Johnson et al., the sample is propelled by gravity or a pressure difference. Again, a color change indicates the presence of analyte. In a further example, U.S. Pat. No. 4,859,421 to Apicella, additional elements in a test kit are described, such as one-way valves that allow only unidirectional flow of reagents. Furthermore, additional reagents for positive and negative controls may be provided. 
     The second class may be characterized as quantitative test systems. Quantitative test systems generally require a specialized instrument, commonly a photometer or fluorimeter. Such quantitative test systems utilize various ways of detection and various ways of how the sample is moved within the test device. In U.S. Pat. No. 4,963,498 to Hillmant et al., for example, a test system is described in which a blood sample is mixed with a reagent and subsequently drawn by capillary action into a flow path. Interactions between the reagents and the sample cause a change in flow rate. The flow rate is measured using a photocell, and the change in the flow rate is then correlated with the concentration of the analyte. In another example, U.S. Pat. No. 3,799,742 to Coleman describes a test system in which a sample is placed into a small test container. The sample is then manually pushed through a filter unit into a cuvette, where a color reaction takes place. The small test container is subsequently inserted into a reading device and the concentration of the analyte is calorimetrically determined. In U.S. Pat. No. 4,673,657 to Christian, a sample is placed into an assay card and forced to a detection zone by a roller bar. A pulse vacuum may firer move the sample repeatedly over the detection zone. The detection zone may specifically bind up to 250 analytes, and the analytes can then be automatically detected and quantified via an optical or magnetic detector. 
     Although various quantitative and qualitative test systems are known in the art, almost all test systems have a number of disadvantages. Typically, the assays performed in such systems are single-step assays, i.e., one sample is mixed with one reagent or set of reagents, and the result of the reaction is then measured. However, many modem diagnostic reactions employ multiple steps prior to the detection reaction, for example reduction of a sample to liberate disulfide bound thiols, or coupled enzymatic reactions to indirectly measure an analyte or secondary reactions for signal amplification. 
     Another disadvantage of many quantitative and qualitative test systems is that reaction of a sample with a substrate, and detection of the analyte, occur in the same location. This often poses problems when additional processing steps are required after the addition of reagents to the sample. Where samples are moved from one location to another within a test system, reproducible test conditions may be difficult to achieve. 
     Yet another disadvantage of known quantitative and qualitative test systems is that many of them utilize squeezable containers for storage and dispensing of reagent solutions. Despite the simple operation of squeezable containers, dispensing an accurate and precise amount of a reagent from a squeezable container is often problematic. Moreover, when an accurate and precise flow rate of a reagent is needed, squeezable containers may produce inaccurate and non-reproducible results. 
     Yet further, while many test systems are supplied with appropriate amounts of reagents, and typically follow relatively simple protocols, a problem frequently persists in that the accuracy and precision of test results become operator i.e. technique dependent. Such measurement is therefore often prone to errors. 
     Thus, many test systems are known in the art to qualitatively and quantitatively determine the presence of an analyte in a sample. However, current test systems tend to limit the complexity of a reaction sequence with which an analyte can be determined. Surprisingly, despite a growing number of new and useful diagnostic systems, there is no test system that permits a relatively quick and simple detection of an analyte in a sample that requires complex test procedures, without using sophisticated instruments. Therefore, there is still a need for methods and test systems that overcomes these limitations. 
     SUMMARY OF THE INVENTION 
     The present invention provides methods and apparatus for automated sample analysis in which a plurality of actuators are involved in moving a sample from one compartment to another, and appropriate reactants are combined with the sample in one or more of the compartments. 
     The actuators are preferably contained in a device that also has a detector, data reduction capability, and a printer. Contemplated signal detectors include a photomultiplier tube, a photodiode, and a charge-coupled device. 
     Steps contemplated to be performed automatically include aliquoting the sample, diluting the sample, contacting at least a portion of the sample with a reagent having a substantially selective binding affinity towards the analyte, a buffer, an acid, a base, or a wash solution. Contemplated reactants include sense and antisense nucleic acids, antibodies, solid-phase substrates, chromophores, and amplifiers. 
     Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings in which like numerals represent like components. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plan view of a disposable diagnostic container according to the present invention. 
     FIG. 2 is a plan view of an alternative disposable diagnostic container according to the present invention. 
     FIG. 3 is a plan view of another alternative disposable diagnostic container according to the present invention. 
     FIG. 4 is a plan view of another alternative disposable diagnostic container according to the present invention. 
     FIG. 5 is a perspective view of an analyzer that cooperates with the containers of FIGS. 1-4 to determine an analyte in a sample. 
     FIG. 6 is a schematic of actuators that can be used in conjunction with the containers of FIGS. 1-4 to determine an analyte in a sample. 
    
    
     DETAILED DESCRIPTION 
     FIG. 1 is a plan view of a disposable diagnostic container  10  according to the inventive subject matter, generally comprising a pouch having a sample inlet port  12 , a plurality of compartments  13 ,  22 ,  26 ,  28 ,  30 , and  32 , as well as passageway  16  coupling the inlet port  12  with compartment  13 , and portals  24 ,  34 ,  36 ,  38  and  40  interconnecting the various compartments. 
     Container  10  is a relatively flat, laminated plastic pouch measuring about 8.5 cm by about 19 cm, and about one millimeter thick, in which the compartments, inlet port, passageway and portals are all defined by heat sealing. The nature and dimensions of the container, arrangement of compartments and interconnections, as well as the contents of the compartments will, of course, vary from embodiment to embodiment, and those skilled in the art will recognize that the embodiment of FIG. 1 is merely exemplary of an enormous number of such possible containers. 
     The size of the container, for example, largely depends on the volume of reactants to be contained, although it is contemplated that practical containers will typically be sized to define a volume in the range of between 50 μL and about 5 milliliters. Suitable containers may have many different shapes, so long as the shape permits contact of at least one side of the container with a plurality of actuators. Preferred shapes are flat, envelope-like shapes, but box-like, round, hemispherical, or even spherical shapes, are also contemplated. 
     The opposing top and bottom sheets forming container  10  may advantageously be formed from a thermoplastic material, including polypropylene, polyester, polyethylene, polyvinyl chloride, polyvinylidene chloride, and polyurethane. Such sheets are contemplated to have a relatively uniform thickness between about 0.05 mm to about 2 mm. The opposing sheets need not be fabricated from the same materials. For example, one sheet may comprise a reflective foil, and the other sheet may comprise a transparent or translucent plastic. The use of foil can help promote temperature stability, and can serve as an additional moisture and oxygen barrier. Foil can also enhance thermal transfer from a heating source to a sample or reagent. 
     Preferred containers are flexible, either in whole or in part. Flexibility as characterized herein is the capability of yielding to a reasonable force by temporarily changing shape without damaging the structure or material. A reasonable force, as used herein, is a pressure, typically below 5 lb/in 2 . For example, a preferred flat, envelope-like container is sufficiently flexible to be wrapped around an inch diameter cylindrical object without breaking or tearing the container. In another example, a portion of a container may advantageously be sufficiently flexible to displace a volume carried within that portion without rupturing the outer walls. The container may furthermore have a plurality of openings. The number of openings may vary considerably between at least one opening and twenty openings or more. Such openings may have a closing mechanism, be sealable or permanently open. Furthermore, some of the openings may be in liquid communication with each other, or may be used as a vent or an overflow. The container is furthermore characterized by having a plurality of compartments. 
     Container  10  also includes attachment holes  42  for mounting on alignment posts in an analyzer  400 . Alternative attachment devices or methods are also contemplated, including hooks, loops and other mounting attachments coupled to the container  10  at appropriate locations. It is further contemplated that container  10  may be devoid of mounting components. 
     One or more labels (not shown) may also be affixed to the container  10 . Labels may indicate identification marks, information relating to the type of diagnostic test being conducted, as well as patient information, test result data, or other information. The label(s) may optionally be removable, and may, for example, be removed from the container  10  to be placed in a patient&#39;s medical file, thereby eliminating the need for transferring data with attendant possibility for error. 
