Abstract:
A microfluidic device for transferring liquid from a first chamber to a second chamber is provided. The device has a first chamber; a second chamber; and a barrier between the first chamber and the second chamber, the barrier having least one opening fluidly connecting the first chamber to the second chamber, the at least one opening being sized such that a retention force, such as surface tension, keeps the liquid in the first chamber. The fluid is transferred from the first chamber to the second chamber when an initiation input such as fluid pressure is introduced to the liquid that is sufficient to overcome the retention force. The device may be a sensor strip.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    the present invention relates generally to fluid transfer mechanisms. Examples of particular embodiments of the invention relate to medical fluid testing mechanisms. 
         [0003]    2. Related Art 
         [0004]    The design of some sensor strips requires two or more chambers wherein fluid can be introduced into one chamber then transferred to a second chamber or additional chambers after a pre-determined time. In particular, immunoassay strips as disclosed in U.S. patent application Ser. Nos. 10/830,841 and 11/284,097 had at least two chambers, a first reaction chamber and a second detection chamber. In use, the liquid was first introduced into the reaction chamber and held there for a predetermined time while immuno-binding reactions proceeded, then transferred to the detection chamber. This timed transfer was achieved by having the detection chamber opening to the reaction chamber but unvented initially, such that when the reaction chamber filled, the opening to the detection chamber was closed off by the liquid. This trapped air in the detection chamber prevented it from filling with liquid. When it was desired to fill the detection chamber, a vent was opened at the end of the detection chamber remote from the reaction chamber, usually by puncturing a layer, whereupon liquid transferred from the reaction chamber to the detection chamber either partially emptying the reaction chamber or drawing sample from a filling reservoir. 
         [0005]    The method given above has a number of potential disadvantages. It can be difficult to close the entrance to the detection chamber in a reliable manner across the range of viscosities of samples encountered when testing whole blood. This means that differing amounts of liquid can enter the detection chamber during filling of the reaction chamber, which can add to the variability of the response. Also, the reliability of a puncturing method can be difficult to guarantee over the life of a meter, with the potential for a needle or blade to become blunt with repeated use. It would therefore be desirable to develop a method for affecting a timed liquid transfer that overcomes these difficulties. 
       BRIEF SUMMARY OF THE INVENTION 
       [0006]    An example of an embodiment of the invention seeks to provide a reliable and robust method for transferring small volumes of liquid between chambers utilizing passive transfer forces. The method involves providing a porous wall between the chambers between which the liquid is to be transferred. The porous wall has pores that are large enough to be substantially filled with the liquid to be transferred, but small enough such that the surface tension of the liquid interface with the second chamber prevents the liquid leaking out of the pores into the second chamber until an initiation step is performed. 
         [0007]    Liquid is introduced into a first chamber such that it wets the porous wall and at least partially fills the pores. The liquid does not, however, enter the second chamber at this point as surface tension prevents it from exiting the opposite face of the porous wall into the second chamber. When it is desired to transfer liquid to the second chamber, an initiation step is performed which overcomes or breaks the surface tension and allows liquid to flow out of the pores and into the second chamber. 
         [0008]    The initiation step is such that it overcomes the surface tension holding the liquid in the pores in the wall and allows the liquid to enter the second chamber. This initiation step can be provided by supplying a pressure pulse to the liquid in the first chamber, creating a vacuum in the second chamber, vibrating the strip, touching a surface to the surface of the porous wall facing the second chamber, or any other method that breaks or overcomes the surface tension. 
         [0009]    Multiple second chambers can be filled from a single first chamber at the same or at different times by inducing the initiation mechanism in the desired second chamber(s) at the desired time(s). In addition, a third chamber could be filled from the second chamber by having at least a portion of a wall of the second chamber porous and in common with the third chamber, with the initiation step being performed on the third chamber when the transfer is required. This can of course be repeated for a subsequent string of chambers in parallel or series. 
         [0010]    Particular embodiments of the invention provide a fluid transfer device for transferring liquid from a first chamber to a second chamber. The device has a first chamber; a second chamber; and a barrier between the first chamber and the second chamber, the barrier having at least one opening fluidly connecting the first chamber to the second chamber, the at least one opening being sized such that a retention force keeps the liquid in the first chamber. The fluid is transferred from the first chamber to the second chamber when an initiation input is introduced to the liquid that is sufficient to overcome the retention force. 
