GB2410086A

GB2410086A - Assay devices having flow block(s) to determine flow of liquids

Info

Publication number: GB2410086A
Application number: GB0400745A
Authority: GB
Inventors: Paul Christopher; Garth Harris
Original assignee: British Biocell International Ltd
Current assignee: British Biocell International Ltd
Priority date: 2004-01-14
Filing date: 2004-01-14
Publication date: 2005-07-20
Also published as: GB0400745D0; WO2005069007A1

Abstract

An assay device having a liquid flow path, flow of liquid along the flow path being initially impeded by a flow block comprising a substance which is suspendable or soluble in the liquid, such that contact of the flow block with the liquid suspends or dissolves at least part of the flow block over a period of time, so as subsequently to permit the liquid to flow along the flow path with less impedance. The flow block may be a hydrophilic substance and may comprise a peptide, saccharide eg sucrose, albumen. The assay device may be a lateral flow device.

Description

1 2410086 C723.00/B Title: Improvements in or Relating to Flow of Liquids

in Assay Devices

Field of the Invention

This invention relates to an assay device, having improved liquid flow control characteristics, and a method for improving the flow of liquid within an assay device.

Background of the Invention

It is well known to perform assays to detect the presence and/or amount of an analyte of interest in a liquid sample. Conveniently such assay methods will comprise the use of an assay device.

A large number of different assay devices are known. One category of device takes the form of a solid substrate, within which are provided small bore "microchannels" for the flow of the liquid sample and/or liquid reagents etc. Such devices may be generically described as "microfluidic" devices. Examples are disclosed in US 5,885,527, US 6,326,211 and WO 01/26813.

A second category of assay device is a "lateral flow" assay device, in which a porous or permeable matrix is present. A liquid sample is brought into contact with the matrix and proceeds to migrate along the matrix as a result of capillary flow. A highly absorbent "wick" or "sink" is typically provided at the end of the matrix. It is also conventional to provide one or more reagents which are deposited on or within the matrix, typically in dried form. Such reagents may be immobilised (i.e. not released upon contact with the liquid sample) or may be releasably deposited (i.e. are released upon contact with the sample liquid and are then free to migrate with the liquid). Examples of lateral flow assay devices are disclosed in a number of publications including, ironer alla, EP 0,383,619, EP 0,291,194.

It is further known to provide means for controlling the rate of advance or movement of liquids within assay devices (including microfluidic and lateral flow devices).

For example, WO 94/26414 discloses a microfluidic device within which fluid communication between adjacent chambers may be prevented by blocking a channel with a heat-meltable or liquifiable gel (comprising, inter alla, a polysaccharide such as agarose or a polypeptide such as gelatin). Application of heat to the gel will cause the gel to melt or liquify sufficiently to allow liquid to flow through the channel.

W O 01/36974 discloses a lateral flow device in which flow of liquid into the device can be prevented by an intervening separation means, such as a thin sheet or film of impermeable material. The separation means can be manually removed from its intervening position, thereby allowing liquid to advance into the permeable matrix.

Various US patents in the name of Biosite Diagnostics, Inc. (e.g. US 5, 458,852) disclose assay devices having "time gates" for delaying fluid flow. The time gate typically comprises a hydrophobic substance, which is impermeable at the outset as a consequence of its hydrophobicity but which is rendered less hydrophobic, after a period of time, by contact with a substance present in the aqueous solution the passage of which is being prevented. The hydrophobic substance may be, for example, polyethylene, polypropylene, polystyrene, polyacrylate, silicon or metal. The hydrophobic substance typically gradually becomes coated with a hydrophilic substance (e.g. a polypeptide, such as BSA) which reduces the hydrophobicity sufficiently to allow the aqueous solution to advance past the time gate.

Summary of the Invention

In a first aspect the invention provides an assay device having a liquid flow path, flow of liquid along the flow path being initially impeded by a flow block comprising a substance which is suspendable or (more preferably) soluble in the liquid, such that contact of the flow block with the liquid suspends or dissolves at least part of the flow block over a period of time, so as subsequently to permit the liquid to flow along the flow path with less impedance.

The assay device may be a fluidic device, a lateral flow assay device, or any other type of assay device having a flow path. The flow path may, for example, comprise one or more channels in a microfabricated device, or may comprise the pores of a porous matrix in a lateral flow.