     Inlet port  12  serves as an entry point for receiving samples or other materials. Many configurations are contemplated, although it is preferable that the entry point uses some sort of common connection mechanism. For example, the entry point  12  in FIG. 1 is a female portion of a Luer lock mechanism. Alternative entry ports may be either simpler or more complex, and may contain a padding that can be punctured or pierced using a needle. 
     Contemplated entry points may also be placed elsewhere on a container other than as depicted in FIG.  1 . For example, a suitable entry point for a solid material may be formed as a simple slot in one of the sheets forming the top or bottom of the container. Such an entry point may be well suited for receiving a relatively solid piece such as a tissue or mineral sample, and may be sealable by a flap or tape mechanism. 
     Compartments  13 ,  22 ,  26 ,  28 ,  30 , and  32  are portions of container  10  that are fluidly separated from other portions of the container during at least some period of time. In general, compartments are separated from one another using at least one continuous element that contacts at least one of the walls of the container. For example, if the container is a cylinder, the continuous element could be a divider that is more or less perpendicular to the longitudinal axis of the cylinder, and contacts the inner circumference of the cylinder. Where the container is a flat bag, the continuous element may advantageously comprise a heat seal between opposing sides, in a form enclosing a defined space. 
     The volume of preferred compartments may advantageously vary between about 3% to approximately 90% of the total volume of the container. Such compartments may be filled with at sample, a reagent, or air, but the compartment may also have essentially no void volume. By way of example, compartment  22  may be designed to contain about 1 ml of a binding reactant, and wash compartment  28  may be designed to hold up to about 5 ml of a solvent solution. 
     At least some of the compartments may advantageously comprise a transparent portion through which a signal can be detected, or the progress of a reaction can be monitored. In such instances it may also be advantageous for an opposing surface to exhibit a reflective surface to improve signal detection. Compartments may also be shielded, for example against heat, light, or other radiation. 
     Compartments may have one or more openings, such as those at portals  34 ,  36 ,  38 , and  40 . Such openings may be in permanent liquid communication with the rest of the container, for example, by an incomplete wall surrounding the compartment. Openings may also be temporarily closed. For example, a breakable seal may form the opening, which separates the compartment from the rest of the container, until an opening force breaks the seal. Typically, the breakable seal is a chevron break point allowing a fluid to pass under about 5-15 psi. In another example, the opening comprises a one-way valve, which permits only a unidirectional flow of material when a pressure difference is applied between the ends of the valve. In yet a further example, the opening may be temporarily closed by a closing force. Typically, the closing force is delivered via a compression pad from outside the container, which effects a temporary physical separation of the compartment from the rest of the container. 
     Passageway  16  and portals  34 ,  36 ,  38  and  40  serve to fluidly connect various compartments and other spaces within the container, and with the external environment. The term “fluidly connect” specifically includes movement of any fluidizable composition, whether a liquid, gas, or fluidized solid. In many instances the fluid will be intended to move in a single direction only, but in other instances it may be advantageous to move at least a portion of a fluid in both forward and backwards directions. 
     In some cases compartments or other spaces may be separated by a barrier for a period of time, and it is contemplated that the barrier will at some point be breached. In such instances the separated compartments or other spaces are considered to be “fluidly connectable.” 
     FIG. 2 depicts an alternative configuration in which a container  100  has an entry slot  12 A instead of an entry port. The slot  12 A is preferably sealable such that a liquid sample placed into container  100  does not leak out. Entry slot  12 A can advantageously be located within a plastic or other ring  15 . Ring  15  can be attached to the container  100  and fitted with an attachable cover (not shown) such that any liquid inserted into entry slot  12 A does not leak out of the container. 