         [0011]    Other embodiments of the invention provide methods of transferring liquid from a first chamber to a second chamber. The methods include providing a first chamber; providing a second chamber; providing a barrier between the first chamber and the second chamber, the barrier having at least one opening fluidly connecting the first chamber to the second chamber, the at least one opening being sized such that a retention force keeps the liquid in the first chamber; and transferring the liquid from the first chamber to the second chamber. The transferring takes place when an initiation input is introduced to the liquid that is sufficient to over come the retention force. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    The foregoing and other features and advantages of the invention will be apparent from the following, more particular description of particular embodiments of the invention, as illustrated in the accompanying drawings wherein like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. 
           [0013]      FIG. 1  shows an example of a first embodiment of the invention; 
           [0014]      FIG. 2  is a sectional view along section line A-A′ of the embodiment shown in  FIG. 1 ; 
           [0015]      FIG. 3  is a sectional view along section line B-B′ of the embodiment shown in  FIG. 1 ; 
           [0016]      FIG. 4  shows an example of a second embodiment of the invention; 
           [0017]      FIG. 5  is a sectional view along section line A-A′ of the embodiment shown in  FIG. 4 ; 
           [0018]      FIG. 6  is a sectional view along section line B-B′ of the embodiment shown in  FIG. 4 ; 
           [0019]      FIG. 7  shows an alternate embodiment of the invention; 
           [0020]      FIG. 8  shows an example of a third embodiment of the invention; 
           [0021]      FIG. 9  is an exploded view of the embodiment shown in  FIG. 8 ; 
           [0022]      FIG. 10  is a graph showing current as a function of time of a first example of the invention; and 
           [0023]      FIG. 11  is a graph showing current as a function of time of a second example of the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0024]    The invention will now be described with reference to a specific two chamber embodiment with a specific initiation step. This embodiment relates to a disposable immunoassay strip using electrochemical detection of the results of an immuno-binding reaction. Note that the terms upper and lower are used for convenience only in the following description, they do not imply anything about the preferred orientation of the device during use, which can in fact be used in any orientation. 
         [0025]    The strip comprises three chambers, a filling chamber, a reaction chamber and a detection chamber. The filling chamber serves to receive the sample and act as a sample reservoir, the reaction chamber contains reagents whereby a probe is selectively immobilized in the reaction chamber to differing extents dependent upon the presence or concentration of an analyte in the sample. The detection chamber contains electrodes and reagents so as to be able to detect the amount of probe transferred with liquid from the reaction chamber and thus detect or quantify the amount of analyte in the original sample. An example of such a strip is shown in  FIGS. 1-3 . The strip  100  has a number of layers which are laminated together using adhesives. The strip has three chambers, a filling chamber  1 , a reaction chamber  2 , and a detection chamber  3 . Layers  10  and  70  are sealing layers that serve to close the faces of chamber  1  to help to form a capillary space. Layer  20  is a layer carrying an electrically conductive upper surface which serves as an electrode in detection chamber  3 . Layers  30  and  50  are spacer layers which have adhesive upper and lower faces. Layers  30  and  50  serve to hold the laminate together and to define the height of the detection and reaction chambers, respectively. A region cut-out of layer  30  shown in  FIGS. 2 and 3  defines the area of detection chamber  3  and the area of the detection chamber electrodes. A region cut-out of layer  50  shown in  FIGS. 2 and 3  defines the area of reaction chamber  2 . Layer  40  is a layer containing pores which serve as pores that connect reaction chamber  2  to detection chamber  3 . Layer  40  has an electrically conductive coating on its lower surface that serves as the second electrode in detection chamber  3 . Layer  60  serves to close reaction chamber  2  and filling chamber  1 . Optionally, layer  60  can carry an electrically conductive coating on its lower surface to serve as an electrode to detect when liquid fills reaction chamber  2 . With this option, liquid fills reaction chamber  2  and the pores in  40 , thus bridging the electrode on the lower face of  40  and that on the lower face of  60 . This bridging can be detected to tell the meter to initiate the test sequence. Layers  10  and  70  can be secured to layers  20  and  60 , respectively, by any suitable method. A preferred method is to use adhesive. In one embodiment, the adhesive can be applied to the lower surface of layer  20  and the upper surface of layer  60 , layers  10  and  70  can then be laminated to these adhesive layers. Alternatively, adhesive can be coated on to layers  10  and  70  and then those layers laminated to  20  and  60 . 
