Abstract:
The present invention is a flow cell and method for use in microfluidic analyses that presents highly discrete and small volumes of fluid to isolated locations on a two-dimensional surface contained within an open fluidic chamber defined by the flow cell that has physical dimensions such that laminar style flow occurs for fluids flowing through the chamber. This process of location specific fluid addressing within the flow cell is facilitated by combining components of hydrodynamic focusing with site specific cell evacuation. The process does not require the use of physical barriers within the flow cell or mechanical valves to control the paths of fluid movement.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a division of U.S. application Ser. No. 11/739,727, filed on Apr. 25, 2007, the entirety of which is hereby expressly incorporated by reference herein. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to microfluidic devices, and more particularly to such devices that are used in the analytical analysis of fluid samples that include a detection device. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the process of analytical analysis of fluid samples (biologic samples, chemicals reagents, and gases) it is common for test samples to be passed through a chamber containing either a detection substrate, or a transparent window allowing the interrogation of the sample by some form of energy or light. It is common for sample fluids to be delivered and removed from these “detection chambers” using a continuous flow of transport fluid entering the chamber from one end and exiting the chamber at another. Thus these chambers are termed detection “flow cells”, and the analysis techniques that utilize them are termed “flow based” detection methods. During flow based analysis, sample fluids to be tested are delivered as discrete volumes, or ‘plugs’, within a stream of continuously flowing buffer passing through the flow cell and over the detection substrate. The accuracy, sensitivity, and applicability of flow based analysis techniques are highly dependent upon the process and characteristics of the sample fluid delivery to, and removal from, the detection flow cell. 
         [0004]    Researchers in a wide variety of fields such as medicinal science and environmental analysis, to name just a few, need to characterize the interactions of biologic molecules found in human, animal, or plant fluids and tissues. These characterizations commonly involve bringing two or more different types of sample molecules into physical contact with each other for a set period of time and then measure if, for example, they have combined to form a molecular complex, or if either has caused a change to the physical structure or function of any of the other reactants. Understanding the kinetics (speed) and affinity (strength) of these molecular interactions are just two of the parameters often measured during these characterization procedures, termed ‘molecular interaction analyses’. Typically when utilizing flow cell based analysis techniques during molecular interaction analysis, a population of one of the interacting molecules is permanently attached, or ‘immobilized’, onto the detection substrate or window within flow cell. Sample containing the other molecule(s) to be investigated are then passed through the flow cell so they have the opportunity to interact with the immobilized molecules and those interactions measured. 
         [0005]    So called biosensors, or “label-free” analysis techniques, commonly utilize detection flow cells and flow based sample delivery methods to “present” test samples to be analyzed to the detection sensor surface or substrate. The use of flow based sample delivery in label-free bio sensor instruments can greatly increase the amount of information these techniques can generate about the molecular interactions being investigated. Biacore instruments sold by GE Healthcare are a well known example of label-free analytical biosensors used in biological research for molecular interaction analysis studies. In the case of Biacore instruments, an optical detection technique called Surface Plasmon Resonance (SPR) is employed to measure mass changes on metal surfaces. These mass changes on the sensor surface result from the addition or subtraction of molecules onto the surfaces due to the interaction of molecules with either the sensor surface itself or another molecule attached to the surface. Other examples of analysis techniques that characterize molecular interactions using label-free detection methods include Dipolar Interferometry, Quartz Crystal Microbalance (QCM), Surface Acoustic Wave (SAW), and micro-cantilevers. Aside from eliminating the additional analysis steps, reagents, and sample preparatory requirements of label based testing methods (MA, ELISA, and Fluorescence techniques), label-free analysis enable the measurement of the molecular interactions under investigation to be recorded as they occur. These real-time analysis capabilities have the potential to provide a great deal of information in addition to confirming the specific binding of target molecules, as is arguably the only capability of label based techniques. Under the proper conditions, real-time, label-free analysis techniques have the ability to determine the speed and strength of molecular interactions, and in some cases, if those interactions resulted in any structural changes to the test molecules. But it has been well documented that these real time analysis capabilities, as well as the accuracy, and sensitivity of label-free detection techniques in general, are highly dependant on the quality of the corresponding flow based sample delivery methods. 
         [0006]    For example, one critical aspect of sample delivery in flow cell based analysis techniques is the fast and efficient transition from one reagent to the next within the flow cell. This need for fast and efficient transition between reagents is most clearly demonstrated when characterizing molecules that exhibit very low binding affinity (weak in ‘strength’) for one another. The association rates (molecules coming together), and dissociation rates (falling apart), termed “kinetic rates”, associated with these low affinity interactions often occur within the first few seconds after the test molecules are brought into contact with one another or separated. Thus, the capability to obtain accurate measurements just after the test molecules have come into contact, and immediately following their separation, is crucial to accurate kinetic rate characterization of low affinity molecular interactions. 
         [0007]    During automated testing procedures using flow cells, it is commonly advantageous for liquid handling devices to transfer the sample volumes to be analyzed from their storage containers or vials to the chamber or detection flow cell as a plug volume pushed through tubing pathways by another liquid termed the running buffer. As the plug volume of sample liquid is pushed through the tubing of the liquid handling unit, mixing between the plug and the running buffer will often occur creating a volume of liquid at the front and back of the sample plug that is a variable gradient of sample and running buffer. As the concentration of this mixture is unknown, including it in the final analysis of the sample can often interfere with the accuracy and sensitivity of testing. 
