WO1997006437A1

WO1997006437A1 - Diagnostic device

Info

Publication number: WO1997006437A1
Application number: PCT/EP1996/003380
Authority: WO
Inventors: Hendrik Sibolt Van Damme; Eduard Gerard Marie Pelssers; Henricus Joseph Ida Theodorus Kaelen
Original assignee: Akzo Nobel N.V.
Priority date: 1995-08-03
Filing date: 1996-07-30
Publication date: 1997-02-20
Also published as: AU6788796A; JPH11510601A; KR19990036069A; EP0843817A1; CA2228485A1

Abstract

The present invention describes a device for the detection or determination of the amount of an analyte in a test liquid in an autonomous way. The device comprises a transparent body provided with a conduit having an inlet and an outlet end. This conduit comprises a first junction branching off in a first branch and a second branch, and a second junction at which the first branch and second branch join. The first branch is provided with a sample inlet comprising assay reagents. The first branch and second branch are arranged such that, in use, a transporting liquid entering the inlet of the conduit is divided in said first banch and said second branch and promulgates a sample in the first branch through the second junction before the transporting liquid arrives at the second junction via the second branch. The outlet end of the conduit is provided with means for detection of the analyte. Optionally the conduit comprises a semi-permeable membrane between the second junction and the detection means to enable a bound/free separation step.

Description

Diagnostic device

The invention relates to a device for performing an assay for the detection or determination of the amount of an analyte in a test liquid.

Numerous assays have been developed for qualitative and quantitative detection of a variety of analytes such as those concerning the biochemical diagnosis of various human and animal disorders. There is a continuing interest to make these assays easier to perform and thus more available to non¬ technical personnel in a wide variety of environments, such as the doctors office, clinics, the home, crisis centres, emergency rooms, ambulances, blood banks, hospital laboratories etc. Current diagnostic instruments are especially intended for large scale testing and are in general rather complex to allow control over sample size, timing of addition of reagents and incubation time.

Such expensive instruments are not suited for small scale testing and in a non-laboratory environment. In these cases testing is mostly performed by manual procedures. Current manual procedures are often too delicate, requiring much skill, and are consequently not easy to perform by laymen or non skilled people. Manual tests especially designed for use by laymen, such as the so-called dipstick tests, exist but these are only suited for qualitative measurements.

The closest state of the art comprises a disposable diagnostic device and method of use described in HO 94/19484 (Biocircuits Corporation). This patent application relates to a device comprising a first and second flow path orthogonal to each other. The first flow path connects a sample port via a transport channel and an incubation area to a waste reservoir. The transport channel and/or incubation area comprises a reagent. The incubation area comprises a signal producing system and is underneath an optically clear window. The second flow path connects an inlet port via a side reagent reservoir and the incubation area to a side waste reservoir. The side reagent reservoir comprises for example a substrate if the signal pro¬ ducing system is enzyme based.

In use an analyte containing sample is introduced into the sample port and drawn by capillary action through the X transport channel into the incubation area. The analyte participates in the signal producing system' in the incubation area. After a certain period of time a buffered wash solution is introduced via the sample port, displacing the sample to the waste reservoir. Then liquid is introduced into the inlet port at a certain point of time, which liquid dissolves the substrate and enters the incubation area where the visualisation reaction takes place. After a predetermined time one or more readings have to be made. The device may comprise a capillary valve for enhanced control over liquid flow through the incubation area. In this way it is prevented that wash solution introduced via the sample port enters the side reagent reservoir or the side waste reservoir. A capillary valve may also be used to control the flow into and/or out of the incubation area.

From the above it is clear that, despite a significant improvement over the prior art, the device according to WO 94/19484 has several drawbacks. It is not able to perform an immunoassay procedure autonomously. That is, a plurality of manual operations are required, said operations being inter¬ rupted by waiting periods. To obviate this problem the patent application again relies on an apparatus. In addition, wash solution is introduced through the same flow path as the sample and thus the wash solution will be contaminated by analyte and reagent due to mixing and desorption of analyte and reagent from the walls of the transport channel, resulting in unsatisfactory washing at the incubation area. In addition, readings have to be performed after a specific predetermined period of time.

The object of the present invention is to provide a device capable of performing an assay, and in particular an immunoassay, requiring less manual operations with a reduced number of waiting periods.

This is achieved with a device for performing an assay for the detection or determination of the amount of an analyte in a liquid sample, characterized in that the device comprises a body provided with a conduit having an inlet end and an outlet end, said conduit comprising a first junction branch¬ ing off into a first branch and a second branch, and a second junction at which said first branch and second branch join, wherein the first branch comprises a εample inlet, and the first branch and second branch are arranged such that, in use, a transporting liquid entering the inlet of the conduit is divided in said first branch and said second branch and promulgates a sample in the firεt branch through the second junction before the transporting liquid arrives at the second junction via the second branch, and the outlet end being provided with detection means enabling the detection or determination of the amount of the analyte in the sample.

