Assay device

A disposable assay device for assaying a sample has a body (10) including a reaction chamber (12) which contains or is adapted to receive an assay reagent sensitive to a component (e.g. nicotine metabolite) being assayed for in the sample. A sample collector/dispenser (20) has a sample collecting chamber (22) closed by an elastic membrane (24) and a downwardly projecting sampling and piercing tube (28), to enable a predetermined quantity of sample to be dispensed into the reaction chamber (12). The body (10) and the collector/dispenser (20) are non-detachably engageable together by engagement of rib (34) on collector/dispenser (20) with lip (18) on the body (10). A seal (32) seals the assembly to prevent leakage of the contents after use.

This invention relates to an assay device and is more particularly 
concerned with a disposable assay device which is relatively safe and 
convenient to use and dispose of, and in which there is a reduced risk of 
operator contamination from either the assay reagents, or the sample to be 
analysed, or the resultant reaction mixture. This invention is 
particularly, but not exclusively, concerned with a disposable assay 
device for assaying for nicotine metabolites in samples of urine for the 
purpose of checking on recent smoking habit. 
BACKGROUND OF THE INVENTION 
The treatment of smoking-related disease is a major expenditure and there 
is a need for a convenient accurate determination of patient's smoking 
habit in order to determine appropriate and effective treatment of 
smoking-related diseases. There is also a demand for an assay device which 
can be used by non-chemists in extra-laboratory situations such as doctors 
surgeries, anti-natal clinics, industrial plants, water works, farms or 
the home. In order to be suitable for this, it is important for the assay 
device to be relatively easy and safe to use and to be relatively safely 
disposed of. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide an assay device which 
meets the above-mentioned demand. 
According to the present invention, there is provided a disposable assay 
device for assaying a sample, comprising a body including a reaction 
chamber which contains or is adapted to receive an assay reagent sensitive 
to a component being assayed for in the sample, a sample 
collector/dispenser for collecting a sample to be assayed and for 
dispensing a predetermined quantity of sample into the reaction chamber, 
said body and said collector/dispenser being non-detachably engaged or 
engageable together, and means for sealing the assembly of body and 
collector/dispenser to prevent leakage of the contents of the reaction 
chamber after use. 
In a first series of embodiments, the sample collector/dispenser is adapted 
to be non-detachably engaged with said body after a sample has been 
collected, and the sealing means acts between the body and the 
collector/dispenser. 
In this first series of embodiments, it is preferred for the sample 
collector/dispenser to include a sample collection cheer defined between 
two relatively moveable parts such that relative movement of the parts in 
one direction causes said predetermined quantity of the sample to be 
dispensed therefrom into the reaction chamber. 
In one arrangement, the sample to be assayed is caused to enter a reservoir 
in which it is retained until being dispensed therefrom. In one aspect, 
the reservoir is defined by the cylinder of a piston and cylinder device 
so that the sample can be aspirated into the reservoir and then dispensed 
therefrom by relative movement of the piston and cylinder. In another 
aspect, the reservoir is filled by immersing it in the sample to be 
collected and causing the sample to be retained by closing the reservoir 
or designing the reservoir so that it retains the sample therein by 
surface tension. In the latter case, the surface tension effect can be 
achieved by providing the reservoir in a relatively narrow bore tube 
having a lower opening and having an upper opening which is openable to 
allow the sample to be collected and dispensed, but closed to retain the 
sample therein. In an alternative arrangement, the surface tension effect 
is achieved by filling the reservoir with a wicking element comprising an 
absorbent material which can be compressed to dispense sample absorbed 
therein. 
The reaction chamber itself may be closed by a pierceable or moveable 
membrane. 
The membrane may be a multi-layer construction and incorporate one or more 
reagents in order to enclose and separate it/them from other reagent(s). 
This multi-layer form of construction is useful for reagents which, when 
mixed together, can be unstable and must be kept separate prior to use. 
In a second series of embodiments, the body and the collector/dispenser are 
permanently non-detachably engaged together and the collector/dispenser 
has an inlet which is opened to collect a sample, said inlet being 
closable by the means for sealing the assembly. In such an arrangement, 
the sealing means preferably comprises a cap which is engageable over that 
part of the collector/dispenser having the inlet so as to seal the 
assembly permanently. 