         [0008]    Thus, it is common for a “cutting” event to be performed on the sample plug volume just prior to its introduction into the analysis chamber. These cutting events typically involve some initial portion of the sample plug volume being directed to a waste just prior to the sample analysis process. Often mechanical valves are used to perform this function but due to limitations in valve technology related to sample waste, valve dimensions, and poor robustness, these structures and methods are not ideal. 
         [0009]    Additionally, as the reagent plug enters the flow cell it pushes assay buffer out, with the reverse occurring at the end of the plug injection. During this process, a period of transition occurs where the flow cell, and thus the detection substrate, is exposed to a concentration gradient or mixture of sample and buffer. During these ‘transition periods’, accurate determination of kinetic rates is not possible as the true concentration of test sample exposed to the detection surface is unknown. Thus, the ability to quickly switch from one fluid to the next within the flow cell during analysis, i.e., the delivery of highly discrete volumes of sample fluid having a clean leading edge without a concentration gradient within a continuous flow of transport fluid, is critical to obtaining as much usable data as possible. 
         [0010]    The vast majority of current flow based sample delivery technologies, even on a micro-fluidic level, do an inadequate job of efficiently transitioning between samples or sample and buffer. It is not uncommon for microliters and even ten&#39;s of microliters of fluid to pass over the detection surface before contacting solution that is 100% test reagent. As typical test volumes can be less than fifty microliters, flowing at ten&#39;s of microliters per minutes, these long transition times severely affect measurement capabilities. The long transition times are mainly due to the physical design of valve technology built into the sample delivery systems, which can often only be effectively utilized at some distance from the flow cell and detection surface. Thus the reagent plug must travel a distance before contacting the detection surface, during which reagent solution mixing will occur. Microfluidic tubing designs employing micro valves have been used with moderate success to overcome this situation as they minimize liquid travel and the micro valves can be located much closer to the detection flow cell. But, due to their design and small size, these valves are costly, often mechanically unreliable, and susceptible to clogging. 
         [0011]    Another critical aspect of sample delivery in regards to kinetic rate analysis is the ability for sample molecules to efficiently diffuse from the sample plug onto the sensor surface as the sample plug passes over. It has been well documented that inefficient transport of sample molecules to the sensor surface, termed “mass transport limitations”, results in inaccurate estimations of kinetics rates. Efficient molecular diffusion from the sample plug to detection surface is facilitated by passing the sample over the detection substrate as quickly as possible (i.e. fast sample flow rates). But when considering the practical applicability of flow cell based analysis techniques, the requirement to pass sample over the detection surface at high rates of speed becomes a liability. 
         [0012]    As the physical nature of molecular interactions often means that sample molecules must be in contact for several minutes to obtain accurate measurements, high sample flow rates during analysis result in the consumption of large volumes of test sample. Historically the most common way to lower sample volume requirements while maintaining high analysis flow rates has been to minimize the size of the detection flow cells. But due to a variety of issues related to the different detection technologies (i.e. size of the detection substrates, electronics, and optics), and the need to interface those technologies with high performance and robust sample fluid delivery systems, there have been practical limitations to the miniaturization of detection flow cells. Thus, with the resource requirements to produce even the crudest biologic samples for testing being very high, and the fact that the new research disciplines such as Proteomics continue to expand the number of samples to be evaluated, there is an ever increasing demand to work with the smallest sample volumes possible. 
         [0013]    The next critical aspect when evaluating the applicability of a technology for molecular interaction analysis is the requirement to simultaneously evaluate large numbers of samples while still meeting the requirements of delivering highly discrete, and small volumes of sample at high rates of flow. This process of simultaneous multi-sample analysis is often referred to as High Throughput Sampling, or HTS. Often, based on the analysis methods used in conjunction with HTS, there is a desire in some instances to handle each sample analysis as a completely independent procedure, and in other instances to handle the multiple analyses using exactly the same procedure and reagents. Thus the ultimate applicability for high throughput analysis comes when the user can switch between “individual” and “common” processing of the multiple sample analyses at any time during the testing procedure. Often these variations in testing procedures represent nothing more than different reagents being applied to different test vessels at certain stages of the testing process. For test methods that employ the analysis of molecules coated onto an array surface, this process of individual and common handling of the multiple individual analyses becomes a process of individual and common “addressing” of different reagent fluids to the different locations of the array. In some steps of the assay procedure it is preferable that the same reagent can be addressed to more than one or all of the target locations on the array. In other cases it is desirable to address a different reagent onto each target location. 
         [0014]    In the past, a variety of techniques based on the manipulation of the process of Hydrodynamic Focusing have been employed in an attempt to address these requirements. The so called, “Hydrodynamic Addressing” and “Hydrodynamic Guiding” techniques, use guide fluid streams to position sample fluid streams over different sections of array surfaces within flow cell chambers. 
         [0015]    One example of a technique of this type is shown in published PCT Publication No. WO/2003/002985, which is incorporated by reference herein and as shown in  FIGS. 1 and 2 , discloses a method of operating an analytical flow cell device comprising an elongate flow cell having a first end and a second end, at least two ports at the first end and at least one port at the second end, comprises introducing a laminar flow of a first fluid at the first end of the flow cell, and a laminar counter flow of a second fluid at the second end. Each fluid flow is discharged at the first end or the second end, and the position of the interface between the first and second fluids in the longitudinal direction of the flow cell is adjusted by controlling the relative flow rates of the first and second fluids. Also disclosed are a method of analyzing a fluid sample for an analyte, a method of sensitising a sensing surface, and a method of contacting a sensing surface with a test fluid. 