Thus, merely introducing liquid, such as washing or transporting liquid, through the inlet results in transport of the sample via the second junction into the conduit, while in addition to this the liquid arriving at the second junction via the second branch remains uncontaminated by analyte and/or reagent. The timing at which the liquid arrives at the second junction depends on the design chosen, not on the person performing the assay, which eliminates human errors.

Preferably the first branch is provided with an absorber element between the first junction and the sample inlet. In use, liquid entering the first branch is absorbed quickly by the absorber element, slowing down or stopping the flow of liquid through the second branch. Air is expelled by the absorber pad, promulgating the sample through the first branch, via the second junction towards the outlet end before the liquid via the second branch reaches the second junction. Thuε, the sample can be promulgated without contact, and thus dilution, with liquid from the first junction. After the absorber pad is saturated liquid is no longer conducted through the first branch. In a preferred embodiment the device according to the invention is characterized in that the sample inlet comprises an outwardly projecting canal allowing the uptake of sample by capillary action into the first branch.

Thus the use of a pipette is obviated. The distal end of the outwardly projecting canal is simply immersed in the liquid sample and capillary forces introduce sample in the device.

Preferably the device is arranged to allow the uptake of a predetermined defined volume of sample, which can for example be achieved using capillary valves, and which will be explained in detail later. This embodiment obviates the use of a pipette or other sampling device requiring training to be used properly.

In a different embodiment the sample inlet is arranged to receive and hold an application rod comprising means for the uptake and release of sample liquid, a defined volume of sample liquid being released into the first branch when the application rod is received by the sample inlet.

This allows for the introduction of a defined volume of sample in the device using an application rod which can be more easily handled, for example for the uptake of sample liquid from a test tube.

Though of particular advantage with the present invention, it should be clear that an application rod may be used for the delivery of a defined sample volume to devices according to the state of the art.

According to a preferred embodiment the inlet end of the conduit comprises a container for transporting liquid.

Thus the use of a pump is obviated, further reducing the operational cost.

To further reduce the number of waiting periods, and allow autonomous operation of the device, a preferred embodiment of the invention is characterized in that said conduit comprises a membrane between said container and the first junction. Introducing washing liquid in the container, prior to introduction of sample, results in a precisely timed assay. The membrane offers a means to control the flow rate of washing liquid and thus the time it takeε for the washing liquid to reach the second junction. Thus, instead of having to add the sample after a particular period after having introduced the washing liquid, it can be added immediately after having introduced the washing liquid and no further attention is needed. The advantage of using a membrane is that, in contrast to for example a constriction in the conduit, it is not easily clogged by some dirt, such as a grain of sand or textile fibre.

According to a preferred embodiment of the invention, part of the wall of the conduit located between the second junction and the outlet end is formed by a semi-permeable mem¬ brane, and an absorber element is arranged immediately adjacent to said semi-permeable membrane outside of the conduit. r

Thus after having arrived at the second junction first, the sample will contact the semi-permeable membrane and only large complexes, comprising analyte bound by the reagent, will be retained while free unreacted reagent and analyte will pass the membrane and be absorbed by the absorber element, together with the sample liquid. Thus a bound/free separation is accomplished. When the washing liquid having followed the second branch reaches the semi-permeable membrane, it can wash the large complexes effectively, especially since it is not contaminated by analyte or reagent from the first branch. After saturation of the absorber element, the large complexes are transported by the washing liquid towards the detection means.

Exemplary embodiments of the invention will now be described in detail with reference to the drawings in which: Figure l is a schematic cross-sectional view of a first embodiment of the device according to the present invention;

Figure 2 shows a cross-section of a part comprising a junction in one embodiment of the invention; Figure 3 shows a cross-section of a modification of the outlet end of the device;

Figures 4a and 4b show a cross-section of the sample inlet of the device;

Figure 5 shows a cross-section of a device according to the invention with multiple branches;

Figure ₆ shows a cross-section of an alternative embodiment of a device according to the invention;

Figure 7 shows a cross-section of a bound/free separation part of the device. Figures ₈-10 show different embodiments of detection means applicable according to the present invention^;

In Figure 1 a device 1 for performing aεsayε, in particular immunoasεayε, is shown comprising a conduit 2 with an inlet end ₃ and an outlet end 4. The conduit 2 comprises a first junction 5 branching of into a first branch 6 and a second branch 7. The first branch 6 and the εecond branch 7 join at εecond junction ₈. The firεt branch 6 compriεeε a εample inlet

9.

In use washing liquid is introduced via the inlet end 3 into the conduit 2. Subsequently an analyte containing sample is introduced into the sample inlet 9. Said washing liquid is transported by capillary action, hydrostatic pressure, gravitational force, a simple pump or otherwise from the inlet end 3 to the outlet end 4. As it is generally preferred to perform assays on small samples, the channels will be small for which reason capillary action will usually be the essential force for transport of liquid. When the washing liquid reaches the firεt junction 5, it iε divided over the two branches 6 and

7. The εample in branch 6 iε promulgated via branch 6b and the εecond junction 8 towardε the outlet end 4. To this end, the branch 6a is preferably provided with meanε that promulgateε the εample mediated by gas in the branch 6 in the conduit 2b. In the embodiment εhown in figure 1 this means compriseε an abεorber element 10. Washing liquid reaching the first junction 5 will be absorbed by the absorber element 10. Gas contained in the abεorber element 10 iε expelled in the process and this gas forces the εample in branch 6 via the εecond junction 8 into the conduit 2b. λε soon as the absorber element 10 is saturated, the flow of washing liquid through branch 6 stops. This prevents washing liquid from passing via branch 6 to the conduit 2b and thus avoids contamination of the washing liquid with the analyte or a reagent present. The absorber element 10 is of porous material such as fibre material like cellulose, nylon, glass, cotton, polyester or acrylic fibres, ceramics or any other material expelling gaε when wetted by the liquid.