The liquid which suspends or dissolves at least part of the flow block may be the sample liquid (containing the analyte of interest). Examples of sample liquids which might be analysed on an assay device include: blood, plasma, serum, sweat, saliva, urine, lachrymal fluid, environmental water samples, and suspensions, solutions or mixtures of food/drink samples. Alternatively, the liquid which suspends or dissolves at least part of the flow block may be a liquid other than the sample liquid, e.g. a buffer, diluent, wash liquid, reagent-containing liquid etc. It is preferred that the liquid is aqueous (i.e. comprises at least 30% v/v water, preferably at least 40%, more preferably at least 50%, and most preferably at least 55%) and that the flow block comprises a substance which is suspendable or (more preferably) soluble in aqueous liquids.

The flow block could, in principle, comprise any suitable substance which can be suspended or dissolved upon contact with the liquid, so as to permit passage of the liquid.

It will be appreciated that various factors can be adjusted to control the extent to which flow of liquid along the flow path is impeded by the flow block. For example, the dimensions of the flow block and/or the concentration or amount of suspendable or soluble substance present in the block can be altered. In some embodiments, for instance, the flow block may comprise a greater or lesser amount of a substance which cannot be suspended or dissolved by the liquid in the assay device, whilst in other embodiments the flow block may comprise of a greater or lesser amount (or even consist entirely) of the suspendable or soluble substance.

The amount of impedance of liquid flow along the flow path in an initial state may be varied. Thus, for instance, there could be total impedance in the initial state (whereby no liquid can flow past or through the flow block), or there may be less than total impedance (i.e. the flow path is not completely blocked, such that a reduced or restricted flow of liquid is possible).

Equally, contact with the liquid might be such that the flow block is completely suspended or dissolved (allowing totally unimpeded flow) or, in the alternative, that during the course of the assay the flow block is only partially suspended or dissolved, such that flow of liquid along the flow path is still at least partially restricted.

Conveniently the flow block is such that, at least initially, there is total impedance to flow of liquid through the block. As explained, the composition and dimensions of the block can be adjusted so as to select the extent and duration of impedance of liquid flow.

Conditions may be selected such that flow may be completely or partially impeded for anything from 2-5 seconds to 2-5 minutes, to up to 45 minutes or more.

It will be apparent to those skilled in the art that the substance or substances employed to form the flow block should preferably be selected so as not to have any significant adverse effect on the development of the final assay result or test signal. For example, substances which are likely to compete for binding to analyte and/or assay reagents are likely to be detrimental to performance of the assay. With the benefit of the present disclosure it will be a matter of routine trial-and-error to identify whether a suspendable or soluble substance will be suitable for use as a flow block in any particular assay system.

The present inventors have found that suitable suspendable or soluble substances include peptides, polypeptides (and proteins) and saccharides. For present purposes a peptide is considered to comprise from two to twenty amino acid residues. A polypeptide is considered to comprise at least twenty-one amino acid residues. The amino acid residues present in a peptide or polypeptide may be commonly occurring residues, or may be unusual residues (such as hydroxyproline, phosphoserine, or carboxyglutamate etc). The peptide or polypeptide may, in particular, comprise a non-amino acid residue component e.g. be phosphorylated and/or glycosylated. In addition, the peptide or polypeptide may be a naturallyoccurring substance or derived therefrom (e.g. by enzymatic or other proteolytic cleavage of a larger precursor), or may be synthetic. For example, methods of peptide synthesis in vitro, and methods of polypeptide expression using recombinant nucleic acid technology, are well now to those skilled in the art.

Generally, polypeptides are preferred over peptides. In addition, it is generally preferable to use a naturally occurring substance, since these are normally readily available in large amounts. Preferred polypeptides include milk proteins, especially caseins (especially bovine or human caseins), and albumens (especially bovine serum albumen or BSA).

For present purposes, the term saccharine is considered to encompass monosaccharides, disaccharides, oligosaccharides and polysaccharides. Many polysaccharides (e.g. cellulose, starch) are of very limited solubility or suspendability in aqueous solvents, and it is generally necessary to use modified polysaccharides which have been chemically treated to improve their suspendability/solubility in water (e.g. by addition of hydroxy- groups or other hydrophilic groups). For this reason, monosaccharides, disaccharides and oligo- saccharides (i.e. containing from 3-20 sugar residues) are generally preferred to polysaccharides, and shorter chain oligosaccharides (3-lO sugar residues) are typically preferred to longer chain (11-20 sugar residues) oligosaccharides. Preferred saccharides include fructose; and especially disaccharides, such as sucrose and maltose.

In some embodiments the flow block may comprise both a saccharide and a peptide or polypeptide (e.g. a combination of BSA and sucrose is found particularly suitable).

In those embodiments where the flow block comprises sucrose, it is generally preferred that the block additionally comprises a further substance.