         [0016]    Another example is found in PCT Publication No. WO/2000/056444 that is also incorporated by reference herein and as shown in  FIG. 3 , illustrates a composition of a liquid ( 26 ) that is caused to interact with a narrow band shaped area at a chosen position on a solid surface within a flow channel ( 12 ) by hydrodynamic focusing of a guided stream of said liquid between two streams of guiding liquid ( 28 ). By altering the ratio of the flow rates of the two guiding liquid streams, the position of the guided liquid stream is changed and further interaction with the solid surface takes place along a second band shaped area. Using two such flow channels it is possible to produce a two dimensional array of interaction sites. 
         [0017]    Still another example is disclosed in PCT Publication No. WO/2006/050617 which is incorporated by reference herein and illustrates in  FIG. 4   a - 4   g  a microfluidic device and its use for the production of micro-arrays, in particular for the detection of protein interactions, is described. The microfluidic device comprises a flow cell part ( 1 ) and a chip part ( 2 ) together forming at least two crossing, preferably perpendicular, closed channels ( 3 ,  4 ), said flow cell part forming open channels providing the bottom wall and at least part of the side walls, in particular three walls of said closed channels ( 3 ,  4 ), said closed channels ( 3 ,  4 ) being connected to at least three fluid providing means for generating at least three fluid flows ( 7 ) and said closed channels ( 3 ,  4 ) being designed and dimensioned such that the flow of at least three aqueous fluids streaming through each of said channels ( 3 ,  4 ) is laminar at least until after said crossing of said channels ( 6 ), said chip part ( 2 ) forming the top wall and optionally part of said side walls, in particular the fourth wall, of said closed channels ( 3 ,  4 ) and having a surface that is activatable by reaction with an activating molecule. 
         [0018]    However, these prior art techniques and structures shown in  FIGS. 1-4   g  are limited to addressing sample fluid streams in single dimensions within the array. Thus, if a surface array is viewed as an x-y grid, these techniques can either address only the entire x-row or the entire y-column with a single reagent. These techniques offer no remedy to address individual x-y locations, or “spots”, on the array independently severely limiting the flexibility of array design. Thus it is desirable when working with array based testing methods to have the ability to address each test location on the array as a completely individual entity in some instances, and in other instances to treat more than one or all of the test locations in the same manner. 
         [0019]    In summary, there remains a considerable need for greater control and flexibility in regards to the volume, speed, and location of reagent presentation to detection surfaces in flow cell based analytical testing technologies. 
       SUMMARY OF THE INVENTION 
       [0020]    According to a first aspect of the present invention, a flow cell device is provided that is capable of operation in a process termed “hydrodynamic isolation” in which highly discrete and small volumes of fluid are presented to isolated locations on a two-dimensional surface contained within an open fluidic chamber that has physical dimensions such that laminar style flow occurs for fluids flowing through the chamber. The device includes a number of reagent inlet ports that are disposed adjacent associated sensor substrates or detection windows. Located between the reagent inlet ports and the detection substrates are reagent evacuation ports. The evacuation ports operate to continuously withdraw a reagent being introduced into a continuous laminar flow of a guide fluid moving along the flow cell through the reagent inlet to enable the reagent to develop a clean leading edge without any appreciable concentration gradient to create problems with regard to the interaction of the sample with the detection substrate(s). Once the clean leading edge of the reagent sample has been created, the vacuum applied to the reagent sample from the evacuation port is stopped, such that the discrete volume reagent sample having the clean leading edge is introduced into the guide fluid flow to move along the flow cell and pass over the detection substrate to interact therewith. Immediately after passing the detection substrate, the reagent sample can be evacuated completely from the flow cell by another evacuation port located downstream from the detection substrate. Thus, the reagent sample is prevented from interacting with any other detection substrate present in the flow cell by removing the reagent sample from the laminar fluid flow moving through the flow cell using a vacuum, without any physical barriers within the cell to divert the fluids, and without the need for mechanical valves, which are difficult to manufacture and break easily. Therefore, the present invention enables discrete volumes of fluids to be injected through a flow cell, or addressed to a specific location within a flow cell, without the need for cumbersome and non-robust valves in the fluid tubing pathways leading up to the fluid inlet ports of the flow cell. This capability enables the design of extremely small array addressing microfluidic devices while maintaining, and in some cases exceeding, the level of functionality of other microfluidic and macrofluidic fluid delivery devices that utilize mechanical valves. 
         [0021]    According to another aspect of the present invention, the flow cell device of the present invention is formed to include a number of detection spots or substrates therein in the form of an array, with a reagent inlet port and a reagent evacuation port associated with each detection substrate. In this manner, the flow cell device is able to simultaneously introduce a number of reagent samples within the flow cell, addressing each of the reagent samples to a specific detection substrate, and preventing the intermixing of any of the introduced reagents with one another or with any detection substrates to which they are not addressed. Also, while the reagent inlet and evacuation ports are located and associated with each detection substrate in the flow cell, in one mode of operation it is possible to selectively operate the reagent inlet and evacuation ports to enable reagent samples introduced at separate reagent inlets to travel with the laminar guide fluid flow over multiple detection substrates to obtain multiple interactions of the sample with separate detection substrates prior to evacuating the reagent sample from the flow cell. 