     FIG. 3 depicts an alternative configuration in which a container  200  includes an overflow compartment fluidly coupled, or fluidly coupleable to compartment  18 . Compartment  18  also contains a volumetric zone  14  that is externally partitionable to define a fixed volume to be used in a diagnostic test. For example, assuming the fixed volume is about 100 μl, fluid-receiving portion  18  can receive an input volume that is greater than about 100 μl, such as 150 μl. In this case, after receiving the 150 μl of sample, volumetric zone  14  can be externally partitioned such that the fixed volume, about 100 μl, is defined and then used for the diagnostic test with the excess volume, about 50 μl, being moved into overflow portion  20 . The excess volume moved into the overflow portion would not be used in the diagnostic test since only the fixed volume of a sample typically is used to perform the diagnostic test. This externally partitionable volumetric zone  14  provides a means for quantitatively analyzing a sample. 
     Partitioning volumetric zone  14  typically involves two steps. The first step involves using at least one movable object such as a compression pad to apply pressure to all the areas around the region defining the fixed volume with the exception of the area providing a fluid connection to the overflow portion  20 . This partially surrounds the region defining the fixed volume while allowing any excess volume to move into overflow portion  20 . The second step involves using at least one movable object such as a partitioning edge to separate the excess volume from the fixed volume. This completely surrounds the region defining the fixed volume. A compression pad and partitioning edge can be made from any material provided the fixed volume can be defined. It is noted that the positioning of the movable objects can be adjusted such that the applied pressure can define any particular volume as the fixed volume. 
     FIG. 4 depicts an alternative configuration in which a container  300  has additional compartments  102 ,  104 ,  106  and  108 . The overflow compartment  20  depicted in FIG. 4 will have the same configuration as depicted in FIG. 3 once a seal is placed along reference line B—B. In this embodiment compartments  102 ,  104 ,  106 ,  108  have portions comprising reagent compartment  22 , reaction compartment  26 , substrate compartment  30 , and wash compartment  28 , respectively. Once a seal is placed along B—B, these compartment portions can become the reagent compartment  22 , reaction compartment  26 , substrate compartment  30 , and wash compartment  28  depicted in FIG.  3 . In addition, compartments  102 ,  104 ,  106 ,  108  have removable delivery portions  110 ,  112 ,  114 ,  116 , respectively. Further, compartments  102 ,  104 ,  106 ,  108  have fluid input ports  118 ,  120 ,  122 ,  124 , respectively. Thus, compartment  102  has a portion that corresponds to binding-reagent compartment  22 , a removable delivery portion  110 , and a fluid input port  118 ; compartment  104  has a compartment portion that corresponds to reaction compartment  26 , a removable delivery portion  112 , and a fluid input port  120 ; and so forth. 
     Container  300  can be fabricated as follows. With reference to FIG. 4, an appropriate fluid is inserted into the removable delivery portion through the fluid input port of each compartment. In an immunoassay, for example, a fluid containing at least one binding pair member can be inserted into removable delivery portion  110  of compartment  102 ; a fluid containing a solid material can be inserted into removable delivery portion  112  of compartment  104 ; a fluid containing a substrate can be inserted into removable delivery portion  114  of compartment  106 ; and a wash solution can be inserted into removable delivery portion  116  of compartment  108 . After inserting the appropriate fluid into each removable portion, the input port of each compartment can be sealed such that the inserted fluids remain within the compartment. This can be accomplished by heat sealing along reference line A—A. To help minimize the number of bubbles introduced into each compartment, each fluid can be positioned proximal to the compartment portion of each compartment before sealing the fluid input ports. To accomplish this, the container can be positioned such that gravity forces each fluid toward each compartment portion. 
     After sealing the fluid input ports, at least a portion of each fluid can be moved from the removable delivery portion of each compartment to the compartment portion of each compartment. Again, to help minimize the number of bubbles introduced into each compartment, each fluid can be positioned proximal to the compartment portion of each compartment before moving the fluids. Any process can be used to move the fluids from the delivery portion to the compartment portion. For example, gravity and/or pressure can be used to move the fluid into compartment portion of each compartment. Once at least a portion of each fluid is moved to the compartment portion of a compartment, that portion can be sealed from the delivery portion of each compartment such that the fluid within the compartment portion remains within the compartment portion. For example, a seal can be placed along reference line B—B. The delivering portion of each compartment can then be detached from the container by any suitable means, such as cutting along reference line B—B. In this case, detachment of the delivery portion of each compartment results in a diagnostic device as depicted in FIG.  3 . 
