DE69305046T2 - Flow control in an enclosed container - Google Patents

Description

This invention relates to a container device used for processing a liquid under closed conditions, detecting the analyte and collecting waste liquids.

It is known to carry out a polymerase chain reaction (PCR) or other forms of DNA amplification in a container device using, for example, a flexible bag. One such method, in which a stream of a designated sample and reactant flows past a detection chamber and into a blind-ending disposal chamber, is described in EP-A-0 381 501.

Although such a device is very effective, the use of a blind-ended disposal chamber can occasionally create a problem. In particular, sufficient back pressure can be created by the incoming stream to disrupt the sequential reactions desired in the detection chamber. For example, the back pressure can place so much stress on the detection chamber that even the beads used to anchor the intended sample can become dislodged.

The most obvious solution to the back pressure caused by a dead-ended disposal chamber is to connect the chamber to the atmosphere. However, this is not acceptable as it violates the first principle of a PCR device, which is that the amplified product must be kept contained.

Accordingly, prior to this invention, it was a problem to ensure that no undesirable back pressure was generated by a disposal chamber that could interfere with optimal results.

It is therefore an object of the present invention to provide a container device which avoids the above-mentioned problems.

More specifically, in accordance with one aspect of the present invention, there is provided a container device for amplifying and detecting nucleic acid material, comprising:

an inlet port through which a sample can be inserted into the device, the device being sealable after insertion of the sample to prevent loss of nucleic acid material;

a reaction chamber in which the replication of the nucleic acid material in the sample can take place;

a detection site for detecting the nucleic acid material; flow means to enable liquid to flow from the reaction chamber to the detection site; and

a disposal chamber downstream of and fluidly connected to the detection point for receiving reactants and material after they pass through the detection point, the disposal chamber comprising a pair of opposed walls;

characterized in that at least one of the opposing walls is provided with fold lines along a bead such that at least one wall has a bistable configuration, wherein in a first configuration at least one wall is folded out near another of the opposing walls, and in a second configuration extends further distally away from the other of the opposing walls to enable the construction of a To reduce pressure in the disposal chamber.

Accordingly, the invention provides the advantageous feature of a containment device having a blind-ended disposal chamber which minimizes the build-up of back pressures as the disposal chamber fills without the contents of the device escaping to the atmosphere.

For a better understanding of the present invention, reference will now be made, by way of example, to the accompanying drawings, in which:

Figure 1 is a plan view of an embodiment of a device constructed in accordance with the present invention;

Figure 2 is a fragmentary sectional view taken generally along the line II-II of Figure 1;

Figure 3 is a sectional view similar to that shown in Figure 2, but showing a second embodiment of a device constructed in accordance with the present invention;

Figures 4 and 5 are plan views similar to that shown in Figure 1, but showing third and fourth embodiments of a device constructed in accordance with the present invention;

Figure 6 is a fragmentary plan view similar to that shown in Figure 5, but showing a fifth embodiment of an apparatus constructed in accordance with the present invention;

Figures 7A and 7B are sectional views taken along line VII - VII in Figure 6, before and after, respectively, sufficient liquid has entered the disposal chamber to expand the folded opposite wall outwardly;

Figure 8 is a fragmentary sectional view taken along the line VIII-VIII of Figure 6; and

Figure 9 is a fragmentary plan view similar to that shown in Figure 6, but showing a sixth embodiment of an apparatus constructed in accordance with the present invention.

The invention will now be described in connection with certain preferred embodiments in which a particular flexible device is processed by a particular processing device for amplification and detection of DNA.

Furthermore, the invention is applicable regardless of the particular construction of the device and/or the processing means and regardless of whether the device is processed in a horizontal or inclined position, as long as there is a disposal chamber which receives the liquid from the detection point, with the risk of building up a back pressure in such a chamber.

Furthermore, it is usable regardless of the liquid content of the device, that is, this invention does not involve or require any particular chemistry or reaction as long as the reaction is contained in a sealed device. The invention is thus independent of any particular liquid reaction occurring in the detection chamber and is not limited to the detection of nucleic acid materials only.