For the detection of the analyte the device l may be provided with one or more reagents. The reagent may be added together with the sample, but it is preferred that the sample inlet 9 comprises the reagent. Thus the reaction εtartε when the εample iε introduced in the device 1. If the reaction requireε more than one reagent these can be included in the εample, the εample inlet or both, as desired.

In an alternative embodiment the reagent is contained in the branch 6b. Thus, the reaction is εtarted only when the εample iε promulgated in branch 6b towards the second junction

8. This allows for very precise control over the onset of the reaction, independent of when washing liquid iε introduced into the inlet end 3. Thiε embodiment is particularly advantageous for fast reactions. ?

These reagents may comprise various components required to perform an assay for detection of an analyte in a teεt liquid, εuch as antigens or fragments thereof, antibodies or fragments thereof, DNA or RNA or fragments thereof, other members of specific binding pairs atc. These componentε may be in an unlabeled and/or labeled form. Suitable labelε include enzymeε, fluoreεcent compoundε, chemilumineεcent compounds, particulate labels such as gold sols and dyestuff sols. Furthermore these components may be coupled to a disperεed solid phase material to enable an adequate separation between the fraction bound to, for example, an antibody, and the unbound (free) fraction. Suitable solid phase materialε are for example polystyrene latices.

The embodiment shown in figure 1 is provided with meanε for the εeparation of large complexes formed between reagent and analyte on the one hand and unreacted analyte and reagent on the other hand. The meanε comprise a semi-permeable membrane 11 forming a part of the wall of the conduit 2b and an absorber element 12 immediately adjacent to said semi-permeable membrane 11 outside the conduit 2b. In this application the meanε will be referred to as a bound/free separator and is described in detail in our pending European application EP 480 497, the description of which is herein incorporated by reference. The semi-permeable membrane 11 is preferably an absolute membrane. This type of membranes is characterized by the fact that liquid flow through the membrane occurs only perpendicular to the membrane surface. In addition theεe abso¬ lute membranes do no contain so-called dead spaceε, aε for instance in tortuous membranes, which can hold a certain amount of the reaction mixture withstanding the εuction action of the absorber element. Reagent from from these dead spaces may dif¬ fuse back into the conduit 2b, thereby decreasing the efficiency of the separation procesε. In order to prevent non-εpecific binding of variouε componentε to the membrane 11, this membrane 11 can be coated for example with a tri-block copolymer of polyethylene oxide and polypropylene oxide, such as F108 Synperonic (ICI Surfactants). In this way especially a solid phase dispersion, such as a disperεion of polyεtyrene latex or gold particles which are very 9 suitable for several types of immunoassay that can be performed using the device according to the invention, can be prevented from attaching to the membrane.

Examples of absolute membranes are track etched mem- branes, such as cyclopore membranes (Whatman, Belgium) and Nucleopore membranes (Nucleopore, USA). Another example of absolute membranes are membranes produced by combined litho¬ graphic and etch techniques εuch aε microεieveε (Aquamarijn Microfiltration, the Netherlandε) . In use the analyte present in the εample will react with the reagents, for example a labeled component and a component coupled to a dispersed solid phase, forming large complexes. The device 1 according to the invention allows control over the time allowed for the reaction to proceed, aε will be discussed shortly, and the reagent containing sample will not arrive at the bound/free separator before the reaction is complete. At the bound/free separator all the sample liquid together with unreacted reagent and analyte, if any, passes through the semi-permeable membrane 11 and is absorbed by the absorber element 12. The large complexes however are retained by the semi-permeable membrane 11. The device 1 is arranged in such a way that the waεhing liquid, coming from the second branch 7, arrives at the semi-permeable membrane 11 only after the complete absorption of the sample liquid. The capacity of the absorber element 12 to absorb liquid is larger than the volume of the sample liquid. Thus, when the washing liquid reaches the semi-permeable membrane 11, it is absorbed by the absorber element 12, resulting in a very effective washing of the retained large complexes and the substantially complete removal of unreacted reagent. It is advantageous if the large complexes are retained in a very small area of the semi-permeable membrane only, the area having reference number Ila. Thus all liquid passes this εmall area which reεultε in a very effective waεhing. To minimiεe contamination of waεhing liquid with reagent, the εemi-permeable membrane ll is located at or near the second junction 8, aε a result of which the walls of conduit 2b will be contacted with and contaminated with free reagent as little as posεible. To prevent the εample liquid/reagent from entering the εecond branch 7 a preεεure barrier, for example an abrupt increase in diameter, increase in hydrophibicity etc., may be provided.