     In FIG. 5 an analyzer  400  generally comprises a main section  410  having a container receiving zone  412  with alignment posts  414 , a door  420 , multiple actuators  430 , a detector  440 , a printer  450 , and an interface  460 . Analyzer  400  is shown with an exemplary workpiece container  200 . 
     The main section  410  houses essentially all of the electronic or other circuitry needed to complete the contemplated tests. Of course, main section  410  can be designed using any suitable shape and dimensions, and can be formed from plastic, metal, or any other suitable materials. 
     Receiving zone  412  cooperates with door  420  to receive container  10  during the contemplated testing. In alternative embodiments a door is not needed at all, and the container can instead be inserted into an access slot. Alignment posts  414  may be configured in any suitable fashion, and can be eliminated altogether. 
     Actuator group  412  is used to deliver one or more forces to the container  10 , with the object of affecting some material with container  10 . Examples of actuators that may form part of group  412  are compression pads, roll bars, or wheels. Contemplated actuators may also have one or more additional functions, including heating, cooling, and delivering a magnetic force. For example, an actuator may heat inactivate an enzyme, or warm a reaction to a desired temperature. In another example, an actuator may be used to concentrate an analyte by binding it the surface of a magnetic bead. Actuators may also be employed to modify a volume occupied by fluids, solids, or air. The fluids may, for example, include a buffer, a sample, a reaction mixture, a reagent solution, etc. The solids may include paramagnetic beads, and the gases may include nitrogen or argon as protective agents, or CO 2  as a byproduct of a chemical reaction. 
     Where an actuator comprises a compression pad, the pad can be made from any material suitable for exerting an appropriate force to a portion of a container, in an appropriate pattern. Typically, a compression pad is a substantially flat surface, and has a shape corresponding to the shape of a compartment or passageway. Where an actuator is employed to otherwise seal a partition, a partitioning edge can be provided, preferably in the form of a wedge or a compression pad having a protrusion. 
     Detector  440  is essentially one, or any combination of signal detectors used to detect a signal generated through use of the container. Contemplated signal detectors include a photomultiplier tube, a photodiode, and a charge-coupled device. It is optional to include detector  440  in analyzer  400 . 
     Printer  450  is used to print information on any combination of human or machine-readable formats, including printing on a paper label or sheet. It is optional to include a printer in analyzer  400 . 
     Interface  460  can be any type of electronic or other means of exchanging information with another device. A typical interface is a common RS232 (serial) data port. 
     Not shown are other options for analyzer  400 , including a scanner than can detect a bar code, or other hand or machine written information included on a label. 
     FIG. 6 depicts further detail of the actuator group  412  described with respect to FIG. 5, and cooperates with the container  200  of FIG.  3 . It should be understood, however, that actuator group  412  could be employed with many different containers besides the specific configuration of container  200 , and that a generic actuator group can be employed with a very large number of containers and corresponding test protocols. 
     With reference to FIG. 6, actuator  412  has a series of compression pads that correspond to the various compartments of a diagnostic device, for example, device  200  depicted in FIG.  3 . Each compression pad can serve to apply external force to a particular region of the device such that fluid is moved. For example, a compression pad can be used to apply 5-50 psi of fluid pressure to a chevron break point within a compartment. Typically, two compression pads correspond to each compartment having a chevron break point. One compression pad is used to move fluid toward the chevron break point while the other is used to apply the force to move fluid through the chevron break point. In addition, the compression pad proximal to the chevron break point can be used to prevent movement of fluid between compartments, if necessary. 
     With reference to FIG. 6, actuator  412  has binding-reagent compartment compression pads V 01 , V 03 . Compression of binding-reagent compartment compression pad V 01  followed by compression of binding-reagent compartment compression pad V 03  can cause a fluid within binding-reagent compartment  22  of device  200  to pass through chevron break point  24  of device  200 . In addition, binding-reagent compartment compression pad V 03  can serve to prevent movement of fluid between compartments. 