         [0022]    According to still another aspect of the present invention, the flow cell is formed with multiple fluid inlets the allow the flow cell to be operated in a manner that allows the guide fluids introduced into the flow cell device through the fluid inlets to be moved across the flow cell through the use of hydrodynamic focusing to enhance the ability of the flow cell to address discrete fluid volumes onto specific spots in the hydrodynamic isolation process. Thus, the reagent samples introduced into the flow cell using the various reagent inlet ports and reagent evacuation ports can additionally be directed to specific detection substrates within the flow cell by the movement of the guide fluid streams into which the reagent samples are introduced prior to being evacuated from the flow cell. 
         [0023]    Numerous other aspects, features and advantages of the present invention will be made apparent from the following detailed description taken together with the drawing figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0024]    The drawing figures illustrate the best mode of currently contemplated of practicing the present invention. 
           [0025]    In the drawing figures: 
           [0026]      FIG. 1  is a schematic view of a first prior art flow cell device; 
           [0027]      FIG. 2  is a schematic view of the first prior art flow cell device of  FIG. 1  including a pair of detection surfaces thereon; 
           [0028]      FIG. 3  is a schematic view of a second prior art flow cell design; 
           [0029]      FIGS. 4   a - 4   g  are schematic views of a third prior art flow cell device; 
           [0030]      FIG. 5  is an isometric view of a first embodiment of a flow cell device constructed according to the present invention; 
           [0031]      FIG. 6  is a top plan view of the device of  FIG. 5 ; 
           [0032]      FIG. 7  is a bottom plan view of the device of  FIG. 5 ; 
           [0033]      FIG. 8  is a top plan view of the device of  FIG. 5  without a guide fluid stream; 
           [0034]      FIG. 9  is a top plan view of the device of  FIG. 5  with a guide stream being introduced into the device; 
           [0035]      FIG. 10  is a top plan view of the device of  FIG. 5  with a continuous laminar guide fluid stream flowing therethrough; 
           [0036]      FIG. 11  is a cross-sectional view of the reagent inlet and evacuation ports of the device of  FIG. 5  prior to introducing a reagent sample; 
           [0037]      FIG. 12  is a cross-sectional view of the reagent inlet and evacuation ports of  FIG. 11  when creating a clean leading edge for the reagent sample; 
           [0038]      FIG. 13  is a cross-sectional view of the reagent inlet and evacuation ports of  FIG. 11  when introducing the reagent sample into the device; 
           [0039]      FIGS. 14 and 14   a  are top plan views of the creation of the clean leading edge for the reagent sample shown in  FIG. 12 ; 
           [0040]      FIGS. 15   a - 15   d  are top plan views of a simultaneous hydrodynamic addressing process for each of the detection substrates of the device of  FIG. 5 ; 
           [0041]      FIGS. 16   a - 16   c  are top plan views of the hydrodynamic addressing process for a second detection substrate in the device of  FIG. 5 ; 
           [0042]      FIG. 17  is a top plan view of a second embodiment of the device of  FIG. 5 ; and 
           [0043]      FIG. 18  is a top plan view of a third embodiment of the device of  FIG. 5 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0044]    Referring now to the drawing figures in which like reference numerals designate like parts throughout the disclosure, a flow cell constructed according to the present invention is illustrates generally at  100  in  FIG. 5 . While shown as a rectangle in the preferred embodiment, the flow cell  100  can have any shape, as long as the dimensions of the chamber  100  induce laminar flow characteristics in the fluids flowing through the chamber  100 , and that the different fluid inlet and outlet or exhaust ports, to be discussed, are located in relation to each other on the chamber  100  such that all the required functions of hydrodynamic focusing and site specific evacuation are possible within the chamber  100 . 
         [0045]    The flow cell chamber  100  is formed by clamping a liquid sealing gasket  102  of known height between two solid surfaces  104  and  106  that form the large walls of the flow cell  100 . Thus, the gasket  102  is formed of a suitably flexible and fluid-impervious material, and forms a single continuous side wall around the periphery of the chamber  100 . However, it is also contemplated that substitute engaging or sealing structures (not shown), can be secured to one or both of the surfaces  104  and/or  106 , such that the gasket  102  is omitted, or positioned on top of one or more of these structures. These structures can take the form of walls formed integrally with one of the surface s  104  or  106 , or other types of suitable members that are attached in a sealing manner to one of the surfaces  104  or  106 . 
         [0046]    The large surfaces  104  and  106  are typically formed of any suitable lightweight and fluid-impervious material, and preferably a plastic material, as is known. Further, one of the large surfaces  104  or  106  of the flow cell  100  is made up of a flat surface into which multiple holes or fluid ports  108  have been cut. In  FIGS. 5-8 , this surface is surface  104 . Fluids are delivered into and out of the flow cell through these ports  108 , and as such this surface  104  is called the fluid delivery surface  104 . There is no requirement all fluid ports  108  must be designed into the same surface  104  or  106  of the flow cell  100 . In the above example, the surface  106  that makes up the opposing large wall or ceiling of the flow cell  100  opposite the surface  104  in which the ports  108  are formed is termed the sensor substrate surface, and can be fitted with either sensor substrates or detection windows  110 . These sensor substrates or detection windows  110  will constitute the sensor spots  110  within the flow cell  100  and represent the spots to be addressed with reagent using the hydrodynamic isolation process. Additionally, while the illustrated flow cell  100  has the sensor spots  110  on the opposing wall  106  of the flow cell  100 , based on the physical dimensions and design of the sensor substrates or detection windows forming the spots  110 , the sensor spots  110  could be located on the same wall  104  of the flow cell  100  as that in which the fluid ports  108  are formed. As the disposition of the fluid ports  108  on the surface  104  will define the areas  111  for sample addressing, it is only required that the sensor spots  110  are located in an optimum position within these addressable areas  111 . 