To restrict the area of the semi-permeable membrane

11 serving as a wash area, the conduit 2b can be provided with a moderate presεure barrier. To increase the absorbance capacity, the conduit 2b may compriεe two abεorber elementε 12 opposite to each other, or the conduit may over a part of its length be surrounded by the abεorber element 12.

To increase the capacity of the absorber element 12 it iε mounted over a long diεtance along the conduit 2b. It iε remarked that eεpecially the firεt liquid entering the abεorber element 12 will contain reagent. Thiε reagent will proceed towards the distal end of the device 1. There is no flow of liquid in the lumen of the conduit 2b until the absorber element 12 is εaturated. When the the abεorber element 12 iε saturated, a high concentration of reagent is present at the distal end of the absorber element 12. If the membrane 11 were permeable at lib, reagent could diffuse back into the carefully waεhed large complexeε containing liquid. Thiε would to a certain extent negate the effect of washing. Therefore the part lib of the semi-permeable membrane may be permeable for gas contained in the absorber element 12 only. Alternatively, the semi-permeable membrane 11 may be made impervious over part of its surface, for example by treating it with chemicals that make the membrane impervious, by covering it, for example with adhesive tape, etc. or a semi-permeable membrane 11 can be used which during manu¬ facture was provided with pores in only part Ila of it. other posεibilitieε to prevent reagent from re-entering the liquid are possible.In the bound/free separator shown in figure 7 the conduit 2a, the first branch 6 and the second branch 7 are diverting from the surface of the semi-permeable membrane Ila. Therefore the use of an impervious part of the membrane is not necessary. Another posεibility iε the uεe of an abεorber element

12 which not only abεorbeε liquid but also binds reagent, for example by ionic interaction.

The εemi-permeable membrane 11 and the abεorber element 12 εhould contact each other over preferably the full εurface of the εemi-permeable membrane 11. Thiε may be achieved uεing physical techniques such as presεure, or by laεting phyεi- cal contact, for example by heat-sealing.membrane 11 and absorber element 12 together.

It should be clear that the features described above for the bound/free separator can also be used in combination with the bound/free εeparation device deεcribed in our pending European application EP 480 497.

When the abεorber element 12 is saturated, the large complexes are transported by the washing liquid to a detection means, in the embodiment shown in figure 1 a detection chamber 13. This detection chamber 13 may contain further reagents 14 and 15, for example enzyme subεtrate and chromogen. In the embodiment εhown the detection chamber 13 iε provided with a tranεparent window 37, allowing visual inspection or εpectrophometric reading εuch aε abεorption measurement, fluorescence measurement, as well as nephelometric measurement etc.

Said reagents 14, 15 may also be provided in the conduit 2c. or alternatively in a third branch (not shown), branching of from conduit 2a or branch 7. These reagents are delivered automatically after the bound/free separation reaction is completed. It should be clear that one or more reagentε may be provided at certain places or delivered at certain places as needed for a particular assay. Thus it is also possible to avoid compounds preεent in the εample for interfering with further reagents such as εubstrates, because the compounds are removed during the bound/free separation before the bound analyte is transported to the further reagent or the further reagent is delivered.

As deεcribed earlier, the sample is introduced into the device 1 immediately after the introduction of the waεhing liquid. The time available for reaction between analyte and reagent dependε on the deεign of the device l. If the operation of the device 1 eεεentially relies on capillary action, the device 1 is preferably provided with a container 16 at the inlet end 3, connected to a filler chamber 17, via a membrane 18. The liquid has to pasε membrane 18 which provideε flow reεistance. Other ways of providing flow resiεtance are poεsible but the use of a membrane 18 is advantageous in that it is not easily blocked by air, dirt such aε a grain of εand etc. Suitable membraneε are thoεe with tranεmembrane fluxes in the order of 1 U to 200 ml/min/cm² at 10 PSI. Hydrophilic membranes are preferred as these are more easily wetted by the liquid. To improve the proper functioning the membrane 18 a tiny absorber element 19 in the filler chamber 17, immediately adjacent to and covering only part of the membrane 18, may be provided, facilitating the initial release of the liquid by the membrane 18. This absorber element is preferably made of glass fibre.

The reproducibility of the time delay depends on the uniformity of the tranε-membrane flux of the type of membrane used. By placing the membrane in a horizontal position and feeding the liquid from below, air inclusion influencing the trans-membrane flux, can be prevented.

Though of particular advantage with the present invention, it is remarked that such a time delay element, comprising a container, membrane and filler chamber, can be used with devices according to the state of the art as well, to provide for the timed delivery of a washing liquid.

The device according to the invention can be con- εidered aε a combination of elementε, each capable of performing one or more assay εtepε, alone or in combination, in an auton¬ omous way. These elements are combined to carry out a complete assay and can be used in different combinations to carry out a different process. The device takes care of complex fluid move¬ ments and makes apparatus with complex mechanics and control software superfluous.

It is posεible to manufacture the different elementε aε separate partε that can be uεed aε building blocks to assemble a device according to the invention. Thus a device can be assembled useful for the desired asεay. It iε also possible that one of the elements, for example the sample inlet element contains different reagentε for different typeε of assay.

As mentioned earlier, small sample sizes are gen¬ erally preferred. This allows the device 1 to be operated uεing capillary forces, obviating costly pumping meanε. The εhape of the conduit 2 may vary, but for capillary action to occur two opposite walls of the conduit 2 are preferably between 0.01 and 2 mm apart.

To prevent the liquid from leaving the device 1, the outlet end 4 should act as a presεure barrier, uεing a εudden increaεe in diameter, hydrophobic coating etc. Further control over period of time for reaction can be achieved in several ways. For example, the conduit 2a and the second branch 7 can be or be made more or less hydrophobic, for example by chemical treatment, coating hydrophobic compounds with for example using Teflon spray, fluor-resin or vaεeline, and other methods known per se, and/or the diameter and length of the conduit 2a can be varied. In addition the conduit 2a and second branch 7 may be provided with delay means 20, aε shown in figure 5, comprising an absorber element 21 and a vent 22. Liquid from the container 16' is absorbed by the absorber element 21 and no liquid is passed on until the absorber element 21 is εaturated with liquid. Gaε contained by the abεorber element 21 is discharged to the atmosphere via vent 22.

To avoid the rise of air getting trapped before absorber element 10, thus preventing (timely) absorption of liquid and consequently impeding transport of the sample into the conduit 2b, the absorber element 10 may be shaped as shown in figure 2. The absorber element extends over a part of the wall of conduit 2a. In the embodiment of the abεorber element 10 shown, it haε alεo been given a larger dimension over part of its length in branch 6, to increase the volume of gas the abεorber element 10 can expell, neceεεary for the promulgation of the εample.

The detection meanε may overlap with the bound/free separator, as iε εhown in figure 3. A transparent device 1 allows visual inspection or quantitative measu ents with a suitable apparatuε.

Aε shown in figuur 4a, the εample inlet 9 may be provided with a filter 23 allowing removal of lumps, dirt or for example blood cells. Reagent may be provided below the filter (not shown) . According to a preferred embodiment the sample inlet 9 can be provided with a reagent 24 containing holding means 25. Thuε a device 1 can be provided with one of a multi¬ tude of holding means 25 each containing different reagents 2 , which offers a choice of asεays to be performed with the device 1. In the embodiment shown the holding means 25 also comprises a filter 23, allowing the removal of cells, such aε red blood cells, dust etc.

According to a preferred embodiment sample liquid is introduced in the device 1 by means of an application rod 26 comprising a handle 27 provided with compressible porous material 28 at one end thereof. This porous material 28 is capable of taking up a defined volume of sample liquid which volume is governed by the amount and capacity of said porous material 28. Suitable compresεible porous materials are for example Porex porous material (EDP x-2124), and preferably 3M foam type 1563. Optionally the compressible porous material 28 can be hydrophilized by plasma treatment or treatment with surfactantε εuch aε a mixture of poly(oxyethylene) εorbitane mono-laurate (Tween 20) and εorbitane-monooleate (Span 80).

The compreεεible porouε material 28 iε dipped into a sample liquid whereby a defined sample volume is taken up. Subsequently the application rod 26 is inεerted in the εample inlet 9 and the porouε material 28 iε compreεεed, whereby thiε defined sample volume is released. To this end the handle 27 engages the sample inlet 9 in an airtight fashion, while a sudden decrease in cross-section of the sample inlet 9 provides a surface against which the porous material 28 is compressed. The released volume of sample should be such that it cannot come into contact with the absorber element 10 in the first branch 6a. In an alternative embodiment the compresεible porouε material 28 can be provided with a reagent, offering the same advantage as the holding means 25 described above.

The reagents for the device 1, holding means 25 and application rod 26 are uεually provided in a lyophilized form, though other forms are possible, for example as a coating obtained by evaporation of a solvent. In a preferred embodiment the substrate and chromogen are added in insoluble, inert, porous padε. Suitable materialε for the padε are for example Nylon membrane (MSI, USA) with a pore size of 0,2 tot 1,0 mm. Said pads do not block the flow of liquid and allow rapid release of reagent into the liquid.

Another embodiment of the device l according to the invention iε shown in figure 5. The device 1 compriseε a conduit 2, container 16, junctionε 5, 8, brancheε 6, 7 and absorber element 10 corresponding to those described with reference to figure 1.

The εaraple inlet 9 compriεeε an outwardly projecting canal 29 allowing taking up sample liquid by capillary action by dipping the distal end of the canal 29 in the sample liquid. The IH volume of sample liquid introduced in branch 6 is defined by means for stopping the flow of liquid by capillary action. Thuε the branch 6a and 6b can each be provided with for example hydrophobic walls over part of the length of each branch 6a, 6b. It should be clear that such a sample inlet can also be used separately or in combination with another device for the uptake of a defined sample volume. In the embodiment shown in figure 5, an abrupt increase in diameter of the branches 6a and 6b pro¬ vides a presεure barrier which can not be overcome, or be over- come quickly, by the sample liquid. However, the presεure built up by gaε expelled by the abεorber element 10, aε discussed in relationship to figure 1, is sufficient to promulgate a defined volume of sample liquid into conduit 2b. A bound/free separator is provided in conduit 2b, comprising a semi-permeable membrane 11 and an abεorber element 12. Waεhing liquid in a container 16', which may or may not be the same as container 16, is delayed by delay meanε 20, discusεed earlier, and arrives at junction 30 with branches 31 and 32, which brancheε 31, 32 join at junction 33, only after the large complexes retained by the bound/free separator have been transported into the branch 31.

The flow of liquid through conduit 2b stops automatically due to the pressure barriers provided in branch 31. Again an absorber element 34 is uεed to promulgate the liquid in the branch 31, containing the large complexeε, into a conduit 35b to a εecond bound/free separator for a second washing step. From this example it is evident that the device according to the invention allows excellent control over the course of the reaction by appropriate design of the device. A third branch as described, or more additional branches, can be uεed to deliver reagentε aε needed for the particular aεsay. Thus the present invention allows a wide range of assays to be performed with a very limited operations to be performed by personell which do not require much training or skill. Miminal inεtructionε εuffice. It is possible to provide a device having one con- tainer and serveral conduits 2', 2" etc. each containing a sample, a blank and/or a εtandard εolution. Thuε adding liquid to the container 16 starts all asεayε at the εame time, allowing for multiple, control and reference measurements

To exemplify the excellent fluid control provided by the present invention the working of the embodiment of a device 1ST according to the invention shown in figure 6 is discuεεed in detail. Waεhing liquid from the inlet end 3 is divided at junction 5. Washing liquid absorbed by the abεorber element 10 expelε gas contained in the absorber element 10, promulgating an asεay mixture, compriεed of εample liquid introduced via εample inlet 9 and reagent, into conduit 2b to the semi-permeable membrane 11. Washing liquid passing through the second branch 7 is divided at a junction 41. Washing liquid pasεing into branch 42 dissolves subεtrate/chromogen reagents 14,15 present in this branch 42. The flow of liquid iε εtopped by the preεsure barrier 44. Waεhing liquid passing from the junction 41 to the second junction 8 flows through conduit 2b and is abεorbed by the absorber element 12, waεhing retained large complexeε in the proceεε. As soon as the liquid reaches the outlet end 4, gas can no longer be discharged to the surroundings. The absorber element 12 however is not yet saturated with liquid and liquid continues to be absorbed. The gas expelled from the absorber element 12 in the process resultε in an increased pressure in chamber 45, as a result of which the disεolved εubstrate is passed via junction 41 to the semi-permeable membrane 11 retaining the large complexes. There, an enzyme reaction may occur, depending on the presence of analyte in the sample. It is clear that the design of the device has to be well-considered. Once designed however, the personnel has no worries over its intricacy and can perform an assay with a mimimum of operations and no control apparatus.

From the above it should be clear that with a device according to the invention gas expelled by an absorber not only can be used to influence the flow of liquid downstream but also upstream.

Figure 8 shows a different embodiment of the detec¬ tion means, the outlet end 104 being provided with a prism 36 which facilitates viεual inspection and/or measurement of the detection reaction. The priεm 36 may have a convex εurface at the diεtal end, in effect forming a convex lenε, to further facilitate inspection of the detection chamber 13. The conduit 2b is provided with reagents 1 , 15 which are for example a εubεtrate for an enzyme, and a chromogen. Here the conduit 2b doubleε aε the detection chamber 13. The embodiment shown in figure 9 is similar to the one shown in figure 8, but here an adsorber 38 is provided that can be inspected, for example viεually. An abεorber element 39 iε provided to facilitate the paεsage of liquid through the adsorber 38. It may also be advantageouε to incorporate a reagent, εuch aε one for stopping an enzyme reaction, in the adsorber 38. The adsorber 38 may concentrate any coloured prod¬ uct formed, increasing the senεitivity of the aεεay. Suitable adsorbers 38 depend on the particular dye used or produced, but Nylon membrane filters (MSI, USA) and preferrably. GF/SE 30 paper (Whatman, UK) appear to be satiεfactory.

Meaεurementε with the device according to the inven¬ tion iε not limited to optical detection, in contraεt, a hoεt of detection methods is possible. Aε an example, figure 10 shows a detection chamber 13 provided with electrodes 40, 40' and reagents 14, 15, allowing electrochemical detection of reaction productε. An enzyme εuitable for electrochemical detection iε glucose-oxidase (GOD) with D-glucose and potassium hexacyano- ferrate as substrates. The device 1 can be made of various materials, pre¬ ferably of transparent plasticε εuch as polystyrene, poly¬ carbonate and polymethylmethacrylate.

Preferably the device comprises two parts la, lb, of which for optical asεayε usually at least one will be of trans- parent plaεtic. If a window iε provided for the detection cham¬ ber 13 the partε la, lb may be opaque.

The partε la, lb are advantageouεly formed by injec¬ tion molding, and contain the conduit 2 and other elementε aε recesses. Advantageously the recesses all lie in one plane, but this iε not required. For assembly, the parts are provided with aborber elements, membranes, reagentε, hydrophobic material to act as presεure barriers etc. as needed for the intended assay. To assure satiεfactory contact between a membrane and an abεorber element, the abεorber may advantageously be placed in a cavity of one of the parts, the depth of said cavity being slightly lesε than the thickneεε of the absorber element.

Using the device εhown in figure 1, a εample can be introduced, εaid εample dissolving solid reagent comprising antibodies directed against the analyte and labeled with an /? enzyme, and latex particles coated with antibodies directed against the analyte, said sample and dissolved reagent plus latex particles forming a asεay mixture. Prior to the introduction of the εample, waεhing liquid iε introduced in the 5 container 16. From there the liquid paεεes the membrane 18 and enters the conduit 2a. At the first junction 5 the liquid iε abεorbed by the abεorber element 10 which expels gas in the procesε. Thiε gaε promulgateε the aεsay mixture via the second junction 8 to the semi-permeable membrane Ila, where the asεay

10 mixture iε abεorbed by the abεorber element 12, in effect stop¬ ping the immunological reaction, which should be completed by then anyway, while the latex particles are retained. After the saturation of the absorber element 10, washing liquid passes through the second branch 7 via the second junction 8 to the

15 semi-permeable membrane Ila. The retained latex particles coated with antibodies which have bound the analyte present in the sample to which in turn antibodies conjugated with enzyme have bound, are washed with the washing liquid until the absorber element 12 is saturated. Then the latex particles are

20 transported by the washing liquid to the detection chamber 13, where subεtrate iε provided for the enzyme. After a prede¬ termined time the colouration of the εolution iε recorded, with the naked eye or uεing an apparatuε. Thuε, the only operationε that have to be performed are: introduction of waεhing liquid,

25 introduction of εample and, after a certain period of time, reading of the detection reaction.

Being cheap to manufacture the deviceε are intended for εingle use, though reuεe iε not excluded.

30 It will be clear that the invention allowε many deεign variations of device according to the invention, allowing many different types of assay including several types of immunasεay to be performed, uεing the timed flow control and improved waεhing provided by the present invention.

■35

The present invention will now be further particularly deεcribed with reference to the following Example.

The device uεed is that depictured in Figure 1.

40 IP

Example

1. Preparation of HIV-l glycoprotein 160 coupled to polystyrene latex particles (solid phaεe coupled σp 160).

To 2 ml of aldehyde activated polyεtyrene latex particles (29.73 μmol aldehydes per m² latex, solid volume fraction = 2%) with a diameter of 723 nm, an amount of 0.4 ml phosphate buffer (500 mM, pH = 7) was added. Subsequently 1.2 ml of a HIV glycoprotein 160 (gp 160) εolution (0.8 mg/ml) waε added, followed by the addition of 0.4 ml of a NaCNBH₃ εolution (250 mM) . The reaction mixture waε then incubated for 4 hourε at ambient temperature. After incubation, the dispersion was centrifuged for 10 min. at 10 OOOxg and the pellet redispersed in a 50 mM glycine buffer, pH 8.5, containing 1 g/1 BSA and 1 g/1 SDS. The latex particles carrying the gp 160, were waεhed twice with thiε glycine/BSA/SDS buffer and subsequently three times with the same buffer but without SDS. Finally the pellet was rediεpersed in said glycine/BSA buffer and stored at 4^*C.

2. Preparation of horse radish peroxidase fHRPi labelled OP 160 fσp 160 conjugatei .

220 mg HRP was diεεolved in 2.81 ml NaH₂P0₄ buffer (12 g/1), pH 7.5, containing 5.85 g/1 NaCl, and incubated during 30 min. with 0.22 ml of a εolution of SPDP in ethanol (12.5 g/1). The obtained HRP-SPDP waε purified on a PD10 column and eluted with a NaH₂P0₄ buffer (12 g/1), pH 7.4, containing 5.85 g/1 NaCl and 1.86 g/1 dinatriumedetaat.2H₂0. To 12 ml of a εolution of gp 160 (2 mg/ml), 0.12 ml of a DTT εolution in water (154 g/1) was added and incubated for 30 min. at ambient temperature. After elution of the gp 160-DTT mixture over a G25 column, the gp 160- DTT was added to 3 ml HRP-SPDP whereupon the mixture was incubated for 120 min. at ambient temperature in the dark. This conjugate waε frozen and εtored below - 50°C.

3. Preparation of immobilized substrate reagents,

a. Preparation of TMB pads. 13

Tetramethylbenzidine hydrochloride (TMB) was dissolved in a solution of 9.1 mg citric acid/ml, 1 mg di-sodium EDTA/ l, 156.5 mg PEG-8000/ml in a concentration of 8 mg TMB/ml. 10 μl of this solution was sprayed per square cm MSI nylon membrane with a E pore size of 0.22 μm. After spraying the pads were dried under nitrogen and subεequently cut into pieces of 10 by 1mm. These pads were then placed and fixed in the detection chamber (13) of the device (l) by slight mechanical preεεure.