         [0026]      FIGS. 4-6  show an alternative embodiment where the cut-out in layer  50  to form reaction chamber  2  is such that the walls of the cut-out fully surround the cut-out to form an enclosed area. This embodiment has the advantage of preventing liquid from reaction chamber  2  wetting around the open edge of reaction chamber  2  to fill detection chamber  3  from its open edge, rather than through the porous wall connecting the two chambers. In this embodiment, the air that is displaced as liquid fills reaction chamber  2  can vent through the holes in the porous wall, allowing the chamber to fill until all the pores in the wall are filled with liquid or reaction chamber  2  is fully filled. Note that it is not necessary for reaction chamber  2  to be completely filled for correct operation, just that there is a sufficient volume of liquid in contact with the reagents in reaction chamber  2  to fill detection chamber  3 . 
         [0027]    Also shown in  FIGS. 4-6  is an embodiment where layers  10  and  70  are not required and instead layers  20  and  60  are extended to form the end walls of filling chamber  1 . Optionally, in this embodiment the conductive layer on the upper surface of  20  can be extended into filling chamber  1 . If this is done, when liquid fills reaction chamber  2  and the pores of layer  40 , electrical connection is made via the liquid between the conductive layer on  20  in filling chamber  1  and the conductive layer on the lower surface of layer  40 . This can be detected electrically as a drop in the resistance or a change in the voltage, or current flowing or capacitance between the conductive layers on  20  and  40 . This can serve as a signal to the meter that liquid has filled detection chamber  3  and thus automatically initiate a pre-determined test sequence. Note that an advantage of this method is that the signal won&#39;t be detected until the liquid in filling chamber  1  is of sufficient volume to touch the opening of reaction chamber  2  and start to fill the pores of layer  40 . Similar to the method disclosed in U.S. Pat. No. 6,571,651, herein incorporated by reference, the capillary dimension of filling chamber  1  is greater than that of reaction chamber  2 , thus filling chamber  1  can empty to fill reaction chamber  2 . So if filling chamber  1  is sized so as to have a volume at least equal to and preferably slightly greater than reaction chamber  2 , then the signal indicating that liquid has been introduced into the device will not be detected until there is sufficient liquid introduced for the device to function as intended. An additional advantage of this is that if at first not enough liquid is introduced into filling chamber  1 , more can be added until enough is present without affecting the intended operation of the device. When the fluid transfer is initiated between reaction chamber  2  and detection chamber  3 , a second change in the electrical conditions between the conductive surfaces on  20  and  40  will occur, due to the wetting of the conductive layer on  20  in the area of detection chamber  3 . This change can be used to confirm to the meter that the fluid transfer has been successfully accomplished. Note that in general the current signal arising from the area of the conductive layer on  20  exposed in filling chamber  1  will be small compared to that generated by the area of the conductive layer exposed in detection chamber  3 , so it will not interfere significantly with the signal from detection chamber  3 . This is so as, in general, there are low concentrations of, or no significant amount of, electroactive species in the native sample that can generate current at the voltages normally applied between the electrodes in detection chamber  3 . Also, the relatively long liquid path between the conductive layer exposed in filling chamber  1  and the conductive layer on  40  gives a relatively high electrical resistance, which tends to reduce the current signal produced. 
         [0028]    An advantage of this embodiment is that only two electrical connections are required to detect all the electrical signals from the strip to complete the test, one connection to the conductive layer on  20  and the second to the conductive layer on  40 . Suitable connectors for this are disclosed in U.S. Pat. Nos. 6,379,513 and 6,946,067, and in U.S. patent application Ser. No. 11/284,136, which are incorporated by reference in this disclosure. 