The disposable assay device of the present invention is preferably supplied 
with the assay reagent therein, most preferably in solid form for use with 
a liquid sample. The assay reagent is preferably one which is designed to 
assay for nicotine metabolites. Most preferably, the assay reagents are 
arranged to assay for cotinine and equivalents thereof using a 
colorimetric assay based on the Koenig reaction (see for example Clinica 
Chimica Acta, 196 (1991) 159-166 or Thorax 1985;40;351-357).

DESCRIPTION OF PREFERRED EMBODIMENTS 
Referring now to FIG. 1a, the assay device illustrated therein comprises an 
open-topped elongated cylindrical body 10 having a reaction chamber 12 
defined at its closed lower end. The reaction chamber 12 contains solid 
assay reagents 14 and is closed at its top by a pierceable membrane 16. 
The reagents may be loose within the chamber 12, immobilized onto the 
walls thereof, or applied to a separate member such as a disc of filter 
paper-like material disposed in the chamber 12. At its open upper end, the 
body 10 is provided with an integral, inwardly directed annular lip 18 
having an upwardly presented lead-in ramp surface 18a and a radially 
directed rear (or lower) abutment surface 18b. 
The assay device further comprises a sample collector/dispenser 20 having a 
sample collection chamber 22 closed by an elastic membrane 24. The 
collector/dispenser 20 has an upstanding wall 26 to facilitate grasping 
thereof without accidental depression of the membrane 24. The 
collector/dispenser 20 further includes a downwardly projecting sampling 
and piercing tube 28 which opens into the chamber 22 through lateral 
apertures 29 in a hollow volume-determining stop 30. The stop 30 is 
disposed within the chamber 22 and serves to limit downward depression of 
the elastic membrane 24. The collector/dispenser 20 has an outwardly 
directed annular seal 32 which cooperates with the internal surface of 
body 10 to provide a sliding seal arrangement between the body 10 and the 
collector/dispenser 20. The collector/dispenser 20 also has an outwardly 
directed rib 34 adjacent its lower end but above the seal 32. The rib 34 
has a lead-in lower ramp surface 34a and an upper surface 34b (as viewed 
in FIG. 1a) which extends radially outwardly. 
The assay device illustrated in FIG. 1a is supplied with the body 10 and 
sample collector/dispenser 20 completely separated. A sample to be assayed 
is aspirated into chamber 22 by dipping the tube 28 into the liquid after 
depressing membrane 24 until it abuts the stop 30, followed by release of 
the membrane 24 to draw the sample into the chamber 22. The 
collector/dispenser is then carried by means of the upstanding wall 26 
taking care not to touch the membrane 24 and fitted into engagement with 
the body 10 so as to adopt the position illustrated in FIG. 1a. Hence, the 
seal 32 is forced past the inwardly directed lip 18 followed by the 
outwardly directed lip 34. The shape of the seal 32 and the provision of 
the ramp surfaces 18a and 34a assist in engagement of the 
collector/dispenser 20 with the body 10 but prevent subsequent detachment 
of these two parts, whilst the seal 32 ensures that a proper seal is 
provided between these two parts. The provision of the lips 18 and 34 also 
assists in providing a seal as well as providing abutment surfaces to 
prevent detachment of the parts without actually destroying either or 
both. 
The collector/dispenser 20 is then urged downwardly from the position 
illustrated in FIG. 1a until the tube 28 has pierced the membrane 16. 
Finally, the elastic membrane 24 is depressed until it abuts against the 
stop 30, thus dispensing a predetermined quantity of the liquid sample 
into the reaction cheer 12. The contents of the reaction cheer 12 can then 
be mixed and the assay results visualised through the wall of the body 10 
which is transparent at least in the region of the reaction chamber 12. 
Instead of visualising the results, e.g. by direct observation of colour 
change in the reaction mixture, it is possible to introduce the assay 
device into a suitable colorimeter or the like to provide an automatic 
reading of colour change or change in turbidity. Once assaying has taken 
place, it will be appreciated that the device can be disposed of with a 
minimum risk of leakage of the contents of the device by reason of the 
sealing of the body 10 relative to the collector/dispenser 20. The 
non-detachable engagement of these parts ensures that access to the 
interior can only be gained actually by destroying one or other of the 
parts and that this can be made quite difficult. 