0 b. Preparation of εodiumperborate-tetrahydrate pads.

Sodiumperborate-tetrahydrate was dissolved in a solution of 44 mg citric acid/ml, 80 mg εodium citrate, 2.6 mg di-εodium EDTA/ml and 156.5 mg PEG 8000/ml in a concentration of 20 mg/ml. 10 μl of thiε εolution waε sprayed per square meter nylon 5 membrane with a pore size of 0.22 μm. The pads were further handled aε decribed for the TMB padε.

4. Preparation of the device containing the assav reagents.

0 The device used is that depictured in Figure 1. The conduit (2a, 2b, 2c) and its branches (6, 7) have a crosε-εection of 2 mm by 0.1mm. The porouε material (28) of the application rod (26) iε capable to take up 30μl of liquid. The εemi-permeable membrane (Ila) used is a Cyclopore membrane (Whatman sa, Belgium), with a 5 pore size of 0.6 μm.

Samples of 15μl containing 5% mannitol, 5% trehalose, 5% NSS, 0.81 g/1 sodiumthiocyanate, 0.91 μg/ml gp 160-HRP conjugate and 0.0425% gp 160 coupled to latex, were applied to holding means (25) aε depictured in Figure 4b. Subεequently theεe εampleε were 0 freeze dried in these holding means, whereupon the holding meanε (25) containg the reagentε (24), were placed in the εample inlet (9) of the device (1) .

5. Assav method. 5

Distilled water is added to the container (16) of the device(l) in a volume of 5 ml, which is indicated by a mark on the wall of the container. Immediately after this handling the application rod (26) is inserted in the εample liquid, whereby 30 μl of sample liquid is taken up. The application rod is then inserted in the sample inlet (9) and the liguid released by pressing.

After 15 min. the colour is read through the window (37) and a slight to blue colour is an indication of a positive sample. The colour of a negative sample is not visually detectable. In this way an amount of 20 ng/ml of human monoclonal anti-gp 41 in serum can be detected.

Claims

1. Device for performing an assay for the detection or determi¬ nation of the amount of an analyte in a liquid sample, charac- terized in that the device compriseε a body provided with a conduit having an inlet end and an outlet end, εaid conduit compriεing a firεt junction branching off in a firεt branch and a εecond branch, and a εecond junction at which εaid firεt branch and εecond branch join, wherein the firεt branch com- priεeε a sample inlet, and the first branch and second branch are arranged εuch that, in use, a transporting liquid entering the inlet of the conduit iε divided in said first branch and said second branch and promulgates a εample in the first branch through the second junction before the transporting liquid arrives at the second junction via the second branch, and the outlet end being provided with detection means enabling the detection or determination of the amount of the analyte in the sample.

2. Device according to claim 1, characterized in that the con- duit is arranged to allow the transporting liquid to flow under capillary action.

3. Device according to claim 1 or 2, characterized in that the first branch iε provided with an absorber pad between the first junction and the sample inlet.

4. Device according to anay of the preceding claims, character¬ ized in that the sample inlet comprises an outwardly projecting canal allowing the uptake of sample by capillary action into the first branch.

5. Device according to claim 4, characterized in that the εample inlet and the first branch are arranged to allow the uptake of a defined volume of sample.

6. Device according to claim 4, characterized in that said sample inlet comprises asεay reagentε.

7. Device according to any of claimε 1 to 3, characterized in that the εample inlet iε arranged to receive and hold an appli¬ cation rod compriεing meanε for the uptake and releaεe of εample liquid, a defined volume of εample liquid being releaεed into the firεt branch when the application rod iε received by the εample inlet.

8. Device according to claims 1 to 3, characterized in that the sample inlet is arranged to receive and hold a filter holder, comprising a filter and assay reagentε, whereby said filter holder is arranged to receive and hold an application rod, a defined volume of sample being releaεed into the first branch when the application rod is received by the filter holder.

9. Device according to any of the preceding claims, character¬ ized in that the inlet end of the conduit compriseε a container for transporting liquid.

10. Device according to claim 9, characterized in that said conduit comprises a membrane between said container and the firεt junction.

11. Device according to any of the preceding claims, character¬ ized in that part of the wall of the conduit located between the second junction and the outlet end is formed by a semi-permeable membrane, and an absorber element is arranged immediately adjac¬ ent to said semi-permeable membrane outside of the conduit.

12. Device according to any of the preceding claimε, character¬ ized in that said detection means compriseε a conduit ending in a detection chamber.

13. Device according to claim 12, characterized in that the detection chamber is provided with reagents for detection of the analyte.

14. Device according to claim 12 or 13, characterized in that the wall of the detection chamber iε provided with an adsorbing material and an absorber is arranged immediately adjacent to said adsorbing material outside of the detection chamber.

15. Device according to any of the preceding claimε, character¬ ized in that the device comprises a third branch to allow fur- ther control over the asεay.