PCT/GB03/03059 (unpublished at the time of filing of the present application) discloses lateral flow assay devices which comprise a nucleic acid amplification reaction zone and which may additionally comprise a "dissolvable barrier" (of which sucrose is the only substance explicitly mentioned). The present application therefore excludes from its scope lateral flow assay devices which comprise a nucleic acid amplification reaction zone and which comprise a dissolvable barrier which consists exclusively of sucrose.

A preferred embodiment of the invention concerns a lateral flow assay device having a flow path, at least part of which is on and/or within a porous matrix or carrier (such as a nitrocellulose membrane, for example), the flow block being formed on and/or within the porous matrix or carrier. Typically, the pores of the matrix or carrier are substantially blocked by the substance.

Methods of depositing substances onto a porous matrix are well known to those skilled in the art. Conveniently, an aqueous suspension or solution comprising one or more (preferably all) of the constituents of the flow block is prepared, and then applied to the desired portion of the porous matrix. Automated apparatus for such application is available. The matrix is then allowed to dry, e.g. by freeze drying, or by drying either at room temperature or at elevated temperature (e.g. 30-50 C), so as to form the flow block.

The present inventors have found, for example, that a suitable flow block may be prepared by applying an aqueous solution of BSA or sucrose to a porous matrix. The concentration of BSA in the solution applied to the matrix may be any suitable value, typically anywhere in the range 0.1 25% BSA, preferably 0.5 - 20%, more preferably 1 - 15% w/v. A concentration of BSA above 25% w/v renders the solution very viscous and difficult to process. The concentration of sucrose in a solution applied to a matrix may also be any suitable value, but typically may be anywhere in the range 0.1 - 60% w/v, preferably l - 50%, and more preferably 10 50% w/v. Again, concentrations above 50 - 60% w/v sucrose make the viscosity of the solution very high and difficult to process. In general, the higher the concentration of substance the greater the impedance to flow and the longer the duration of time over which flow of liquid along the flow path is prevented or impeded.

As mentioned previously, the dimensions of the flow block may also be adjusted in order to control the amount of impedance to flow offered by the flow block. Typically, in a lateral flow assay device, the flow block will extend across the entire breadth of the flow path. The length of the flow block along the flow path (i.e. in the direction of flow) may be of any desirable size, but typically will be anything from 0. lmm to 20mm, preferably 0.5 - 15mm, and more preferably 1 - lOmm.

In all other types of assay device (e.g. containing a conduit or channel), a porous matrix, bibulous member or the like containing the flow block may be introduced into the channels, or the channel or conduit may be formed around a pre-formed flow block. For example, a bibulous member comprising a flow block as detailed above may be provided in an impermeable, disk-like housing, with an aperture provided on each of the flattened faces, which apertures may then be placed in liquid flow communication with a channel, conduit or other flow path component.

The inventors have further found that dissolution or suspension of the suspendable/soluble substance in the flow block can be improved by inclusion in the suspending/dissolving liquid of a surfactant. A large number of suitable surfactants are known, including for example TweenRTM (e.g. Tween 20 etc) and the like. The presence of such a surfactant improves the uniformity with which the flow block is degraded, leading to greater reproducibility. The surfactant may be included at a suitable concentration (e.g. 0.01 - 10%, preferably 0.1-5% v/v) in the sample liquid or else added subsequently e.g. as a component in a wash buffer, or carrier liquid. The surfactant may even be provided, pre- dispensed, on or within the assay device. For instance, the surfactant may be dried and releasably deposited on/in the device e.g. within a porous matrix. It is even possible that the surfactant could be deposited at the leading edge and/or within the flow block, and is released upon contact with the liquid which suspends or dissolves at least part of the flow block.

Accordingly, an assay device in accordance with the invention may additionally comprise a source of surfactant, typically deposited in dried and mobilizable upon contact with a liquid. The surfactant may be provided in the region of the flow block.

An assay device in accordance with the invention may comprise a single flow block or a plurality of flow blocks. The plurality of flow blocks could be provided along a single flow path or, if the assay device comprises two or more flow paths, a flow block may be provided in each of two or more of the flow paths.

Where a plurality of flow blocks are provided along a single flow path, they can be used to assist performance of a plurality of assay steps in a sequential manner e.g. by allowing a first assay step to proceed to completion (or nearer to equilibrium) before the reactant(s) contact a reagent, label or the like which takes a part in a second assay step.

An assay device in accordance with the invention may comprise one or more flow blocks at any point along the flow path. For example, the flow block will typically be formed on/in a nitrocellulose strip or other porous material portion and may be located towards one or other (or even both) end regions of the strip and/or centrally located. Alternatively (or in addition), a flow block could be provided outside a strip portion of the device e.g. in a porous pad adjacent to a strip portion.

In particular WO 00/77524 discloses lateral flow assay devices which operate using a bidirectional flow mechanism, and corresponding methods. Generally, the prior art device has a sample addition zone on a lateral flow assay strip which is located between a test zone (which comprises a binding agent for the analyte of interest) and an absorbent wick having a relatively high absorption capacity for the sample liquid compared to the rest of the assay strip. The volume of sample, and relative positioning of the components and their absorption characteristics, are arranged so that sample applied to the sample addition zone tends to flow initially to (and past) the test zone. The direction of flow is then reversed, and the liquid flows back through the test zone towards the absorbent wick. It has occurred to the present inventors that devices of the sort disclosed in WO 00/77524 are particularly amenable to inclusion of a flow block of the sort contemplated and disclosed in the present specification. Thus, for example, it may be desirable to place a flow block between a sample addition zone and an absorbent wick (the sample addition zone being itself positioned between the absorbent wick and a test zone). The flow block would initially direct sample towards the test zone, but with gradual dissolution or degradation of the flow block facilitating the reverse in direction of fluid flowback towards the wick.

Alternatively (or in addition) it may be desirable to position a flow block distal from a test zone. Further details and other embodiments are described in the following example.

In a second aspect, the invention provides a method of performing an assay, the method comprising use of an assay device in accordance with the first aspect. In particular, the method may comprise the step of contacting the assay device with a surfactant, which step may be performed prior to and/or during performance of the assay. In a third aspect the invention provides a method of making an assay device in accordance with the first aspect of the invention.

For the avoidance of doubt it is expressly stated that any feature described as "preferred", "desirable", "advantageous" or the like may be utilised in any embodiment of the invention in isolation or in combination with any one or more of the other features so described, unless the context dictates otherwise.

The invention will now be described by way of illustrative example and with reference to the accompanying drawings, in which:

Examples

Example 1 - Lateral Flow Assay Device with a flow block comprising BSA A lateral flow assay device was constructed with a flow path along a multicomponent porous matrix. The is illustrated schematically in Figure 1. The device comprised a sample application pad 2 (Ahlstrom 222 membrane from Ahlstrom, Edinburgh EH1 14DH, UK), a glass fibre flow block pad 4 (detailed below), a glass fibre dye pad 6 (containing a conventional water-soluble food-dye), an HE 075 nitrocellulose membrane 8 (from Millipore (UK) Limited, Watford WD18 8YH, UK), and a Whatman WE 1.5 cellulose/glass fibre wick 10 (Whatman pie Maidstone, ME16 OLS, Kent, UK). Each component was held in contact with adjacent components on a plastic backing sheet 12, coated with adhesive.

The flow block pads 4 were prepared as follows: Solutions of BSA were prepared in ultra pure water, with a concentration of 2% w/v BSA, 5% or 10%. Small sections of the glass fibre sheets were placed into trays and the BSA solution poured over, in order to completely saturate the sheets. A control sheet saturated with pure water (0% BSA) was also prepared. The soaked sheets were removed from the trays and any excess solution allowed to drain off. The sheets were then placed in a controlled atmosphere (<20% humidity) and left to dry overnight. Once dry the sheets were cut into strips or pads of 5, 10 or 15mm length and placed in foil pouches with a desiccant and the pouches sealed until ready for use.

To assess the effectiveness of the flow blocks, the assembled assay devices were positioned so that the sample application pad 2 was contacted with the well of a microtitre plate containing 15011 of water. The time taken for the liquid front to reach an end point (lOmm along the nitrocellulose) was noted. Progress of the liquid was observed by inclusion of a dye, in a dye pad adjacent to the flow block pad. The results are shown in Table 1 below.

Results: Flow block length (mm) ho BSA Time to End Point O 0'59 2 2'12 5 3'35 6'04 *a 2 3'15 5'45

DNR *a

2 4'07 5 DNR

DNR Key:

DNR = did not run - the barrier had not been breached after lOmin therefore the runs were terminated.

a* = poor flow through the untreated glass fibre pad (possibly due to high hydrophobicity of the untreated glass fibre).

Conclusions:

An increase in BSA concentration leads to an increase in the delay for the liquid front to reach the end point.

As the barrier pad increased in length, longer it took for the liquid front to reach the end point.

Example 2 - Lateral Flow Assay Device with a flow block comprising sucrose A lateral flow assay device was constructed with a flow path along a nitrocellulose membrane (either HF90 or HF135, both from Millipore). A sample application pad was applied to the nitrocellulose membrane and an absorbent wick provided at the other end.