         [0047]    When the flow cell  100  is formed, the liquid sealing gasket  102  encloses the all fluid ports  108  and sensor spots  110  within the flow cell  100 . While the flow cell  100  illustrated contains only two sensor spots  110  on the sensor substrate surface  106 , it is contemplated that the flow cell  100  can be formed in a manner to include a sensor substrate surface or surfaces  106  containing hundreds and even thousands of sensor spots  110 . 
         [0048]    In the first embodiment of the flow cell  100  shown in  FIGS. 5-10 , the fluid delivery surface  104  is designed such that two main inlet ports  112  are positioned at one end of the fluid delivery surface  104 , and a single outlet, or main exhaust port  114  is positioned at the opposing end of the fluid delivery surface  104 . During operation of the flow cell  100 , continuously flowing guide fluid streams enter the cell through the main inlet ports  112  and, in most instances of operations, will exit the cell  100  through the main exhaust port  114 . This design ensures that all fluids entering the cell  100  will flow in a direction from the end of the flow cell  100  where the main inlet ports  112  are located towards the end of the flow cell  100  where the main exhaust port  114  is located. When describing its position within the flow cell  100 , the exhaust port  114  is said to be located downstream of the main inlet ports  112 . Additionally, the number of inlet ports  112  and outlet ports  114  can be altered as desired, so long as at least one inlet port  112  and at least one outlet port  114  are present to ensure proper movement of the fluids through the flow cell chamber  100 . 
         [0049]    In this embodiment of the flow cell  100  having only two (2) sensor spots  110 , four (4) additional fluid ports  108  are formed within the fluid delivery surface  104 . These additional ports  108  are positioned between the main inlet ports  112  and the main exhaust port  114  also formed in the fluid delivery surface  104 . In a particularly preferred embodiment, these additional ports  108  are aligned along the central axis  116  of the longest dimension of the flow cell  100 , i.e. down the middle of the cell  100 . Two of these ports, termed sample or reagent inlet ports (RIPs)  118  and  120 , are located downstream of the main inlet ports  112 , and just upstream of their respective addressable areas  111  within the flow cell  100 . The three other fluid ports  122 ,  124  and  126  are termed sample or reagent evacuation ports (REPs). REP  122  and REP  124 , are each positioned immediately downstream of their corresponding RIP  118  and  120 , respectively, such that any fluid entering the flow cell  100  from either RIP  118  or  120  will first pass over the corresponding REP  122  or  124  before contacting any downstream sensor spot(s)  110 . REP  126  is located just downstream of the general area of the upstream sensor spot  110  and just upstream of RIP  120 . REP  126  allows two independent samples or reagents to be passed over the upstream and downstream sensor spots  110  simultaneously without any mixing of the reagents using the process of hydrodynamic isolation within the flow cell  100 , as described below. 
       Hydrodynamic Isolation Process 
       [0050]    A. Control of Sample Fluid Stream Using Hydrodynamic Focusing 
         [0051]    A key component of the process of hydrodynamic focusing, as it relates to the present invention, is the ability to control the position and size of a stream of fluid  128  passing through a microfluidic flow cell  100  under conditions of laminar flow, using two or more guide fluid streams  130  and  132 . 
         [0052]    It is known that when two or more independent streams of fluid flowing under conditions of laminar flow, i.e., the streams each have a low Reynolds number, are in direct contact with each other and flow in the same direction, i.e. parallel to one another, there will be no mixing of the fluid streams other than by diffusion. Also, by varying the rates of flow of the different fluid streams in relation to each other, the size and position of the various streams can be altered. (“Biosensors and Bioelectronics Vol. 13 No. 3-4, pages 47-438, 1998”). In the case where two guide fluid streams  130  and  132  flow on either side of central fluid stream  128 , the width of the central fluid stream  128  can be controlled by manipulating the flow rates of the guide fluid streams  130  and  132  in relation to the central fluid stream  128 . For example, by changing the rate of flow of the central fluid stream  128  in relation to that of the guide fluid streams  130  and  132 , the width of the central fluid stream  128  can be narrowed by decreasing the central stream flow rate, or expanded by increasing the central stream flow rate. Also, by changing the flow rate of one of the guide fluid streams  130  or  132  in relation to the other, the position of the central fluid stream  128  within the flow cell  100  can be shifted from a central location towards either side of the flow cell  100 . 