     Actuator  412  also has volumetric zone compression pads V 03 , V 04 , V 07 , V 10 . Volumetric zone compression pads V 03 , V 04 , V 07  can serve to surround partially an area that defines a fixed volume of sample. Volumetric zone compression pad V 10  can serve to move a fluid from one compartment to another. In addition, actuator  412  has a partitioning edge V 08  that can serve to define a fixed volume. Partitioning edge V 08  can prevent fluid from moving between, for example, fluid-receiving portion  18  and overflow portion  20  of device  200 . 
     Actuator  412  also contains a reaction compartment compression pad V 09 . In addition to being able to move fluid from a reaction compartment, reaction compartment compression pad V 09  can rotate such that the magnetic force created by permanent magnet V 15  also rotates. A movable magnetic force can be used to move paramagnetic particles within a reaction compartment such that assay kinetics are increased. In addition, a magnetic force provided by permanent or electro-magnet can be used to hold paramagnetic particles in a particular location. 
     In addition, actuator  412  has substrate compartment compression pads V 06 , V 11 ; wash compartment compression pads V 05 , V 12 ; and waste-receiving compartment partitioning edge V 02 . These compression pads can be used to move fluid while waste-receiving compartment partitioning edge V 02  can be used to prevent fluid movement between, for example, reaction compartment  26  and waste-receiving compartment  32  of device  200 . 
     An analyzer apparatus can have any type of signal detection mechanism including, without limitation, a photomultiplier tube, photodiode, and charge-coupled device. With reference to FIG. 5, analyzer apparatus  400  has a photomultiplier tube  414 . In addition, shutter  416  can be used to protect photomultiplier tube  414 . 
     The analyzer can be programmable such that the compression pads and partitioning edges apply particular external force at particular times during the diagnostic test. In addition, the analyzer apparatus can have an alignment means (e.g., a plurality of pins) for positioning the diagnostic device. Further, the analyzer can have pressure sensors on either side of each compression pad and partitioning edge. These sensors can be used to determine and regulate the amount of pressure being applied. In addition, these sensors can be used to determine whether each compression pad and partitioning edge is working properly during operation. 
     The following methods are examples of operations during a test. These methods involve using device  200  with reference to the actuator components depicted in FIG.  6 . The number zero (0) means “off” or no external force applied and the number one (1) means “on” or external force applied. 
     
       
         
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 V01 
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                 To measure a fixed volume of a sample within volumetric zone 20 as a function of 
               
               
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                 To mix a sample with binding reagent compartment 22, incubate, and mix with 
               
               
                 magnetic particles within reaction compartment 26: 
               
             
          
           
               
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                 To wash the paramagnetic particle: 
               
             
          
           
               
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                 To add substrate from substrate compartment 30: 
               
             
          
           
               
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     Method of Use 
     In general, a sample is deposited into inlet port  12  under pressure, and travels to sample compartment  13 . Excess sample beyond the capacity of compartment  13  spills over into a spillage compartment  20 , which serves to aliquot the amount of sample in compartment  13 . A first reactant from compartment  22  is added to the sample, and after appropriate incubation the sample is shunted to reaction compartment  26 . Reaction chamber  26  may contain additional reactants, and still further more reactants can be added from substrate or other reactant compartment  30 . At one or more points in the processing stage the sample can be washed by a wash fluid from wash compartment  28 . Waste material is forced into waste compartment  32 . During these processes, various reactions take place with respect to an analyte within the sample, and a color or other detectable signal is produced that corresponds to the amount or existence of analyte. The signal is “read” through one of the side walls of compartment  26 . 