As shown in FIG. 1b, the tube 28 may include a constriction 28a adjacent 
its lower end to prevent premature loss of liquid prior to dispensing. 
As shown in FIG. 1c, the piercable membrane 16 is a multi-part membrane 
composed of upper and lower membranes 16a and 16b which are supported in 
spaced relationship by an annulus 16c. A chamber 17 is defined between the 
two membranes 16a and 16b for a further reagent 14a which is to be kept 
separate from the reagent(s) 14 illustrated and described above in 
relation to FIG. 1a. When the membrane 16b is ruptured by piercing tube 28 
of collector/dispensor 20, the reagent 14a is mixed with the reagent(s) 14 
and with the sample being assayed which is dispensed through the piercing 
tube 28. 
As shown in FIG. 2, where similar parts to those of the embodiment of FIG. 
1a are accorded the same reference numerals, instead of providing an 
elastic membrane 24, collector/dispenser 20 may resemble a convention 
syringe and therefore include a slidable piston 36 for aspirating liquid 
samples through tube 28 into chamber 22 and for dispensing the contents of 
the chamber 22 into the reaction chamber 12 after piercing the membrane 
16. Manually depressible piston rod 38 is provided for this purpose and 
may include a localised weakened region 40 to enable the rod 38 to be 
snapped off after use to prevent the device from being opened. 
Alternatively, a latch 42 may be provided for latching the piston rod 38 
in the closed position. Removal of the piston 36 is prevented by circular 
barbs 41 around the open end of the cylinder of the collector/dispenser 
20. 
In FIG. 3, tube 28 is mounted on the piston 36 and communicates with 
chamber 22 through an upper hole 44 at the underside of piston 36. The 
chamber 22 is defined by a cup-shaped body 46 whose upper surface is 
welded to the underside of a cap 48. A lateral hole 50 passes through the 
wall of the body 46 adjacent the underside of the cap 48. In this 
embodiment, lip 18 with upwardly directed ramp surface and downwardly 
directed radial abutment surface projects externally of the body 10 around 
the open end thereof. Cap 48 has inwardly directed seal 39 which forms a 
sliding seal with the external cylindrical surface of body 10 and which 
coacts with the lip 18 to prevent disengagement of the cap 48 from the 
body once the two parts have been engaged together. 
A liquid sample to be assayed can be aspirated into the chamber 22 by 
dipping tube 28 into the sample with the body 46 and piston 36 mutually 
arranged so that piston 36 lies against the base of the body 46. In this 
condition, downward pressure on cap 48 causes movement of the body 46 
relative to the piston 36 and aspiration of the sample through the tube 28 
until the piston 35 clears the whole 50. Once this has happened, a reduced 
pressure no longer exists within the chamber 22 and so aspiration ceases. 
When tube 28 is removed from the liquid, it substantially drains. The 
collector/dispenser 20 is then carefully manoeuvred into position to 
engage the cap 48 with the body 10 so that initially the parts adopt the 
mutual positions illustrated in FIG. 3. Subsequently, the piston rod 38 is 
depressed in order to cause the tube 28 to pierce the membrane 16. This 
action brings the hole 44 to the bottom of the chamber 22, thus allowing 
the sample contained in the chamber 22 to dispense through the tube 28 
into the reaction chamber 12 for mixing with assay reagents 14 therein. 
Referring now to FIGS. 4a and 4b, the assay device illustrated therein has 
collector/dispenser 20 formed with cap 48 carrying rod 38 with sample 
collection chamber 22 in its lower end. A vent hole 60 fitted with a 
hingedly mounted flap valve 62 is provided at the top of the chamber 22. 
The cap 48 has a porous vent plug 64 therein formed of a suitable 
hydrophobic porous material, such as Zitex (Norton Performance Plastics, 
New Jersey, USA) which allows air to pass but not low pressure liquids. 
In use, the lower end of rod 38 is dipped into the liquid sample to be 
assayed so that the chamber 22 is disposed below the surface of the 
sample. The liquid sample is permitted to enter the chamber 22 because air 
therein is vented through the vent hole 60, as permitted by the flap valve 
62. However, when the collector/dispenser 20 is removed from the sample, 
the valve 62 closes and prevents escape of the sample from chamber 22. The 
collector/dispenser 20 is then engaged with the body 10 to adopt the 
position shown in FIG. 4a. In this condition, seal 39 within cap 48 has 
been forced past lip 18 and is sealingly slidable over the body 10. The 
cap 48 is then depressed so that the lower end of rod 38 is caused to 
puncture the membrane 16. Once the device has been moved into the 
condition illustrated in FIG. 4b, the flap valve 62 has abutted against 
the part of the membrane 16 surrounding the rod 38 and is thereby deformed 
so as to open the vent hole 60 which then allows the sample within chamber 
22 to be dispensed under the action of gravity into the reaction chamber 
12 and mixed with the assay reagents 14. In the position illustrated in 
FIG. 4b, the seal 39 has moved past a further lip 66 which is similar in 
construction to lip 18. Mutual engagement of the seal 39 and lip 66 
retains the device in the condition illustrated in FIG. 4b in a 
non-detachable manner. Pressure which builds up within the cap 48 as a 
result of depression of the latter from the position illustrated in FIG. 
4a to that illustrated in FIG. 4b is relieved by venting through the plug 
64. In place of plug 64, a one-way valve may be provided (not shown). 
Either form of venting device may be used, if desired, in any of the other 
embodiments described herein. 
In the place of flap valve 62, a non-return valve may be provided in simple 
form comprising merely a band of paper held around the outside of the 
conduit. Both the flap and the band may be of a fibrous material such as 
the type of paper commonly used for making paper towels. 
In FIG. 4c, flap valve 62 is replaced by a plug 68 of porous material such 
as cotton wool so to act in a similar way to a paper band. Alternatively, 
the plug 68 may be formed of a compressed foam, such as polyvinyl alcohol, 
which expands when wetted and which projects from the side of the rod 38 
so as to be removed automatically upon depression of the cap 48 into the 
position illustrated in FIG. 4b. 
In FIG. 4d, the rod 38 containing chamber 22 terminates in a castellated 
end which allows easier piercing of the membrane 16 and which enhances the 
surface tension effect, thus further protecting the sample within chamber 
22 from premature dispensing. As an alternative, a constriction may be 
provided at the bottom end of chamber 22 in a similar manner to the 
constriction 28a provided in tube 28 (FIG. 1b). 
In FIG. 4e, the rod 38 is provided with grooves 69 at the lower end. These 
provide a means of sampling the material to be analysed. The grooves 69, 
in the case of a liquid sample, provide retention due to surface tension 
forces and substantially define the volume of sample therein. The rod 38 
has a pointed lower end 70 to assist in piercing of the membrane. In this 
construction, the reagents would normally be in liquid form to allow the 
sample to dispense from the grooves. Furthermore, this embodiment may be 
used for sampling semi-solids such as faeces. In this case, the membrane 
coacts with the grooves to remove excess sample and hence the sample 
volume is defined principally by the volume of the grooves. 
Referring now to FIGS. 5a and 5b, the assay device illustrated therein has 
collector/dispenser 120 permanently connected with body 110. Tube 128 is 
integrally joined with hollow piston 136 and permanently communicates with 
reaction chamber 112 in which solid assay regents 114 are provided. If 
desired, a perforated plate or plug may be provided in piston 136 to 
prevent the assay reagents 114 from falling into tube 128 but to allow 
permeation of the sample into the reaction chamber 112. The lower end (as 
viewed in FIG. 5a) of body 110 is provided with an inwardly directed 
annular lip 18 of similar construction to that described above in relation 
to FIG. 1a, and an outwardly directed annular rib 19 to similar 
construction. The rib 18 permits the piston 136 to be forced into the body 
110 during initial assembly of the assay device and to retain it 
thereafter. 
The lower end of tube 128 is provided with opposed liquid inlet holes 170 
which are closed by respective lobes 172 of a non-return valve body 174 
inserted into the lower end of tube 128. In use, a liquid sample to be 
assayed is aspirated by immersing the apertures 170 in the liquid and then 
withdrawing the body 110 upwardly relative to tube 128. If desired, 
markings may be provided on the outside of the tube 128 to indicate the 
level to which the sample has to be aspirated in order to enable the 
required quantity of sample to be taken. Alternatively, the piston 136 may 
cooperate with internal stops (not shown) in the body 110 to provide a 
pre-set amount of aspiration. The lobes 172 prevent loss of sample from 
the tube 128. Thereafter, the whole assembly is inverted to adopt the 
position illustrated in FIG. 5b, and a cap 90 is snap-fitted into position 
so as to seal the whole assembly in a non-detachable way. The cap 90 
completely overlies the rod 128 and has a seal 139 which coacts with rib 
19 in a similar way to that in which the seal 39 coacts with lip 18 in the 
embodiment of FIG. 3. 
In the embodiment of FIGS. 6a and 6b, body 110 has chamber 112 containing 
solid assay reagent 114 closed by a liquid-permeable support plate 92 
which separates the reaction chamber 112 from a compressible absorbent pad 
94 provided within the body 110 at the open end thereof. The assay device 
of FIGS. 6a and 6b further comprises cap 90 with inwardly directed lip 32 
and plunger 96 with seals 96a and 96b. 
In use, body 110 is inverted so as to contact the exposed portion of the 
absorbent pad 94 with the sample to be assayed which is drawn into the 
absorbent pad until the latter is saturated therewith. The body 110 is 
then withdrawn and inverted to the position illustrated in FIG. 6b. Cap 90 
is then snap-fitted onto body 110 and pressed firmly down so as to 
compress the absorbent pad 94 and thereby force a predetermined quantity 
of the sample to be assayed through the liquid-permeable plate 92 and into 
the reaction chamber 112 to be mixed with the assay reagent 114 therein. 
The seals 96a and 96b ensure that the assembly is fully sealed and the 
engagement of the lip 32 first with the lip 18 and finally with the lip 66 
ensures that the cap 90 is non-detachably engaged with the body 110 for 
safe disposal of the assay device after use. 
FIGS. 7a and 7b show a similar embodiment to that described above in 
relation to FIGS. 6a and 6b. In this embodiment, however, the absorbent 
pad 94 is secured to the end of plunger 96 on cap 90 and is compressed 
against liquid-permeable plate 92 in body 110 to dispense a predetermined 
quantity of the liquid sample to be assayed into reaction chamber 110. 
In FIGS. 8a and 8b, a collector/dispenser 20 similar to that illustrated in 
FIG. 2 is utilised. In this embodiment, however, body 10 is of generally 
L-shaped form and is provided with a slide valve arrangement comprising a 
slidable valve plate 200 carried on support 202 and cooperating with an 
annular seat 204 around a sample entrance hole 206 aligned with tube 28. 
The plate 200 is provided with a passage 208 therethrough which, in the 
condition illustrated in FIG. 8a, is closed by support 202. The plate 200 
is moveable by means of a compressed foam pad 210 formed, for example, of 
polyvinylalcohol foam which expands when wetted. It is to be appreciated 
that, in the position illustrated in FIG. 8a, the valve plate 200 
completely closes the chamber 112 from the remainder of the body 10 and 
that the pad 210 is shown in its compressed (i.e. unwetted condition). The 
assay reagents 114 are in dry solid form. 
When a predetermined quantity of liquid sample to be assayed has been 
dispensed from the collector/dispenser 20 by depression of piston rod 38, 
the sample passes through hole 206 and travels along a shallow passage 212 
to wet the compressed pad 210. This expands the pad 210 and moves the 
plate 200 to the left as viewed in FIG. 8a until it adopts the position 
illustrated in FIG. 8b where the entrance hole 206 is sealed by the plate 
200 and where the sample to be assayed can enter the reaction chamber 112 
through hole 208 when the device is tipped. It will be appreciated that, 
at no stage before, during or after use of the device, is external access 
to the reagents 114 permitted. As with previously described embodiments, 
the collector/dispensor cannot be removed. 