         [0029]    An alternative electrical connection method for this device is shown in  FIG. 7 . The connection device  200  is illustrated with reference to the present invention, however it is to be understood that this aspect of the invention is applicable to any device when it is desired that connection be made to surfaces that are in close proximity and face one another. The numbered elements in  FIG. 7  with numbers common to the other figures denote the same element in the present illustration. In this embodiment of this aspect of the invention,  FIG. 7  shows the end of the strip opposite to the end onto which filling chamber  1  opens. According to this embodiment, layer  20  is extended beyond spacer layer  30  and layer  40 . Layer  20  carries an electrically conductive layer  70  on its upper surface and layer  40  carries an electrically conductive layer  90  on its lower surface. It is desired to make separate electrical connection to layers  70  and  90 . Layer  40  is extended beyond spacer layer  30 . An additional layer  110  is introduced into the space between layers  20  and  40  that extends beyond  30 . Preferably the thickness of layer  110  plus a conductive layer  80  and any adhesive layers that may be present is to equal to or slightly greater than the thickness of layer  30 . Layer  110  is electrically conductive at least on its upper surface  80  however is not electrically conductive through its full thickness, such that electrical connection can be made between  80  and  90  but not between  90  and  70  via  110 . Layer  110  and conductive layer  80  carried thereupon extend beyond the edge of  40 , thus by bringing  80  and  90  into electrical connection, electrical connection can be made to  90  via  80  in the area of  80  that extends beyond  40 . Preferably an adhesive layer is present between the lower surface of  110  and  70  to fix layer  110  in position. A conductive adhesive can optionally be placed between layers  90  and  80 , in at least a portion of where they overlap, to help ensure good electrical connection. Alternatively the port containing the pins or similar devices to connect the strip to an external electrical circuit can be configured such that when the strip is inserted into the port, a face or faces of the port push against the upper surface of  40  to push  90  into connection with  80 . 
         [0030]    An embodiment of the invention using a device with three active chambers is shown in  FIGS. 8 and 9 .  FIG. 8  shows a top view of a device with three active chambers and a filling chamber.  FIG. 9  shows an exploded view of this embodiment showing the various layers. The strip  700  comprises a filling chamber  800 , a first reaction chamber  510 , a transfer and reaction chamber  310  and a second reaction chamber  520 . Perforations  410  in layer  400  serve to connect first reaction chamber  510  with transfer and reaction chamber  310 . Perforations  420  in layer  400  serve to connect transfer and reaction chamber  310  with second reaction chamber  520 . In use, sample is added to filling chamber  800  until it fills to the opening to  510  at the end of filling chamber  800 , whereupon the sample fills first reaction chamber  510 . The air that is necessarily displaced during this filling process will vent through the open ends of second reaction chamber  520 , via perforations  410  and  420 . One or more reagents and reagent layers can be dried into first reaction chamber  510  to do a sample pre-treatment step, for example. After the desired time in first reaction chamber  510 , the transfer of fluid to transfer and reaction chamber  310  can be initiated by the means disclosed, such as pushing on layer  200  from below until it contacts the lower surface of layer  300  at the perforated area  410 . When this is initiated, treated sample will flow from first reaction chamber  510  to fill transfer and reaction chamber  310  until perforations  420  are blocked with liquid sample such that air can no longer vent through perforations  420 . Optionally, a second set of reagents can be dried into transfer and reaction chamber  310  if desired to perform a second reaction of the sample, such as a binding reaction as discussed below. Note that is it advantageous, but not necessary, for reactions to take place in all chambers. For example, first reaction chamber  510  could correspond to the reaction chamber of the embodiment shown in  FIGS. 1-7  and second reaction chamber  520  could function as the detection chamber. In this case, transfer and reaction chamber  310  acts purely as a transfer chamber which separates first reaction chamber  510  and second reaction chamber  520  laterally as well as by perforated area  420 . This can be advantageous in some applications in minimizing vapor from the fluid in first reaction chamber  510 , when filled, from diffusing to second reaction chamber  520  and wetting the reagents prematurely. 
         [0031]    After the sample fluid is present in transfer and reaction chamber  310  for the desired time, a further transfer of sample from transfer and reaction chamber  310  to second reaction chamber  520  can occur via perforations  420 . This transfer can be initiated, for example, by pushing on the upper surface of layer  600  above perforations  420  such that the lower surface of layer  600  comes into contact with at least some of perforations  420 . Further reagents can be dried into second reaction chamber  520  to further treat or react with components of the sample. For example, the results of any reactions carried out in transfer and reaction chamber  310  and first reaction chamber  510  can be detected in second reaction chamber  520  and converted into a usable signal, either optical, electrochemical or for some other suitable method. 
         [0032]      FIG. 9  gives more detail of how the various chambers in this embodiment are formed. Layers  200  and  600  are the lower and upper closing layers, respectively, whose functions are to close faces of the capillary spaces in the strip, provide layers to contact the perforated layer to initiate fluid transfer if initiation is performed in this manner, and to serve as supports on to which one or more other layers may be placed. Examples of other layers are layers of conductive material to form electrodes and electrical connection tracks and dried reagent layers that may be required to process the sample in the various chambers. 