     As used herein, the term “sample” refers to any solid, fluid or gaseous material, which contains at least a portion that can be tested for an analyte. Contemplated solid samples include organic materials, inorganic materials or a mixture of organic materials and inorganic materials. Contemplated organic materials include macromolecules, and assemblies of macromolecules, cells, and tissues. Examples are drugs, viruses, bacterial or eukaryotic cells, and vertebrate tissues. Contemplated inorganic materials include salts, complexes or mixtures thereof, for example, mineral salts and mineral compositions. Liquid samples preferably include water or chemically homogeneous fluids, but may also include mixtures of various liquids with other liquids or components, for example water, petroleum, or coffee. Especially contemplated herein are liquids that comprise complex mixtures of a fluid phase and dissolved or undissolved solids. Examples are bodily fluids, wastewater, beverages and so on. Gaseous samples may include relatively pure gases, but also complex mixtures of relatively pure gases with other gases or vapors. Examples are ambient air and air with various organic contaminants including NO 2 , CO, benzene and so forth. 
     As used herein, the term “analyte” refers to any component in a sample that is to be analyzed. Analytes are generally at least partially soluble in a solvent, or at least miscible in a fluid. Analytes may be an organic, organometallic, inorganic, or any reasonable combination thereof. Contemplated organic compounds range from complex compounds to very simple compounds. For example, analytes of interest include proteins, growth factors, hormones, transmitters, enzymes, clotting factors, IGF- 1 , bacteria, virus, yeast, acteylcholine, caffeine, benzo(a)pyrene, and dioxin, drugs, calmodulin and Pb-tetraethyl, alkali metal and alkaline earth metal ions such as K + , Na + , Ca 2+ , Mg 2+ , as well as salts. 
     As used herein, the term “reactant” refers to any composition that can react with a component of a sample, or another reactant, in performing a determination. This includes binding reagents, solid-phases, solvents, wash compositions, signal generators, and so forth. In general, practically any reactant that can be utilized at a lab bench test can also be employed in connection with the containers and devices contemplated herein. Reactants may be contained separately, or in combination, in the various compartments as appropriate for a given test protocol. 
     One particularly contemplated class of reactants includes test reagents. For example, reactant compartment  22  may contain a fluid that comprises at least one binding pair member. A binding pair member can be any molecule that specifically binds another molecule to form a binding pair, including an antibody or an antigen that specifically binds that antibody. Other contemplated binding pair members include antibody fragments having specific antigen binding capacity, receptors and ligands, sense and anti-sense nucleic acids, metal ions, chelating agents, and aptamers. 
     In many tests, reagent compartments such as compartment  22  will contain more than one of the reactants for the test being performed, and in the case of assays involving binding, such reactants will often comprise more than one binding pair member. For example, reagent compartment  22  may advantageously contain a first binding pair member and a second binding pair member each having specificity for a different epitope present on an analyte to be detected. In addition, the first binding pair member can be conjugated to a molecule that allows for analyte detection and the second binding pair member can be conjugated to another binding pair member such that an analyte-multiple binding pair member complex can be captured. For example, the fluid within reagent compartment  22  can contain two different antibodies that each bind analyte X present within a sample. The first antibody can be conjugated with an enzyme such that the amount of enzymatic activity can be correlated with the amount of analyte X. The second antibody can be conjugated to biotin such that any complex containing analyte X and the antibodies are captured by streptavidin. It is to be understood that any particular combination of binding pair members can by used to conduct a particular diagnostic test. In another embodiment, a labeled antigen may be used, for example, in competitive assays. 
     Another contemplated class of reactants includes labels that allow for analyte detection. Once again, as with other aspects of the inventive subject matter, virtually any label that can be employed in a bench test can also be employed in conjunction with the teachings herein. For example, labels can include acridinium esters, isoluminol derivatives, fluorophores, enzymes, and any combination thereof, and enzymes such as alkaline phosphatase, peroxidase, xanthine oxidase, and glucose oxidase can be coupled to a binding pair member to detect the presence of an analyte. 