         [0053]    As stated previously, the process of hydrodynamic isolation preferably incorporates the use of two guide fluid streams  130  and  132  to control the width and position of a central reagent sample fluid stream  128  introduced into, and flowing within the flow cell  100 .  FIGS. 8-10  illustrate of the action and flow path of the two guide fluid streams  130  and  132  within the flow cell  100  of the present invention. The guide fluid streams  130  and  132  each enter the flow cell  100  though one of the main inlet ports  112  located at the upstream end of the flow cell chamber  100 , and exit the flow cell  100  through the main exhaust port  114  located at the downstream end of the chamber  100 . The main inlet ports  112  are optimally positioned along the same x-axis coordinate within the flow cell  100 , and are spaced equidistant from the central y-axis of the flow cell  100 , along which the others ports  108  present in the cell  100  are preferably aligned. The two guide fluid streams  130  and  132  utilized in the preferred embodiment of the present invention are intended to flow at equal rates of speed at all times during the use of the flow cell  100  in the hydrodynamic process. Due to the laminar nature of the flow of the two guide fluid streams  130  and  132 , these streams do not mix because the surface tension for each fluid stream  130  and  132  at the interface  134  of the streams  130  and  132  forms a barrier between the fluid streams  130  and  132  along the interface  134 . However, in certain circumstances it is also contemplated that only one guide fluid stream  130  or  132  can be used in the flow cell  100  of the present invention, such as when only one sensor spot  110  is present in the flow cell  100 . 
         [0054]    During the use of the flow cell  100  in the hydrodynamic isolation process, a reagent sample fluid stream  128  enters the flow cell through one of the RIPs  118  or  120  located on the central axis  116  of the flow cell  100  and downstream of the main flow cell inlet ports  112 . The width of the reagent sample fluid stream  128  is determined by its flow rate relative to that of the guide fluid streams  130  and  132 . During all stages of sample analysis within the flow cell  100 , the flow rate of the sample fluid stream  128  is maintained equal to, or less than, the rate of flow of the guide fluid streams  130  and  132  to ensure proper control of the sample fluid stream  128  by the guide fluid stream  130  and  132 . 
         [0055]    B. Site Specific Sample Fluid Evacuation 
         [0056]    Looking now at  FIGS. 11-16   c , as stated previously, the process of hydrodynamic isolation involves site specific evacuation used in combination with the previously described hydrodynamic focusing to provide the overall function of the hydrodynamic isolation process within the flow cell  100 . To facilitate site specific evacuation, the REPs  122 - 126  described previously are formed in the fluid delivery surface  104  forming a component of the structure of the flow cell  100 , and are positioned along the same central axis  116  as that of the RIPs  118  and  120 . The REPs  122  and  124  are located downstream of their corresponding RIPs  118  and  120 , and upstream of the main fluid outlet port  114  for the flow cell  100 . Evacuation of all or a portion of the sample fluid stream  128  within the flow cell  100  is performed by a process of applying suction to the sample fluid stream  128  through the REPs  122  and/or  124  whereby the sample fluid stream  128  is physically removed from the flow cell  128  through the corresponding REP  122  and/or  124  at a rate preferably equal to, or greater than, the rate of flow of the sample fluid stream  128  that is to be evacuated. 
         [0057]    The size of the areas  111  which can be addressed by the sample fluid stream  128  downstream of the particular RIP  118  or  120  from which it is introduced into the flow cell  100  is controlled by two factors. These factors are: 1.) the distance between the RIP  118  or  120  and any active downstream REP  122  or  124 , or the main exhaust port  114 ; and 2.) the width of the sample fluid stream  128  as defined by the flow boundaries created by the guide fluid streams  130  and  132 . Therefore, the number of locations, or addressable areas  111  within the flow cell which can be independently addressed with different sample fluid streams  128  is dependant upon the number of RIPs  118 ,  120  and corresponding REPs  122 ,  124  formed in the fluid delivery surface  104  of the flow cell  100 . 
         [0058]    By way of example, in the “2-Spot” flow cell  100  forming the first embodiment of the present invention, best shown in  FIGS. 5-7 , location specific fluid addressing is possible at two separate locations  111  within the flow cell  100 , as well as over an area that is the combination of these two areas  111 . To enable this addressing capability, as discussed previously, the fluid delivery surface  104  of the flow cell  100  is formed with two RIPs  118  and  120 , and three REPs  122 - 126 . These RIPs  118 - 120  and REPs  122 - 126  are aligned along the central axis  116  of the flow cell  100  and downstream of the main inlet ports  112 . A pair of REPs  122  and  124  are each located immediately downstream of each RIP  118  and  120  to facilitate the injection of the sample fluid streams  128  associated with each of the RIPs  118  and  120 . (See  FIGS. 6 and 7 ) Another REP  126  is formed in the fluid delivery surface  104  between the REP  122  and the RIP  120 , such that the REP  126  is associated with the RIP  118  and enables the evacuation of the sample fluid stream  128  that has passed over the upstream detection spot  110  prior to this stream  128  passing over RIP  120 , REP  124 , and the downstream detection spot  110 . 
         [0059]    i.) Addressing Upstream Spot Only or Upstream and Downstream Spots 
         [0060]    To address either the upstream spot  110 , or both the upstream and downstream spots  110 , the hydrodynamic isolation process begins with the two streams of guide fluid  130  and  132  being introduced into the flow cell  100  through the fluid inlets  112  to flow at the same rate of speed, passing the guide fluid streams  130  and  132  through the interior of the flow cell  100 , and then discharging the guide fluid streams  130  and  132  from the flow cell  100  through the main fluid outlet port  114 . While the initial charging of the flow cell  100  with the guide fluid streams  130  an  132  can be done with these fluid streams  130  and  132  in any suitable manner, it is essential that once a sample or reagent fluid stream  128  is ready to be introduced into the flow cell  100 , the guide fluid streams  130  and  132  must continuously flow through the flow cell  100  at an equal rate of speed. 