         [0033]    Layer  500  is an upper spacer layer. Portions of layer  500  are either cut away or otherwise formed to define the area of first and second reaction chambers  510 ,  520 . Layer  500  can be formed from a substrate with adhesive coated on both sides or may be just a layer of adhesive that has been formed or laid down with the areas that will correspond to first and second reaction chambers  510 ,  520  left free of adhesive. If an adhesive coated substrate is used, the areas corresponding to first and second reaction chambers  510 ,  520  could be formed by punching or otherwise removing those areas. 
         [0034]    Layer  400  is a layer that acts as a barrier between, and comprises the perforations necessary to complete the fluid transfers between, first reaction chamber  510  and transfer and reaction chamber  310  and transfer and reaction chamber  310  and second reaction chamber  520  when required. The perforations can be formed as described elsewhere in this disclosure. Layer  300  is a second spacer layer with an open area that serves to define transfer and reaction chamber  310 . This can be constructed by the methods given above for layer  500 . Filling chamber  800  can be formed by first laminating or otherwise joining layers  300 ,  400  and  500  with areas  310 ,  510  and  520  and perforations  410  and  420  pre-formed in the respective layers, and then punching through the tri-laminate to form the cut-out for filling chamber  800 . Alternatively, the regions of  300 ,  400  and  500  that correspond to filling chamber  800  can be formed separately in the layers and then the layers laminated such that the cut-out regions align to form the side walls of filling chamber  800 . The end faces of filling chamber  800  are closed when  200  and  600  are laminated to the upper and lower surfaces of the tri-laminate comprising  300 ,  400  and  500 . 
         [0035]    Referring to the embodiment illustrated in  FIGS. 1-6 , to function as a dry strip immunoassay, reagents can be dried into reaction chamber  2  and detection chamber  3  during fabrication. The reagents in reaction chamber  2  comprise a probe linked to a binding agent (hereafter termed the conjugate) and a binding target to which the binding agent can bind, where the species carrying the binding target, or the binding target itself, can be prevented from entering detection chamber  3 . For example, the conjugate can consist of an enzyme such as PQQ dependent glucose dehydrogenase (GDHpqq) linked to an antibody to an analyte of interest. The target binding site can then be the analyte of interest tethered to magnetic beads. The magnetic beads can be prevented from entering the detection chamber by means of a magnet confining them to reaction chamber  3 . Alternatively, the beads need not be magnetic but be large enough such that they cannot fit through the pores in layer  40 , such that this prevents them from entering detection chamber  3 . When there is analyte in the sample, the free analyte can bind to the binding site on the conjugate and therefore block the conjugate from binding to the immobilized target sites. The conjugate therefore remains free in solution and so able to transfer to detection chamber  3 . In this embodiment it is desirable that the conjugate and the target binding site are not mixed before the sample contacts the reagents. To achieve this, the conjugate can be dried onto the lower face of  60  and the species carrying the target binding sites onto the upper face of  40 . If the target binding sites are located on magnetic beads a permanent or electromagnet placed next to the upper face of  60  can draw the beads up to mix with the conjugate after sample has filled reaction chamber  2  and freed the magnetic beads from the initially dry layer. In addition, the magnet serves to prevent the beads entering detection chamber  3   
         [0036]    Detection chamber  3  also contains reagents dried down during strip fabrication. These reagents are those necessary to translate the presence of the probe into a current that can flow between the electrodes in detection chamber  3 . In this embodiment of the invention where the probe is an enzyme, a substrate and electrochemically active mediator for the enzyme can be incorporated. Alternatively, the substrate for the enzyme can be incorporated into reaction chamber  2 . This has advantages where the substrate can take some time to become active. When the probe is GDH, glucose is a suitable substrate, however the GDHpqq is only active with β-D-glucose. D-glucose in the dried state is predominately in the form of α-D-glucose, which proceeds to mutarotate to β-D-glucose once it dissolves. Thus it is advantageous to dissolve the glucose in the sample in reaction chamber  2  so that it can mutarotate while the binding reactions are taking place. 