     Another contemplated class of reactants includes solid-phase materials, including polypropylene, polyester, polystyrene, polyurethane, nylon, styrene, glass fiber, and thermoplastic. Such solid-phases can be employed in substantially the same manner as employed in ordinary lab procedures. In some classes of tests, for example, a solid-phase may be employed to bind a diagnostically useful compound such as streptavidin. Of special interest are various beads or other particles, and especially paramagnetic particles, which may advantageously be coated with a binding member to bind a target substance. The paramagnetic particles can then be moved under the influence of a magnetic force to separate the bound target substance from the remainder of a sample. A particularly useful application of paramagnetic particles involves the separation of plasma from whole blood. In an exemplary process, whole blood can be combined with a first antibody that has a high specificity for a red blood surface antigen, and subsequently combined with paramagnetic beads to which a second antibody is bound. The second antibody binds to the first antibody, and the red blood cells can be gently pulled away from the remaining plasma under the influence of a magnetic field. 
     It is specifically contemplated that a solid phase may be moved from one compartment to another. Beads may be moved in that manner, as can a “puck” that alters fluid flows within or between compartments. 
     Reactants may also comprise a solvent or other simple fluid. The fluid may be used for many purposes, including maintaining the stability of a reactant, or to fluidize a substance that would otherwise be in a solid state, or for use as a wash. Contemplated fluids for these purposes include preservatives, detergents (e.g. CHAPS, Tween-20, Triton X-100, cholate, and SDS), proteins (e.g. BSA), saline, phosphate-buffered saline, tris-buffered saline, water, and compatible aqueous organic solvents. 
     Another particularly contemplated class of reactants is a filter material. All of the known filter materials are contemplated, including nitrocellulose, steel wool, and so forth. 
     A very large number of test protocols can be accomplished in accordance with the teaching principles set forth herein. In addition to the tests referred to herein, multiple tests can be run on a single sample by aliquoting portions of the sample to multiple reaction chambers, and additional compartments can be added to accommodate additional reagents. Agitation, heating and other operations can be accomplished by the appropriate actuators, and time delays of anywhere from a fraction of a second, to one or more minutes can readily be accommodated. Thus, the teachings herein should not be read as limiting the application to any particular assay or protocol, or to any particular container or detector. 
     EXAMPLE 
     Diagnostic Assay 
     With respect to FIG. 1, an operator selects a container  10  adapter for an appropriate test, and inserts a 100 μL sample (calibrator, controls, or patient samples) into entry port  12 . The sample passes under pressure to compartment  13 . 
     The container  10  is then placed in an analyzer  400 , and employing various actuators the analyzer  400  takes control of the testing protocol. First, passageway  16  is sealed, preferably by a sealing actuator compressing the opposing top and bottom sheets of the container  10  at appropriate places. Compartment  18  is then squeezed to aliquot a specific desired volume of sample, with excess sample passing into compartment  20 . The connection between compartments  18  and  20  is then actuator sealed. 
     Using another actuator, 100 μL of an antibody solution containing biotinylated monoclonal anti-PSA antibody and polyclonal alkaline phosphatase-labeled antibody is passed from compartment  22  into compartment  14 . After addition of the antibody solution, the sample is incubated for 5 minutes at 37° C. 
     Using another actuator the sample is passed to compartment  26 , in which was stored 25 μL-100 μL of a homogenous suspension of streptavidin-coated paramagnetic particles. Using one or more actuators, a shaking or vibrating motion is imparted to the sample, and further incubation takes place for an interval, such as 2 minutes at 37° C. 
     Using other actuators, about 1.0 ml of a wash solution is passed from wash compartment  28  into compartment  26  to wash the sample. Further incubation is allowed to take place, during which the paramagnetic particles sediment. Sedimentation may be enhanced using a magnetic force from a permanent magnet. 
     Using other actuators, about 50 μL-100 μL of a chemiluminogenic substrate (ImmuGlow) is added to compartment  26  from compartment  30 . 
     Using other actuators, about 100 μL-300 μL of wash is added to the sample in compartment  26  from compartment  31 , and agitated for several seconds. The wash cycle is repeated three to four times. 
     Chemiluminescence is measured after a specified interval, for example, 15 seconds following addition of the substrate. Determination of the unknown is computed using a standard-dose-response curve. Depending on the test, additional measurements can be made at intervals, such as a minute or longer. 
     Thus, specific embodiments and applications of methods and apparatus for performing tests have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims.