WO2006032853A2

WO2006032853A2 - Reagent holder and testing assembly incorporating a reagent holder

Info

Publication number: WO2006032853A2
Application number: PCT/GB2005/003590
Authority: WO
Inventors: Stephen Peter Fitzgerald; John Victor Lamont; Sonya Ferguson; William James Montgomery
Original assignee: Randox Laboratories Ltd
Priority date: 2004-09-20
Filing date: 2005-09-20
Publication date: 2006-03-30
Also published as: WO2006032853A3; GB0420878D0

Abstract

A reagent holder (1) comprises a liquid reagent reservoir (2) defined by a tubular wall having an exit opening (5). A plug (10) is movable between a closed position, in which it prevents reagent exiting the reservoir (2) through the exit opening (5), and an open position, in which it allows reagent to exit the reservoir through the exit opening. A plunger (11) sealingly engages the reservoir wall such that a reagent is retained between the plunger (11) and the plug (10). When he plunger (11) is moved towards the plug (10), the liquid reagent is compressed and is moved to its open position in which reagent is dispensed through the exit opening (5).

Description

REAGENT HOLDER AND TESTING ASSEMBLY INCORPORATING

A REAGENT HOLDER

The invention relates to a reagent holder typically for the storage of chemical reagents required for use on, for example point of care diagnostic systems and medical diagnostic machines such as assay systems including immunoassay systems.

The established method for the storage of chemical reagents as required for use on medical diagnostic machines is a plastic bottle. Such containers are used for multi- test or bulk storage and come in a variety of shapes and sizes. They are typically located in a rotary carousel to enable a relatively simple method of location and delivery for aspiration of the contents via automated pipetting. A typical example is shown in WO-A-98/00236.

These methods have proved very successful to date and have therefore become the universally preferred method of reagent handling. In recent years, attempts to improve on the model have involved the development of combination packaging whereby chemistries related by test are grouped into hybrid multi-celled packages. One known alternative to a carousel involves the arrangement of such multi-celled containers on an XY matrix. In use, the fluids from these containers are required to be delivered into reaction vessels often referred to as "cuvettes". Some cuvettes are of a disposable type while others are reusable but these require relatively intense automated cleaning.

Some significant shortcomings of the current methods can be identified:

• The capacity of carousel storage is limited and chemistries for all tests are not accessible. Many systems employ a changeover of carousel content to facilitate the full spectrum of tests.

• Despite some methods to re-seal or cover reagent containers, all bulk containers have a relatively short "on system" life once the container has been opened for the first time. Typically 8 to 10 weeks.

• In operation, the machine is required to move the carousel to select the required container, aspirate a measured dose into a pipette and deliver an accurately metered dose into a reaction vessel or "cuvette". This activity requires dedicated robotic hardware and machine working area.

• There is a significant proportion of chemical wasted comprising non-usable (dead) volumes remaining in a container.

WO-A-02/058845 discloses an alternative approach in which a reaction vessel is provided, which is preferably disposable, having a number of upper holding chambers for containing chemicals needed for the process. A plug selectively opens and seals the upper inlet of each chamber, each chamber having a lower outlet. The problem with this system is that it is relatively complex and therefore costly to manufacture.

US-A-5971953 describes a syringe having a bore which is divided during use into upper and lower chambers for containing medicinal contents that are to be first mixed and then dispensed. A first lower piston occupies a position generally in between the upper and lower chambers. The first lower piston engages a plurality of ribs at an enlarged diameter bypass portal section of the syringe housing with multiple longitudinally extending channels after the user applies pressure to a second upper piston in the bore and above the upper chamber. Continued downward movement of the two pistons causes the two components to mix to be dispensed from the distal end of the syringe. The ribs have recess portions that form a dampening slot for engaging the periphery of the first lower piston as it travels distally. This enables the diluent fluid to completely mix with the dry drug product before the second, upper piston engages the lower piston to force it down during administration of the reconstituted product to the patient . This syringe has a rather complex construction, particular with regard to actuation of the pistons.

In accordance with a first aspect of the present invention, a reagent holder comprises a liquid reagent reservoir defined by a tubular wall having an exit opening; a plug movable between a closed position, in which it prevents reagent exiting the reservoir through the exit opening, and an open position, in which it allows reagent to exit the reservoir through the exit opening; and a plunger sealingly engaging the reservoir wall such that a reagent is retained between the plunger and the plug, in use, wherein when the plunger is moved towards the plug, pressure is applied to the liquid reagent so that the plug is moved to its open position in which reagent is dispensed through the exit opening. This new reagent holder provides a much simpler structure and thus is much simpler to manufacture and can be made as a disposable item. Movement of the plug is controlled through movement of the plunger via the liquid reagent which is incompressible and thus no additional control components are required. Furthermore, the quantity of the reagent can be carefully predetermined enabling the holder to be utilized once, in its entirety, to carry out the required reaction.

In accordance with a second aspect of the present invention, a testing assembly comprises a reagent holding device having at least one reagent holder which contains a predetermined quantity of a reagent, the reagent holder having an exit opening which is sealed; a reaction vessel which is permanently, or preferably removably, fitted to the reagent holding device and into which the exit opening opens, the reagent holder including a dispense mechanism which, when actuated, breaks the seal and dispenses the predetermined quantity of reagent into the reaction vessel. Again, this aspect of the invention leads to a very- simple structure and enables a self-contained assembly to be produced which avoids the need for carousels and the like.

In particular, and with reference to the problems outlined above, the invention has a number of significant advantages. The reaction vessel could include an assay device e.g. a single biochip well with substrate on which an array of different test sites has been spotted. Each multi-site biochip in a reaction vessel could have 15-20 or more assays and use one set of reagents dispensed from the holder above. A different group of tests i.e. on another multi-site biochip would be supplied in a separate reagent holder/reaction vessel with its own particular and different reagents. There could in principle be for example 6-10 or more different groups of tests. Thus, different reagents are generally required for different single tests and different multi-site test substrates.

Each multi-site biochip and group of tests thereon will have one set of reagents. The individual reagents having common chemicals etc. or an appropriate mix of chemicals etc. to work with the individual assays on the biochip. A machine which separately carries out 15-20 assays may need to maintain up to 60 and possibly 100 different (volumes of) reagents.

The Randox™ Biochip uses an additional two reagents to provide the luminous response for test completion measurement. If₇ according to an aspect of the invention, each Biochip is pre-packed with its respective chemistry then the user does not have to manage/handle this inventory nor does the machine need to store, aspirate and deliver these fluids to the reaction vessel/cuvette.

The invention enables an anaerobic pre-measured dose of each chip reagent chemistry to be packaged and stored, for example, with the relevant Biochip, for an optimum period. The shelf-life of the packaging will significantly exceed that of an open bottled chemistry as used in current practice. Existing analyser equipment spends much of its time extracting and delivering measured doses of reagent chemistries into reaction vessels/cuvettes. This process also involves the requirement for washing and servicing the hardware employed. The pre-packaged approach removes all of the complexity requiring only a simple mechanical displacement to take place.

The mechanical methods employed to extract reagent chemistry from bottles cannot use all of the fluid shipped/contained in the bottle. In percentage terms, up to 5% may be left unusable. If all of the 60 and possibly up to 100 reagents stored on-board a machine are not consumed within eight to ten weeks then the reagent volumes cannot be used and must be disposed of. In contrast, with the invention, the shelf-life of the pre-packed dosing units is typically expected to exceed one year.

Preferably one, and most preferably both, of the plug and plunger are a friction fit against the wall of the reservoir. Alternatively, the plunger could be provided by a screw thread or other mechanical linkage to the wall of the reservoir but this adds to the complexity. In either case, the plunger and plug should also be a vacuum or gas tight fit.

A particular advantage of the slidable plug is that it can then be automatically moved from its closed position to its open position upon movement of the plunger towards the plug. This is particularly appropriate when using an incompressible fluid in the reservoir.

In some cases, the exit opening could be provided at one end of the tube of the reservoir, axially aligned with the direction of movement of the plunger and plug, the plug allowing reagent to pass beside it to the exit opening when in its open position. In other cases, the exit opening is provided alternatively or additionally in a side facing portion of the reservoir wall since this again simplifies construction of the device.

The exit opening may have parallel sides to help guide the fluid but this could be disadvantageous in certain cases because fluid may adhere to the walls. Thus, the walls could diverge towards the exit to reduce this problem.

Furthermore, where the exit opening includes an opening axially aligned with the direction of movement of the plunger and plug, preferably the axially aligned opening extends through a tip of the tubular wall, the tip being tapered inwardly towards the axially aligned opening. The use of a more pointed tip, possibly combined with diverging exit opening walls, can help drop detachment as the contact area between fluid and tip is reduced, also reducing the overall attachment forces and encouraging break-off in order to minimize the surface energy of the liquid. Where a side exit opening is provided, liquid reagent may be initially forced out (or squirted) at high speed

(pressure) directly away from the side of the holder. This may be acceptable in some configurations. However, this may be mitigated by shaping of the sidewall just at the exit e.g. if the outer edge of the sidewall protrudes over and down with respect to the inner face of the sidewall

(similar to a shop front canopy) . Alternatively, a downwards-extending cover feature may be used to ensure that the fluid is caused to move downwards. The above assumes that the holder is mounted vertically. Sideways ejection even from a vertically mounted holder may be acceptable, even desirable in some situations, depending on reaction vessel configuration, assay technique, agitation method etc. In addition, it should be noted that with suitable speed and/or positional control of the plunger or plunger actuator force feedback, the motion of the plunger may be controlled in order to control the rate of flow, for example to minimize the severity of squirting, or provide a slow, gradual addition of reagents.

The plunger and/or plug are preferably made of elastomeric material. This is particularly advantageous in the case of the plunger since it enables a delivery needle or the like to be inserted through the plunger so that the reservoir can be filled.

The testing assembly preferably comprises one or more reagent holders made in accordance with the first aspect of the invention.

The reaction vessel may be push fitted or otherwise connected to the reaction holding device, for example by a screw threaded connection or the like. As explained above, the testing assembly may be provided with a combination of reagent holders which provide measured quantities of appropriate reagents to carry out a particular experiment such as an assay.

We also provide a reaction vessel assembly for use in an assay, the assembly comprising a reaction vessel in which is located a surface on which molecules are bound, the reaction vessel having one or more apertures in its outer wall; and a waste collection vessel- within which the reaction vessel is located to collect waste fluid which exits through the aperture(s) .

The surface may be provided by a sidewall, base or top of the reaction vessel (depending on configuration) , but preferably by a biochip located in the reaction vessel.

Some examples of testing assemblies and reagent holders according to the invention will now be described with reference to the accompanying drawings, in which:-

Figures IA and IB are a perspective view from one side and a longitudinal section respectively of a reagent holder with a plug loaded; Figures 2A and 2B are similar to Figures IA and IB but illustrating the loading of a plunger; Figures 3A and 3B are views similar to Figures 2A and 2B but illustrating the plug and the plunger at their pre¬ load positions;

Figures 4A and 4B are views similar to Figures 3A and 3B after reagent has been loaded into the holder;

Figures 5A and 5B are views similar to Figures 4A and 4B respectively at the commencement of a dispense operation;

Figure 6 is a perspective view from below of a multi- dose housing including five reagent holders similar to those shown in Figures 1 to 5;

Figure 7 is a perspective view of a tooling form for bung support during filling;

Figure 8 is a perspective view of the multi-dose housing of Figure 6 seen from above and without reagent holders;

Figure 9 is an exploded view of the testing assembly;

Figure 10 is a cross-section through a second example of a reagent holder; Figure 11 is a perspective view of a third example of a holder;

Figure 12 is a perspective view of the Figure 11 example fitted with a cover;

Figure 13 is a view similar to Figure 12 but illustrating an alternative cover; and,

Figure 14 is an exploded perspective view of a reaction vessel assembly.

The reagent holder shown in Figures 1 to 5 either alone or in combination with other reagent holders located in a multi-dose housing provides the requirements for a pre-packaged test in which measured doses of reagents can be supplied to a reaction vessel such as a disposable cuvette. In some cases, a multi-site test substrate such as a biochip may be provided in the reaction vessel. The reagent holder shown in Figures 1 to 5 comprises a single injection moulding of a plastics material, such as polyethylene, as shown at 1 defining a cylindrical, internal reservoir section 2 having a relatively wide diameter. Alternative means and materials include metal casting and drilling. The reservoir section 2 having a typical diameter of 6mm is coupled via a conical section 3 to a narrower cylindrical portion 4 of the injection moulding defining a dispense nozzle and having a typical diameter of 2.5mm. The narrower, cylindrical portion 4 has a rectangular slotted section 5 removed to define an exit opening. This section or slot 5 extends radially from the nominal centre of the moulding through the wall such that the longest side profile is aligned with the axis of the moulding 1.

To complete the structure, two further parts are required namely a plug 10 and a plunger 11. The plunger 11 and the plug 10 are typically manufactured from a compliant/compressible elastomeric material such as a rubber, silicon or TPE (Thermo Plastic Elastomer) such as Santoprene™. The method of assembly will now be described. Initially, the plug 10 is inserted through the wider reservoir section 2 into the narrow portion 4 and pushed down to the closed end as shown in Figure IB. The geometry of the plug 10 is such that it is of a shorter length than that of the slot 5. The plunger 11 is then introduced into the reservoir section 2 and pushed fully into the conical section 3. It will be noted that the plunger 11 has a conical section 12 which generally corresponds to the shape of the conical section 3 such that these sections will meet progressively to ensure that substantially all entrained air is expelled towards the exit opening or dispense nozzle 5. Pressure is maintained on the plunger 11 by means not shown.

The plug 10 is slidable, a friction fit and a fluid seal within the portion 4 of the moulding 1. The plunger 11 is also a friction fit and fluid seal in the wider reservoir section 2 of the moulding 1. Next, a tooling form (to be described with reference to Figure 7) is introduced into the slot or exit opening 5 from the direction of the closed end of the slot so as to displace the plug 10 towards the plunger 11 until they meet. This is shown in Figure 3. The geometry of the plug 10 and plunger 11 is such that the abutting surfaces form a seal around the periphery of the smaller diameter portion 4 leaving a small central volume 13 in the form of two opposing cones. The relative length of the dispense nozzle 4, the length of the exit opening 5 and the height of the plug 10 are such that the plug 10 effectively occludes and seals the narrow diameter portion 4 with respect to the exit opening 5 thus effectively closing the opening.

In the next stage, a dosing needle (not shown) is introduced through the upper end of the moulding 1 into the reservoir section 2 and through the centre of the elastomeric plunger 11 stopping such that the tip of the needle is located within the small volume 13.

In an alternative approach, the needle may be pre- introduced into the plunger 11 prior to the insertion of the plug 10. In this situation, the dosing needle is introduced just through the centre of the plunger 11 prior to insertion of the plunger into the moulding of the reservoir. The plug 10 is pushed down to the bottom of the reservoir. The plunger 11 plus needle is inserted into the reservoir and pushed down to the bottom to push gas/air out. A tool is inserted to position the plug 10 to seal the outlet. A vacuum is introduced in the needle and residual space between the plug and plunger before feeding in fluid through the needle.

The required fluid or liquid dose (including any empirically assessed offset volume) is then accurately measured and injected through the needle. With the plug 10 still constrained by the tooling, the plunger 11 is allowed to move up the reservoir section 2 under the hydraulic pressure of the volume of fluid being pumped. This is shown in Figure 4 where the plug 11 has been displaced upwardly to define a region 15 in the reservoir section 2 filled with reagent. This liquid is sealed within the reservoir section 2 by the plug 10 on the one hand and the plunger 11 on the other. As can be seen in Figure 4B, the plunger 11 includes sealing features 16 which seal against the wall of the reservoir section 2. As an alternative, an 0-ring could be used.

The tooling is then removed and the measured dose of fluid can be retained for extended periods within the region 15 until the dispense process is initiated.

When the entrained volume of fluid is to be dispensed, the plunger 11 is displaced mechanically by means not shown so that it slides towards the plug 10. As pressure is applied to the plunger 11, the incompressibility of the trapped liquid body in the region 15 will hydraulically transfer the pressure onto the surface of the plug 10. Since the plug is only a friction fit in the narrow portion 4, it is free to be displaced downwardly within the narrow portion 4 and this motion continues until the upper surface of the plug 10 passes the upper edge of the slot or exit opening 5. At this point, pressurized fluid is able to escape through the exit opening 5 (Figure 5) . Once the plunger 11 is fully displaced, all the measured dose must have passed the plug periphery via the slot 5 into the reaction chamber or cuvette located below the holder.

It will be understood that careful control of the respective geometries and relative motions between the plunger, plug and injection needle should ensure near zero volumes of gaseous contaminant. If the filling is carried out under a controlled atmosphere the gas involved can be ensured to be chemically inert with respect to the dosing fluid.

Figure 10 illustrates an alternative construction for the reagent holder. In this case, the holder comprises a single injection moulding 100 of plastics defining a central, cylindrical internal reservoir section 102 within which are slidably mounted an elastomeric plunger 111 and an elastomeric plug 110. A reagent to be dispensed is located in the region of the reservoir 102 between the plunger 111 and the plug 110 as in the previous example.

An internal slot 115 is provided in part of the internal wall of the holder 100 opening into the reservoir 102. The longitudinal dimension of the slot 115 is greater than that of the plug 110. When the holder is retaining the reagent, the plug 110 is located at least partially upstream of the slot 115 towards the plunger 111. Depression of the plunger 111 forces the stored liquid reagent and in turn the plug 110 downwards until the plug just passes the top of the slot 115 as shown in Figure 10. Fluid then flows through the slot 115 past the plug 110. When the plunger contacts the plug 110 it is then forced further downwards pushing any fluid in the lower part of the holder down and out through the tip.

The tip 120 of the holder 100 may be shaped (narrowed) , coated or its material chosen in order to optimise the break-off of drops. It is also preferable if the sides of the exit opening diverge so as to reduce problems due to capillary action and the like.

Figure 11 illustrates a modified form of the holder shown in Figure 1 in which the lower section 4 ' of the holder has an exit opening 5' which opens both radially and axially. The exit opening 5' has diverging side walls 120 and a tip 122 which tapers inwardly towards the axis of the holder. The use of this pointed tip 122, particularly when combined with the diverging walls 120, helps drop detachment as the contact area between liquid and tip is reduced, also reducing the overall detachment forces and encouraging break-off in order to minimize the surface energy of the liquid.

As mentioned above, when the plug 10 is pushed down until it just reaches the exit opening or slot 5', liquid is likely to be initially forced out at high speed in a radial direction. Although this may be acceptable in some configurations, it may not be in others. In those latter cases, the portion 4' of the holder may be provided with a cover member 130 (Figure 12) to prevent significant radial movement of the reagent which is then confined to an exit substantially axially.

An alternative cover member 130 ' is shown in Figure 13 which constitutes an extension of the wider portion 1 of the holder.

As a further alternative, the cover member 130 or 130' could be refined into a fluid guide member (not shown) , which is arranged to direct reagent to a dispense tip.

Normally, the plug will be retained within the holder 100. However, in some cases, it will be possible for the plug 110 to be completely ejected along with the liquid reagent. If the presence of the plug 110 could create problems e.g. damage to reaction sites, then the plug could be removed by various means including magnetic attraction if it was manufactured of a suitable magnetic material or contained a magnetic core within a polymeric material . Although the reagent holder described could be used in this form by itself, it is particularly convenient to provide a testing assembly including one and preferably more than one such reagent holder. An example of a testing assembly in the form of a cuvette holder will now be described. The assembly comprises a cylindrical housing 20

(Figures 6 and 8) having an internal structure, most clearly seen in Figure 8, defining five reagent holder support apertures 22, each having an opening 23 in their base through which the narrow section 4 of each holder protrudes. In typical examples, housings similar to that shown in Figures 6 and 8 can be designed to hold between two and six reagent holders. An advantage of having only one or two diameters for the reagent holders is minimization of the number of different plunger sizes required.

In use, empty reagent holders of the type shown in Figures 1 to 5 are first located in respective apertures 22, as shown in Figure 6. A tooling form as shown in Figure 7 is then offered up to the lower end of the housing 20 so that respective blocks 30 of the tooling form 32 locate in corresponding slots or exit openings 5. This forces the respective plugs 10 to the position shown in Figure 3B.

The respective plungers 11 are then inserted into the upper ends of the tubular portions 2 and each reagent holder is then loaded with the appropriate volume of reagent in the manner described above.

The tooling form 32 is then removed.

The housing 20 is then secured at its lower end to a reaction vessel in the form of a cuvette 40 (Figure 9) , a biochip 42 being secured to the base of the cuvette. The cuvette 40 can be secured to the housing 20 by means of a push fit or screw connection or the like.

When the experiment such as an assay is to be performed, the operator or automated/semi-automated equipment (not shown) depresses the appropriate plungers 11 at appropriate times fully into the reservoir sections 2 so that the corresponding reagents are fully dispensed into the cuvette 40, the assay then taking place in a conventional manner.

The surface geometry of the housing 20 can be designed to allow for the provision of a top and/or base sealing foil or film should this feature carry additional benefits with respect to sterility or anaerobic storage conditions.

The lower detail of the housing 20 can be designed/adapted to suit a required cuvette or arbitrary vessel to receive the measured dose(s) .

In addition to the fluid dispense volumes, the housing 20 can provide a through hole access 26 to allow the addition of sample, washing fluid or further/additional reagents. Additional blind cavities may be provided to enable a pre-mixing or pre-dilution to take place within the vessel body. A volume/location may also be incorporated to enable storage/transit of non-fluid parts such as disposable pipette tips.

It is envisaged that this system can be taken to both immunoassay as well as clinical chemistry testing providing a common vehicle to combine both test formats on a single machine. The pre-packed tests still represent a stored volume but this does not limit test access as with a carousel. For any given stored volume, there will be a balance between test variety and test quantity. Advantages of the new approach include:

• No imposed limitation to the variety (or complexity) of test chemistries that may be accessible.

• Extended shelf-life due to single use - likely to be 1 to 2 years depending on specific reagents.

• Simplification of machine mechanics by removal of the carousel and the associated pipetting hardware. • Elimination of reagent waste.

• Simplification of test/consumable inventory requirements - reagent and pipette handling.

• Reagents supplied with matched biochips in same package - less operator or machine matching of batches of reagents and other consumables, if required for example to maintain calibration, i.e. possibly eliminate recalibration if new reagents are selected unnecessarily.

• Reduction in inventory monitoring e.g. number of barcode readers that may be required within or used in conjunction with the analyser instrument.

• The complete unit can be disposable either as a one "piece" unit or in separate parts depending on the assay detection/measurement technique employed, the reagents, and sample under test. By inversion of the holder in an appropriate manner then fluid could be sucked into the reservoir, provided the plunger is of a push/pull i.e. can be mechanically withdrawn or may be retracted by vacuum. Withdrawal of reagent from the reaction vessel may be required for waste storage and/or subsequent processing of biochip or other substrates within the reaction vessel and/or optical or other measurements of within the reaction vessel for assay applications.

Various relative positions of reagent holders and reaction vessels are possible. The outlets of the reagent holders may be above the reaction vessel at the sides of the vessel, or even in the base of the vessel. The reagent holders could be positioned below the reaction vessel and the plungers moved upwards; or to the side of the reagent vessel.

Figure 14 illustrates a modified form of reaction vessel in which the vessel 40 is surrounded by an outer waste chamber 45. In this case, the vessel 40 is provided with apertures or channels 140 in its side wall which open into the waste vessel 45 when the reaction vessel 40 is fully inserted therein. A biochip 150 is held in the reaction vessel 40 under lips 155 (although it could be glued) . If the reaction vessel and biochip are spun or tilted, liquid reagent will flow radially outward from the biochip through the apertures or channels 140 in the side wall and into the waste chamber 45.

In other examples, molecules could be bound directly to walls or base of the vessel 40 and the biochip omitted.

It will be understood that the walls of the reaction vessel and reagent holder(s) may need to be made of an opaque non-transparent material if light sensitive reagents are employed or the material becomes light sensitive when mixed for an assay application.

Claims

1. A reagent holder comprising a liquid reagent reservoir defined by a tubular wall having an exit opening; a plug movable between a closed position, in which it prevents reagent exiting the reservoir through the exit opening, and an open position, in which it allows reagent to exit the reservoir through the exit opening; and a plunger sealingly engaging the reservoir wall such that a reagent is retained between the plunger and the plug, in use, wherein when the plunger is moved towards the plug pressure is applied to the liquid reagent so that the plug is moved to its open position in which reagent is dispensed through the exit opening.

2. A holder according to claim 1, wherein the plug is a friction fit against the wall of the reservoir.

3. A holder according to claim 1 or claim 2, wherein the reservoir wall defines a relatively wide, tubular section in which the plunger is located, the wide section communicating with a relatively narrow tubular section in which the plug is located.

4. A holder according to claim 3, wherein the wide and narrow sections are connected via a conical section.

5. A holder according to claim 4, wherein the plunger is shaped to fit into the conical section.

6. A holder according to any of the preceding claims, wherein the plug is made of an elastomeric material.

7. A holder according to any of the preceding claims, wherein the plunger is made of an elastomeric material.

8. A holder according to any of the preceding claims, wherein the plunger is a friction fit against, and slidable along, the wall of the reservoir.

9. A holder according to any of the preceding claims, wherein the exit opening is provided in a side facing portion of the reservoir wall.

10. A holder according to claim 9, further comprising a cover section extending over the radially outwardly opening portion of the exit opening.

11. A holder according to any of the preceding claims, wherein the exit opening includes an opening axially aligned with the direction of movement of the plunger and plug.

12. A holder according to claim 11, wherein the axially aligned opening extends through a tip of the tubular wall, the tip being tapered inwardly towards the axially aligned opening.

13. A holder according to any of the preceding claims, wherein the sides of the exit opening diverge.

14. A testing assembly comprising a reagent holding device having at least one reagent holder which contains a predetermined quantity of a reagent, the reagent holder having an exit opening which is sealed; a reaction vessel which is permanently, or removably, fitted to the reagent holding device and into which the exit opening opens, the reagent holder including a dispense mechanism which, when actuated, breaks the seal and dispenses the predetermined quantity of reagent into the reaction vessel.

15. An assembly according to claim 14, wherein the reaction vessel contains a multi-site test substrate such as a biochip.

16. An assembly according to claim 14 or claim 15, for use in a predetermined experiment, the predetermined quantity of reagent corresponding to that required for the experiment.

17. An assembly according to any of claims 14 to 16, wherein the reagent holding device includes a plurality of reagent holders.

18. An assembly according to claim 17, wherein each reagent holder contains a different reagent.

19. A testing assembly according to any of claims 14 to 18, wherein the or each reagent holder is constructed in accordance with any of claims 1 to 13.

20. A testing assembly according to any of claims 14 to 19, or a reagent holder according to any of claims 1 to 13, wherein the reagent is suitable for use in an assay experiment .

21. A testing assembly according to at least claim 19, further comprising a filling tool which is adapted to be removably fitted to the reagent holding device so as to hold the or each plug in its closed position to enable the corresponding reservoir to be filled with reagent.

22. A reaction vessel assembly for use in an assay, the assembly comprising a reaction vessel in which is located a surface on which molecules are bound, the reaction vessel having one or more apertures in its outer wall; and a waste collection vessel within which the reaction vessel is located to collect waste fluid which exits through the aperture (s) .

23. An assembly according to claim 22, wherein the surface is provided on a biochip located in the reaction vessel.

24. A testing assembly according to claim 19 or claim 20, wherein the reaction vessel is constructed according to any of claims 21 to 23.