         [0037]    Any fluid containing a probe that enters the detection chamber will dissolve the dried chemicals and the chemicals and the probe begin to react. In the case of GDHpqq as the probe, glucose is a suitable substrate and ferricyanide is a suitable mediator. When GDHpqq, glucose and ferricyanide are mixed the GDHpqq will oxidize the glucose and be reduced in the process, the GDHpqq will then be reoxidised by ferricyanide, which forms ferrocyanide in the process. The ferrocyanide can then be oxidized at the anode in the detection chamber to produce a measurable current. This current can be related to the rate of production of ferrocyanide, which in turn can be related to the concentration of GDHpqq in the detection chamber, which in turn can be related to the concentration of analyte originally in the sample. 
         [0038]    Optimally, the chemistry dried into the detection chamber should be dried onto the upper surface of  20 . This prevents liquid filling the pores of  40  coming into contact with the dried reagents prematurely. Additionally, the dissolving of the chemistry on  20  in the reacted sample liquid when the chemistry contacts the liquid filled pores of  40  (as set out below) helps to encourage the transfer of liquid into the detection chamber. 
         [0039]    In order for the GDHpqq to be detected in the detection chamber liquid from the reaction chamber must be transferred to the detection chamber after a pre-determined time when the binding reactions in the reaction chamber have proceeded to the desired point. When the liquid fills the reaction chamber, the hydrophilicity of the pores in  40  are such that they also fill with liquid at this point. However, for the liquid to exit the pores at the face of  40  facing the detection chamber it would have to increase the area of the air/liquid interface, which the liquid surface tension opposes. Therefore the liquid tends to fill to the base of the pores and stop. In this embodiment, in order to break the surface tension layer  20  is pushed from below in the region of the detection chamber. This distorts  20  such that its upper surface comes into contact with the lower surface of  40 . At the point(s) of contact, the liquid can now exit the pores in  40  without increasing the air/liquid interface area by directly wetting the upper face of  20 . However, as the pushing mechanism is withdrawn and the upper face of  20  moves away from the lower face of  40 , the solution that wetted  20  moves away with it and draws more liquid through the pores of  40  in order to minimize the ratio of air/liquid interface to liquid volume as the surfaces move apart. This process draws liquid through the pores until eventually the detection chamber is completely filled. Note that both the reaction chamber and the detection chamber should be open to the atmosphere when they are being filled for correct function, so that air can be displaced and vented during the filling processes. In the embodiment shown in  FIGS. 1-3 , a venting function is provided by the reaction and detection chambers opening to the sides of the strip  100 . In the embodiment shown in  FIGS. 4-6 , the detection chamber is vented through its openings to the sides of the strip and the reaction chamber is vented through the open sides of the detection chamber via the pores in  40 . 
         [0040]    Also, in order for this embodiment to function optimally it is desirable that the filling of the detection chamber does not result in the emptying of a chamber with similar capillary dimensions, as the two forces can oppose one another and create a slow or incomplete fill. In the strip  100  shown, the filling chamber has a larger capillary dimension than either the reaction or the detection chamber. Thus when the detection chamber fills, the filling chamber will empty if there is no excess liquid attached to the filling chamber. Since the filling chamber has a larger capillary dimension than the detection chamber, the filling of the detection chamber will be less impeded. Alternatively, if the filling chamber has the same capillary dimensions as the detection chamber then the detection chamber should be more hydrophilic than the filling chamber in order to affect the transfer of liquid. In general, the value of (γ d,SL −γ d,SA) ΔA   d +(γ FSA −γ f,SL ) ΔA f  should be considered, where γ is the surface tension, ΔA is the change in the wetted area of a chamber, the subscripts d and f refer to the detection and filling chambers, respectively, SL refers to the solid-liquid interface and SA refers to the solid-air interface. 
         [0041]    The invention has a number of advantages over the related art. A pusher mechanism rather than a piercing mechanism can be used to initiate fluid transfer, which should add robustness to the system. Also, the chambers can be stacked one upon the other, leading to miniaturization and manufacturing advantages. Also, multiple chambers can be stacked and offset, with multiple pusher mechanisms, as exemplified in  FIGS. 8 and 9 , thus allowing multiple chambers in either parallel or series to be filled as desired times, increasing flexibility. Also, electrode areas in the detection cell can be more conveniently defined since a cut-out region in  30  can entirely define the electrode areas. 
         [0042]    Examples 1 and 2, below, are given as examples of embodiments of the invention and should not be considered as limiting in any way. 