         [0000]    To address the upstream spot  110 , or the combination of the upstream and downstream spots  110  with a sample fluid stream  128 , the sample fluid enters the flow cell  100  through RIP  118 . 
         [0061]    As best illustrated in  FIGS. 11-15   d , in the hydrodynamic isolation process, a portion of the sample plug volume or fluid stream  128  is directed to waste just prior to analysis. The flow cell  100  is designed such that a REP  122  or  124  is always located between a RIP  118  or  120  and the downstream spot  110  where addressing of the sample fluid stream  128  is to occur. Thus, as the leading edge  136  of the sample fluid stream  128  enters the flow cell  100  through the RIP  118 , it is immediately directed over its corresponding REP  122 , where the leading edge  136  can be evacuated from the cell  100 . (See  FIGS. 12 and 15   b ). 
         [0062]    Additionally, as the sample fluid stream  128  enters the flow cell  100 , its width and flow path are controlled by the guide fluid streams  130  and  132 , forcing the sample fluid stream  128  to flow along the central axis  116  of the cell  100 . (See  FIG. 14   a ) The rate of flow of the sample fluid stream  128  relative to that of the guide fluid streams  130  and  132  is set to a velocity such that the width of the sample fluid stream  128  is at least equal to, and preferably narrower than, the orifice of the downstream REPs  122  or  124 .  FIGS. 14 and 14   a  illustrate how the combination of the hydrodynamic focusing provided by the guide fluid streams  130  and  132 , and the site specific evacuation provided by the REP  122  ensures the initial sample-buffer mixture present at the leading edge  136  of the sample fluid stream  128  will not come in contact with any other areas of the flow cell  100 . While the preferred embodiment calls for the REP  122 - 126  to be at least as large as the corresponding RIP  118 ,  120 , it is possible for the REP  122 - 126  to be made smaller than the RIP  118  or  120 , so long as the rate of evacuation through the REP  122 - 126  is sufficient to withdraw all of the sample fluid flow  128  through the REP  122 - 126 . Also, for those flow cells  100  designed to address only one spot  110 , only a single RIP  118  is required with a single corresponding REP  122  for evacuation of the leading edge  136  of the stream  128 . This is because the remainder of the stream  128  can simply be evacuated from the flow cell  100  along with the guide fluid streams  130  and  132  at the main fluid outlet  114 . 
         [0063]      FIGS. 11-13  illustrate in more detail how this process of valveless switching employing the REPs  122 - 126  is used to redirect sample fluid streams  128  without the need for in-tubing valves or mechanical barriers in the flow cell  100 . Away from the flow cell  100 , a volume of the sample fluid, or a sample plug is transferred into some form of sample handling unit which will push the sample fluid through a tubing pathway (not shown), using a flow of running buffer, until it reaches a sample loop  138  just prior to the flow cell  100 . As the sample fluid volume  128  fills the sample loop  138  and approaches the RIP  118  in the flow cell  100 , evacuation through the REP  122  located just downstream of the RIP  118  is initiated. The sample fluid stream  128  enters the flow cell  100  at a flow rate that is extremely slow relative to that of the guide fluid streams  130  and  132 . This slow rate of flow confines the size of the sample fluid stream  128  formed in the flow cell  100  such that it is at least equal to or smaller than the diameter of the corresponding REP  122 , as described previously. (See  FIG. 14   a ). Also the rate of evacuation of the sample fluid stream  128  through the REP  122  is such that the entire sample fluid stream  128  is removed from the cell through the REP  122 . After the sample-buffer mixture at the leading edge  136  of the sample fluid stream  128  has been evacuated to waste, evacuation through the REP  122  is stopped, and the sample fluid stream  128  is allowed to flow to other areas of the flow cell  100 . (See  FIGS. 13 and 15   c ). Once past the REP  122 , the path and size of the sample fluid stream  128  is then controlled by its rate of flow relative to that of the guide fluid streams  130  and  132 . Once the sample fluid stream  128  has interacted with and passed the upstream spot  110 , the REP  126  is activated as the sample fluid stream  128  approaches to evacuate all of the stream  128  in a manner similar to that done for the leading edge  136  upon injection of the stream  128 , to prevent the stream  128  from coming into contact with the downstream spot  110 . (See  FIG. 15   c ). 
         [0064]    Additionally, in some situations when sample plugs are pushed through the tubing pathways of the sample handling unit, one or more air bubbles (not shown) will be used to separate the sample plug from the running buffer. These air bubble separators can greatly reduce sample-buffer mixing during transfer, but often they can cause major interference in the detector response signal if allowed to come in contact with the detection substrate or spot  110 . The process of valveless switching using the hydrodynamic isolation process in the flow cell  100  as previously described can be used to redirect these air bubble separators to waste prior to sample analysis within the flow cell  100 . 
         [0065]    To address the sample fluid stream  128  over the combination of both the upstream and downstream spots  110 , termed a “non-evacuation” event, as best shown in  FIG. 15   d , the sample fluid stream  128  enters through RIP  118  and is allowed to flow to the main exhaust port  114  of the flow cell  100 . The sample fluid stream  128  is not acted upon by any of the REPs  122 - 126 , except during the evacuation of the leading edge  136  of the stream  128  as described previously, such that the stream  128  exits the flow cell  100  at the main fluid outlet port  114 , along with the guide fluid streams  130  and  132  due to the pressure differential created by the force of the fluid streams  128 - 132  filling the enclosed flow cell  100 . In this case the “spot” in the flow cell  100  that is addressed by the sample fluid stream  128  extends from RIP  118  all the way to the outlet port  114 , as best shown in  FIG. 15   d . Additionally, in a flow cell  100  adapted for this method of operation, the RIP  120 , and REPs  124  and  126  can be omitted from the flow cell  100 . 