As shown in Figure 1, the reaction cuvettes 10 usable in the present invention include those having an inlet opening 22 for patient injection of sample fluid, the inlet opening being connected to a PCR reaction chamber 26 via a passage 21. A seal 46 temporarily blocks the flow from the chamber 26. When the seal 46 is ruptured, the liquid is fed via passage 44 into the detection chamber 40 having locations 41 which preferably contain beads anchored in place which complex with any intended analyte passing by them from chamber 26 and then with the reactants coming from the other reactant chambers. These other chambers are chambers 30, 32, 34 and optionally additional chambers 36 which lead to chamber 40 via passages 48, 50 and 52 respectively. Each of these passages is temporarily sealed at 56 and contains an appropriate reactant liquid (and possibly residual air).

The details of the chemicals usable in all chambers and of the positions 41 are explained in more detail in the above-mentioned EP-A-0 381 501. However, since the invention of EP-A-0 381 501, the number of chambers required has been reduced. Thus, chambers 26, 30 32 and 34 preferably comprise:

The chamber 26 may contain, in addition to the patient's fluid, which is later added by the user, all of the usual reagents necessary for PCR amplification, which are held there by a temporary seal 25. This includes primers bound to one member of a binding pair, the other member appearing in the chamber 30, described below. A useful example of the binding member bound to a primer is biotin. (The seal 25 is ruptured by injection of the sample.) Alternatively, the reagents may be injected with the sample, thus eliminating the seal 25.

The chamber 30 preferably contains an enzyme bound to a complexing agent such as avidin, which is one member of a binding pair, the other member of this pair being bound to the intended analyte in the reaction chamber 26. as described above. Streptavidin-horseradish peroxidase (hereinafter referred to as streptavidin-HRP) is therefore a reagent that can be used in chamber 30.

The chamber 32 preferably contains a washing solution as a reactant.

Chamber 34 preferably contains a signal precursor and a color stabilizing agent, which may be useful. Accordingly, for example, a solution of a leuco dye, which is a common substrate for the enzyme in chamber 30, can be used as the reactant solution in chamber 34.

The remaining chambers 36 are preferably eliminated, along with their passageways, but may optionally be added. Therefore, if a second wash is desired prior to adding the leuco dye to chamber 34, such a wash is provided through chamber 34, and the leuco dye is moved to chamber 36, and so on.

Chamber 40 supplies chamber 42 via passage 58. Chamber 42 is a waste collection chamber to which the invention is particularly applicable, as described below.

A roller 60 illustrates the external pressure device used to sequentially rupture each of the chambers and sequentially convey the contents of the respective chambers into the detection chamber 40. The roller 60 advances along the path 62 of width "A".

The distances P1, P2 and so on between the exit points are preferably the same for each burstable chamber.

Sealing of the inlet port 22 is accomplished by folding over the upper left corner of the cuvette, Figure 1, to bend down the passage 21, as described in US-A-5,154,888.

According to the present invention, the disposal chamber 42 is intended to contain any excess liquids flowing past the detection points in the chamber without creating back pressure due to the lack of a drain. This is accomplished by forming a disposal chamber 42 that includes opposing walls 70, 72, Figure 2, that provide the major portion of the interior surface area (as opposed to the side walls 80) of the chamber, i.e., at least 51% of the total surface area. At least the wall 72 has sufficient fold lines 74 to provide the wall 72 with a bistable configuration. The fold lines are formed in at least one of the opposing walls of the pair 70, 72 so that a nip projects from the plane of the opposing wall. The fold lines and the crease can either be a continuous closed loop or represent the major portion of a closed loop, for example at least 50% of the loop that would be formed if the fold lines and the crease extended all the way around. Furthermore, the fold lines and the crease can be located either on the perimeter or slightly inside the perimeter of the disposal chamber.

As shown in Figure 1, the fold lines 74 form a closed loop which most preferably describes a pattern, Figure 1, which is congruent with the overall shape and the inside of the perimeter of the chamber 42 defined by the side walls 80. The walls 80 connect the walls 70 and 72, Figure 2, to form a sealed enclosure of the chamber except for the incoming passage 58. As shown, the shape is roughly a rectangle. Other shapes are also obvious.

The bistable configuration is also evident. Initially, wall 72 is folded as shown by the solid line so that it is near wall 70. However, as liquid moves into chamber 42, wall 72 snaps outward along fold line 74 to assume the phantom position, thereby relieving any back pressure that is created. In reality, back pressure is initially built up to a point sufficient to snap wall 72 outward, at which point the pressure in chamber 42 becomes negative until more liquid is entered.

Optionally, there may be more than one fold line (not shown), for example to form concentric shapes, which in turn allow further extension of the wall; for example, on the inside of line 74, another fold line could be included, describing a concentric rectangle.

Optionally, an expansion pad 90 is included which, when wetted, has a tendency to expand, thereby assisting in the process of displacing the wall 72 to its outer position away from the wall 70. Such a pad may be any conventional sponge, such as a commercially available cellulose sponge, which is dried to a compressed state.

As a further alternative embodiment, Figure 3, both walls of the disposal chamber may have fold lines so that both walls have a bistable configuration. The parts similar to those previously shown bear the same reference number suffixed with the distinguishing letter "A".

Accordingly, the disposal chamber 42A is constructed as in the embodiment of Figure 2, except that the wall 70A has a fold line 74A' similar to the fold line 74A of the wall 72A. The solid line positions are of course the folded configuration in which the two opposite walls are close together, whereas the phantom positions represent the expanded configuration in which the walls are apart from each other. Greater expansion is possible if both walls are so equipped. As before, there may optionally be a cushion 90A, preferably glued to one or the other of the walls 70A or 72A, if present.

The paths followed by the passages 44, 48, 50 and 52 need not be as shown, nor do they need to extend so far from the path 62 of the roller 60. Instead, the passages may be arranged so that the majority of their path length (at least half) lies within the path 62 of the roller, Figure 4. The parts similar to those previously described bear the same reference number to which the distinguishing letter "B" is affixed.

Accordingly, the cuvette 10B has an inlet port 22B and, as described with reference to the previous embodiments, all of the chambers 26B, 30B, 32B, 34B, 36B, 40B and 42B have passages 44B, 48B, 50B and 52B, respectively, which provide fluid connecting the upstream chambers to the chambers 40B and 42B. The disposal chamber 42B has the fold line 74B to allow at least the wall 72B to snap outward to release the back pressure. However, unlike the previous embodiments, the major portions of the paths of passages 48B and 50B are parallel and closely adjacent to the path of passage 44B which provides fluid from chamber 26B, so that the application of roller pressure along path 62B of width "A" will cause the roller at a particular point to move each of the named passages along at least half its length Such reach through the roller allows better control of the emptying of the respective passages. That is, as long as the roller clamps off each passage, including passage 44B, which occurs up to point "X", there can be no "counterflow" into that passage which could interfere with the proper sequential delivery of the reactants to the detection sites.

Optionally, each of the chambers 30B, 32B, 34B and 36B may be provided with a side fill port 100 so that as each chamber is filled the fill will exit to line 102, eliminating any air, and then the opposing walls will be sealed together through the liquid at 104, as is conventional. This will ensure that no air bubbles are pushed from the outer roll into the chamber 40B where they could interfere with the liquid phase reactions occurring.

Each passageway in the embodiment of Figure 4, however, has a considerable length from its respective rupturable chamber to where it meets the other passageways just upstream of chamber 40b. This is the feed portion of each passageway. However, it is not necessary that this be so. The length of the feed portion of the passageway from its chamber to the junction with other passageways may be minimized to such an extent that the length is less than the maximum diameter of the rupturable chamber from which it extends, Figure 5. The parts similar to those previously described bear the same reference numeral to which the distinguishing letter "C" is attached.

Accordingly, the cuvette 10C has an inlet opening 22C and all of the chambers 26C, 30C, 32C, 34C, 36C, 40C and 42C, as described with reference to the previous embodiments, have passages 44C, 48C, and 50C, respectively, which provide the fluids which the upstream underlying chambers to chambers 40C and 42C. Disposal chamber 42C includes fold line 74C to allow at least wall 72C to snap outward to relieve back pressure.

Unlike the previous embodiments, however, each passage 48C and 50C connects to passage 44C such that the length L of the passage from its respective rupturable storage chamber to the connection is less than the maximum dimension D of its storage chamber. (As will be shown, because of the teardrop shape of the chambers, the dimension is measured from the future exit opening of the chamber to an opposite point closest to the next upstream chamber.) In fact, the most preferred length L is less than half the dimension D for the respective chamber. Such an arrangement also minimizes the backflow of a reactant from an upstream chamber into the passage length L prior to expulsion of the contents of the storage chamber through the length L. This in turn minimizes undesirable side reactions which might occur between reactants along the path length L rather than in the chamber 40C where they are desired.

As before, the roller 62C preferably covers the majority of the path lengths of the passages.

Optionally, an air vent path 200 may be provided from the reaction chamber 26C back into the sealed portion of the bag, for example into a blind-ended storage area 202 of the bag, to minimize the buildup of back pressure that could prevent the uptake of sample from the opening 22C via the passage 21C. The path 200 is also sealed, as are all flow lines and chambers, from venting to the atmosphere to provide prescribed containment against leakage of the amplified nucleic acid material that could cause contamination by transfer could cause.

The inlet port 22C and passageway 200 are preferably closed and sealed after injection of the samples as in the previous embodiments by folding over the corner, all as described in the aforementioned US-A-5,154,888.

It is not necessary that the fold line of the disposal chamber providing the bistable configuration leave a gap on the inside of the perimeter, or that the fold line crease forms a completely closed loop. An alternative to this is shown in Figures 6 to 8, where the parts corresponding to those previously described bear the same reference number, to which the letter "D" is appended.

Thus, the cuvette 10D, Figure 6, is constructed as described in the previous embodiments, except that the disposal chamber 42D in the opposite wall 72D, Figure 7A, has a fold line 74D, Figure 6, which forms a kink or bend that is not connected to itself to form a closed loop and is located at the periphery of the chamber rather than at a distance on the inside thereof. Accordingly, the fold line 74D is formed into parts 174 and 176 which represent the major portion of the periphery, or a major portion of what would form a closed loop if it were to expand to connect both parts 174 and 176 together. ("Major proportion" as applied to fold line 74D means at least 50%, since lesser proportions would not allow wall 72D, Figure 7A, to move outwardly far enough upon entry of liquid L, Figure 7B.)

When the liquid enters the chamber 42D, the wall 72D eventually springs out of its folded configuration or position due to its bistable construction, Figure 7A, into its expanded second configuration or position, Figure 7B. Only the portion 17B of the wall 72D which clampingly seals the opposite wall 70D, Figure 8, remains unexpanded.

The side wall 80D is not affected by the incoming liquid. That is, as in the previously described embodiment, it does not expand sideways from its original position shown in Figure 7B, which in fact it cannot do since it is sealed at 180 to the opposite wall 70D.

For example, the entire periphery of the chamber 42D from the sealing wall 72D to the wall 70D at the portions 180, Figure 7A, is permanently sealed at these locations, except for the passage 58D, Figures 6 and 8.

Yet another example is shown in Figure 9 in which the same reference numerals are used for similar parts, except for the distinguishing letter "E". Thus, as in the previous embodiments, the cuvette 10E features a disposal chamber 42E with fold lines 74E in one of its paired opposite walls 72E which forms the majority of the interior surface of the chamber. In this case, however, the fold lines form a folded crease, generally in the shape of an "H", comprising a cross member 190 and legs 192 and 194. The linear extent of the crease, defined as (L1 x 4 x L2), is such that it encompasses at least 50% of what would be present if the lines 74E formed a closed loop around the periphery. The outward expansion of the wall 72E will, of course, reach its maximum value along the cross member 190 when liquid enters the chamber 42E.

Claims (7)

1. Container device (10; 10A; 10B; 10C; 10D; 10E) for multiplication and detection of nucleic acid material, comprising: an inlet opening (22; 22B; 22C) through which a sample can be inserted into the device (10; 10A; 10B; 10C; 10D; 10E), the device (10; 10A; 10B; 10C; 10D; 10E) being sealable after insertion of the sample to prevent loss of nucleic acid material; a reaction chamber (26; 26B; 26C) in which the replication of the nucleic acid material in the sample can take place; a detection site (40, 41; 40B; 40C) for detecting the nucleic acid material; Fluid means (44; 44B; 44C) to enable a liquid flow from the reaction chamber (26; 26B; 26C) to the detection point (40; 41; 40B; 40C); and a disposal chamber (42; 42A; 42B; 42C; 42D; 42E) downstream of the detection point (40; 41; 40B; 40C) and fluidly connected thereto for receiving reactants and material after passage through the detection point (40, 41; 40B; 40C), the disposal chamber (42; 42A; 42B; 42C; 42D; 42E) including a pair of opposing walls (70, 72; 70A, 72A; 70B, 72B; 72C; 70D, 72D; 72E); characterized in that at least one of the opposite walls (72; 70A, 72A; 72B; 72C; 72D; 72E) is provided with folded lines (74; 74A, 74A';74B;74C; 74D, 174,176; 74E) along a bead such that the at least one wall has a bistable configuration, the at least one wall (72; 70A, 72A; 72B; 72C; 72D; 72E) being buckled proximate the other of the opposing walls (70; 70A, 72A; 70B; 70D) in a first configuration and extending further distally away from the other of the opposing walls (70; 70A, 72A; 70B; 70D) in a second configuration to reduce the buildup of pressure in the disposal chamber (42; 42A; 42B; 42C; 42D; 42E) by movement of the at least one wall (72; 70A, 72A; 72B; 72C; 72D; 72E) from the first configuration to the second configuration. 2. The device of claim 1, wherein both opposing walls (70A, 72A) have folded lines (74A, 74A') and the bistable configuration. 3. Apparatus according to claim 1 or 2, wherein the disposal chamber (42; 42A; 42B; 42C; 42D; 42E) further comprises chamber side wall portions connected to the opposing walls (70, 72; 70A, 72A; 70B, 72B; 72C; 70D, 72D; 72E) to give the chamber a predetermined shape in plan view, and wherein the folded lines (74; 74A, 74A'; 74B; 74C; 74D, 174, 176; 74E) form a shape that is congruent but smaller than the predetermined shape. 4. Device according to claim 1 or 2, wherein the folded lines (74; 74A, 74A'; 74B; 74C) form a bead which represents a closed loop. 5. The device of claim 4, wherein the loop forms a shape which is concentric with the overall shape of the disposal chamber (42; 42A; 42B; 42C). 6. The apparatus of claim 1 or 2, further comprising at least one storage chamber (30B, 32B, 34B, 36B; 30C, 32C, 34C) and an associated liquid passage (48B, 50B, 52B; 48C, 50C) extending between the storage chamber (30B, 32B, 34B, 36B; 30C, 32C, 34C) and the detection site (40B; 40C) along a curved path, with at least half of the liquid passage being arranged parallel to and closely adjacent to at least half of the fluid (44B; 44C) such that a roller applied to the contents of the reaction chamber (26B; 26C) and each storage chamber (30B, 32B, 34B, 36B; 30C, 32C, 34C) can be moved along a path (62B; 62C) which also covers at least half of the passage (48B, 50B, 52B; 48C, 50C) and the fluid (44B; 44C). 7. Apparatus according to claim 6, wherein each passageway (48C; 50C) exiting from each of the chambers (30C, 32C, 34C) connects the others at a location upstream of the detection site (40C), the length (L) of the passageway (48C; 50C) from each storage chamber (30C, 32C, 34C) to the site being less than the maximum dimension (D) of the storage chamber (30C, 32C, 34C) so that the possibility for backflow of the reaction material within the passageway length is minimized.