In FIGS. 9a and 9b, an arrangement which is somewhat similar to that 
described above in relation to FIGS. 5a and 5b is illustrated. In this 
embodiment, instead of a predetermined quantity of sample to be assayed 
being aspirated into collector 20, a predetermined quantity of sample is 
taken without application of reduced pressure. In this device, a sleeve 
300 has a hollow plunger 302 slidable therein. The distal end of plunger 
302 (i.e. that end remote from chamber 112) has a transverse bore 304 
therethrough. Above and below the bore 304 (as viewed in FIG. 9a), the 
plunger 302 has lands 306 and 308, respectively, with grooves 310 and 312 
therein. The grooves 310 and 312 have their lower surfaces chamfered and 
their upper surfaces extending substantially radially of the respective 
lands 306 and 308. As shown in FIG. 9a, groove 310 cooperates with a 
correspondingly shaped lip 314 provided internally of sleeve 300. In this 
condition, transverse bore 304 is disposed externally of sleeve 300 so 
that it can be completely filled with sample to be assayed when the distal 
end of the device is immersed in the sample. When this has taken place, 
the plunger 302 is manually lifted so as to bring the groove 312 into 
engagement with the lip 314. Such movement is permitted because of the 
relative shapes of the grooves 310 and 312 and the lip 314, but movement 
in the opposite direction is not permitted. It will thus be appreciated 
that a predetermined quantity of the sample to be assayed is thereby 
collected, such quantity corresponding to the volume of the transverse 
bore 304. When the device is inverted to the position shown in FIG. 9b, 
the sample in the bore 304 is free to enter the reaction chamber 112 by 
passing through a channel 316 defined within sleeve 300 and into a further 
transverse bore 318 defined at the opposite side of land 306 no transverse 
bore 304. 
The distal ends of the plunger 302 and sleeve 300 are sealed closed by cap 
90 in an non-detachable way by virtue of the provision of inwardly 
directed sealing lip 32 at the open end of cap 90 which seals with the 
outer peripheral surface of sleeve 300 on which outwardly directed 
chamfered lip 18 is provided. 
In FIG. 10a, collector/dispenser 20 comprises slidable sleeve 400 with 
castellations 402 at its upper end provided for a similar purpose to that 
described above in relation to FIG. 4d. Within sleeve 400 is defined 
chamber 22. The sleeve 400 is detachably supported by a downwardly 
directed conical member 404 carried on actuating rod 406. A predetermined 
quantity sample to be assayed is taken by immersing the sleeve 400 and 
conical member 404 in the sample and then transferring it to the body 10. 
Downward pressure on the plunger 406 causes the conical member 404 to 
pierce the membrane 16 and engagement of the sleeve 400 with the pierced 
membrane 16 and its support causes separation of the conical member 404 
from the sleeve 400 and thereby dispensing of the sample within chamber 22 
into the reaction chamber 12 for mixing with the assay reagents 14. 
The upper end of the body 10 (i.e. that end which is not illustrated) is 
non-detachably closed and sealed by a cap which may be similar to those 
described herein above in relation to, for example, FIGS. 3 or 4a and 4b. 
In FIG. 10b, an arrangement similar to that illustrated in FIG. 10a is 
provided and similar parts are accorded the same reference numerals. 
However, in FIG. 10b, spillage is minimised and sample volume determined 
by the provision of a siphon arrangement 408 through which chamber 22 is 
initially filled with the sample. 
In FIGS. 11a and 11b are shown two ways of enabling colorimetric assays. In 
FIG. 11a, body 10 in the region of reaction chamber 12 is completely 
transparent and so can be traversed by light rays emanating from a 
convenient light source 500. The lights rays may be condensed by lens 502 
to enter detector 504 for producing an output which gives an indication of 
the absorbance resulting from passage of the light rays through the 
reaction mixture in reaction cheer 12. 
In FIG. 11b, a similar arrangement is shown except that the light rays are 
reflected from a mirror 506 so that each light ray passes twice through 
the reaction mixture so that the effective optical path length through the 
latter is doubled. It is within the scope of the present invention, 
however, to observe the change in optical properties of the reactants by 
eye rather than automatically by a machine. 
In the case where the above-described assay reagents are used for assaying 
nicotine metabolites in urine, the sample to be assayed is urine and the 
assay reagents comprise: 
______________________________________ 
1. Citric acid (2M/Sodium citrate 
150 .mu.l, 
(1.5M)/buffer pH 4.7) 
2. Potassium cyanide (20%) 
50 .mu.l, 
3. Chloramine - T (20%) 
50 .mu.l, 
4. Thiobarbituric acid (10%) 
500 .mu.l, 
______________________________________ 
to which is added 500 .mu.l of urine (Modification of Peach et al--Thorax 
1985;40:351-7).