         [0066]    ii.) Addressing Downstream Spot Only 
         [0067]    As illustrated in  FIG. 16   a - 16   c , to address the sample fluid stream  128  across only the downstream spot  110 , the sample fluid stream  128  enters the flow cell  100  through RIP  120  in the manner described previously regarding the introduction of the sample fluid stream  128  through the RIP  118 . (See  FIG. 16   b ) As the sample fluid stream  128  enters the flow cell  100 , its width and flow path are controlled by the guide fluid streams  130  and  132  forcing the sample fluid stream  128  to flow along the central axis  116  of the flow cell  100  and over the downstream spot  110 . After passing the downstream spot  110 , the sample fluid stream  128  then exits the flow cell  100  through the main fluid outlet port  114  along with the guide fluid streams  130  and  132 . (See  FIG. 16   c ). 
       Hydrodynamic Isolation In Multi-Spot Arrays 
       [0068]    While the first embodiment of the present invention illustrates the use of the flow cell  100  in a hydrodynamic isolation process to address sample fluid streams  128  over two separate sensor spots  110 , and the combination of those sensor spots  110 , in a second embodiment of the present invention illustrated in  FIG. 17 , the flow cell  200  is constructed with having multiple addressable sensor spots  210  forming a spot array  250 . The flow cell  200  has a greater length than the flow cell  100 , and correspondingly a longer central axis  216  than the previous embodiment for the flow cell  100 , such that the cell  200  can be formed with the array  250  including multiple addressable sensor spots  210  and corresponding sets of fluid ports  208 , i.e., RIPs  218  and REPs  222  and  226 , along the longer central axis  216 . The number of separately addressable spots  210  in the array  250  within the flow cell  200  is determined by the total number of RIPs  218  and corresponding REPs  222  and/or  226  provided in the fluid delivery surface  204  of the flow cell  200 . 
         [0069]    In addition, the width of the flow cell  200  can be extended, such that multiple copies of the array  250  can be repeated in a grid-like pattern  240 , with each added set of fluid ports  208  further including additional fluid inlets  212  and fluid outlets  214  to create a large array of individually addressable  210  within a single open flow cell  200 .  FIG. 17  illustrates a top down view of a thirty-two (32)-spot array configuration for the flow cell  200 . However, it is also contemplated that flow cells  200  having an array  250  including any number of spots  210  could be formed as well. 
       Two-Dimensional Hydrodynamic Isolation 
       [0070]    Looking now at  FIG. 18 , a third embodiment of the flow cell  1000  of the present invention is illustrated in which the flow cell  1000  is capable of location specific addressing of sample fluid streams over a two (2) dimensional sensor spot array  1050  formed in the flow cell  1000 . The flow cell  1000  includes sensor spots  1010  oriented in a grid-like pattern  1040  to form an array  1050 , similarly to the flow cell  200 , with a corresponding set of fluid ports  1008 , i.e., fluid inlets  1012 , fluid outlet  1014 , RIPs  1018  and REPs  1022 ,  1026 , oriented along each column of the spot array  1050 . However, the flow cell  1000  also includes an additional set of fluid ports  1008 ′ disposed along each row of the spot array  1050  and oriented generally perpendicular to the set of fluid ports  1008  disposed along the columns of the array  1050 . The various apertures forming the row sets  1008 ′, i.e., the fluid inlets  1012 ′, fluid outlet  1014 ′, RIPs  1018 ′, and REPs  1022 ′,  1026 ′, function identically to the corresponding members in the column sets  1008 , such that sample fluid streams can be addressed to individual spots  1010  of the array  1050  in either the rows of spots  1010  or columns of spots  1010  formed in the array  1050 . 
         [0071]    As stated previously, one advantage of the design of the flow cell of the present invention is the ability to address fluids over multiple locations individually or concurrently in an open cell format by using the configuration of the ports formed in the flow cell in conjunction with hydrodynamic focusing employing the guide fluid streams. The ability to address individual spots is further enhanced in the flow cell  1000  as a result of the multiple guide fluid streams  1030 ,  1032 ,  1030 ′ and  1032 ′ that are positioned within the flow cell  1000  at ninety (90) degrees with respect to one another. By varying the flow rates for each guide fluid stream  1030 ,  1032 ,  1030 ′ and  1032 ′ in the flow cell  1000 , it is possible to move sample fluid streams not only along the rows and columns of spots  1010  of the array  1050 , but in virtually any direction, e.g., diagonally, across the array  1050  to address selected spots  1010  on the array  1050 . In conjunction with this ability, it is also contemplated that additional sets of ports can be formed in the flow cell  1000 , such as a set of ports oriented forty-five (45) degrees with respect to each of the rows and columns of the array  1050 , to enable more direct introduction and movement of sample fluid streams along directions other than along the rows and columns of the array  1050 . In short, the flow cell  1000  expands the ability to address sample fluid streams to specific sensor spots  1010  by enabling concurrent fluid addressing events over a wider variety of combinations of addressable spots  1010  within the array  1050 . 
         [0072]    Various alternatives to the present invention are contemplated as being within the scope of the following claims particularly pointing out and distinctly claiming the subject matter regarded as the invention.