       Example 1 
       [0043]    0.007 inch thick Melinex 329 was sputter coated with a thin layer of palladium to give an electrical resistance of 10 Ohms/sq to form layer  20 . 0.002 inch thick Melinex 329 was coated with ca. 22 microns of heat activated adhesive ARCare-90503 (Adhesives Research Inc) on both sides to serve as layers  30  and  50 . The adhesive tape was supplied with siliconised PET release liners on both faces. 
         [0044]    A 0.004 inch thick web of PET was perforated by laser cutting through holes in lines in the down-web direction. The holes were conical in shape with the larger end being 150 micron diameter and the smaller end being 45 microns in diameter. The average hole density was 8.2 holes/mm 2 . After perforation, one side of the web was sputter coated with gold to give an electrical resistance of 10 Ohms/sq. 
         [0045]    The double sided adhesive tape was laminated to both sides of the perforated PET leaving the gaps as shown in  FIG. 1  which would form the reaction chamber  2  and the detection chamber  3 . The palladium coated Melinex was then laminated to the lower face of layer  30  to form layer  20 . Clear PET film was laminated to the upper face of layer  50  to form layer  60 . Filling chamber  1  was then formed by punching through layers  20  to  60  and laminating adhesive coated PET film layers  10  and  70  to close the faces of filling chamber  1 . 
         [0046]    Conjugate and derivatised magnetic beads were prepared as per US Patent Application Publication No. US-2006-0134713-A1, herein incorporated by reference. The conjugate comprised an antibody to CRP (C-Reactive Protein) joined to at least one GDHpqq. The surface of the magnetic beads were modified to comprise CRP. This CRP served as the immobilized binding site for the conjugate. The magnetic beads were prevented from entering the detection chamber by a permanent magnet placed near the reaction chamber. 
         [0047]    The conjugate was dried onto the lower face of layer  60 . In some strips beads were dried on the upper face of layer  40 . A mixture of potassium ferricyanide, glucose and buffer was dried on the upper surface of layer  20 . During testing a permanent magnet was placed adjacent to the upper face of layer  60 . This served the dual purpose of preventing beads (if present) entering the detection chamber and attracting the beads towards the layer of conjugate to promote mixing of the two once the sample was introduced into the reaction chamber. 
         [0048]    In use, sample was introduced into filling chamber  1  until it filled across to touch the entrance to reaction chamber  2 , whereupon reaction chamber  2  also filled with sample. Sixty seconds was then allowed to elapse. After sixty seconds, a metal rod was pressed against the lower surface of  20  such that  20  was deflected up until the upper surface of  20  came into contact with liquid filling the holes in layer  40 , whereupon liquid flowed through the holes in  40  to completely fill detection chamber  3 . When liquid bridged the space between the electrode on the upper face of  20  and that on the lower face of  40  the meter initiated an electrochemical test sequence, where it made the lower electrode +300 mV relative to the upper electrode for 16 seconds. 
         [0049]      FIG. 10  shows plots of the typical current response for strips filled with 0.1 M HEPES buffer in water with and without the presence of CRP labeled beads in the test solution. When no beads are present, maximal conjugate should be transferred to detection chamber  3 . When an excess of CRP labeled beads over conjugate is present in the solution the conjugate is substantially immobilised on the beads leading to a minimal transfer of conjugate to detection chamber  3 . In this case the lower electrode was at +300 mV with respect to the upper electrode during the sixteen seconds the potential was applied. 
         [0050]      FIG. 11  shows typical current responses for strips with conjugate and beads dried into them when tested blood serum containing either zero or 250 micrograms/milliliter of CRP. In this case the upper electrode was at +300 mV with respect to the lower electrode. 
       Example 2 
       [0051]    The invention is also pertinent to a sensor with a single, larger punched hole in layer  40  rather than a series of small, laser formed holes. For example, a 1.5 mm diameter male/female punch was used to create a hole in layer  40 . When liquid filled chamber  2 , it stopped just past the edge of the hole. When layer  20  was pushed against the hole, the liquid entered chamber  3 . 
         [0052]    The invention is not restricted to the number of holes per sensor or the range of hole diameters described in Examples 1 and 2. 
         [0053]    The invention is not limited to the above-described exemplary embodiments. It will be apparent, based on this disclosure, to one of ordinary skill in the art that many changes and modifications can be made to the invention without departing from the spirit and scope thereof.