The membrane was coated on the reverse surface with a plastic backing layer.

The sample application pad was formed from a number of materials, the combined effect of which was to filter out red blood cells from a sample (60111) of human blood applied to the pad. The plasma exiting the lower surface of the pad is free to migrate along the nitrocellulose membrane. However, between the sample pad and the wick was formed a flow block comprising a narrow band of sucrose deposited in the nitrocellulose.

The flow block was prepared using a solution of sucrose (25, 30, 35 or 40% w/v) in ultra pure water, and then striping the solution onto the mebrane using a conventional reagent dispensing machine. The membranes were allowed to dry for 1 hour in an incubator at 37 C under normal atmospheric conditions. The resulting dried sucrose stripe on the membrane is visible to the naked eye.

The time taken for the plasma to breach the flow block was observed and noted as the "sucrose dissolution time". The results are shown below. \

Sucrose dissolution time(s) Sucrose cone. (%) HF135 HF9O \\\ 25 41 29 \\ 30 40 37 \\ 35 62 54 \ 40 132 125 Example 3 - Flow Block Location The incorporation of a flow block into an assay device, as in the present invention makes possible new approaches to lateral flow assays, or can be used to improve known lateral flow\assay devices. \

For Instance, conventional lateral flow assays typically permit sequential reactions in which sample first contacts a labelled binding reagent and then an immobilised capture reagent. Further, once fluid flow has commenced the reaction normally proceeds to completion. In some circumstances however, it would be highly desirable for fluid flow to be temporarily halted or at least impeded, in order to optimise the assay.

Various embodiments of assay devices in accordance with the invention and incorporating a flow block are detailed below.

Example 3.1 - Bi-directional Sample Flow Assays In a first embodiment, illustrated schematically in Figure 2. Referring to that Figure, sample is added to a sample addition zone 20 located relatively centrally on a nitrocellulose assay strip 22. The sample then enters the strip 22 by capillary action and flows in two directions, towards a test zone 24 and towards a wick 26. The flow of the sample into the device may be controlled by adjusting the sample volume or viscosity. Alternatively the system allows excess sample to be added with only a controlled amount of sample allowed to entr the test zone 24. The volume of sample entering the test zone is controlled by the absorption volume of the nitrocellulose up to the point where a flow block 28 is located.

The block 28 stops the flow of sample after the test zone with excess sample flowing towards the wick end of the system. This system therefore controls the amount of sample that enters the test zone. Following sample addition a reagent may be added or alternatively the system may be incubated for a period of time.

A pad 30 may optionally be provided in fluid flow communication with at least part of the strip 22. The assay device may comprise a reagent (e.g. in dried, typically Iyophilised, form) which may be located in the pad 30 and/or on the strip 22, which reagent is mobilisable upon contact with a liquid, so as to flow into the test zone 24. Thus, for example, reagent may be mobilised by application of water, buffer, or other suitable liquid to the pad 30. Alternatively, a reagent solution may be applied to the device (e.g. at pad 30). Mobilised reagent, or reagent applied to the device, can flow past the flow block 28 towards the wick 26 (and hence flow into the test zone 24) as a result of degradation of the flow block 28, by suspension or solution of the flow block in the liquid sample and/or by suspension or solution in the liquid which mobilises the reagent.

To facilitate degradation of the flow block 28, a surfactant may be provided (e.g. contained within the liquid used to apply, or mobilise, the reagent, or else provided on the assay device and, conveniently, mobilised by the sample liquid and/or the liquid used to apply or mobilise the reagent). The reagent will typically comprise a detectable label (or other assay-signal generating means.

Such subsequent application of liquid to the pad 30 allows unbound or unreacted sample to be washed out of the test zone 24 towards the wick 26.

An alternative bi-directional sample flow embodiment is illustrated schematically in Figure 3. Equivalent components in Figure 3 are allocated common reference numerals with the components shown in Figure 2. The embodiment illustrated in Figure 3 is generally similar to that shown in Figure 2. However, if desired, an excess volume sink 32 may be added so that the system allows excess sample to be added, with only a controlled amount o sample allowed to enter the test zone. The volume of sample entering the test zone is controlled by the absorption volume of the nitrocellulose strip 22 up to the point where the block 28 is located. The block 28 stops the flow of sample after the test zone 24 with excess sample flowing towards the excess volume sink 32. This system therefore controls the amount of sample that enters the test zone. Following sample addition, reagent may be added or alternatively the system may be incubated for a period of time. The reagent addition can be made via, for example, pad 30. Reagent is added (or buffer which may reconstitute further reagents) before flowing into system. This reagent passes the block 28' e.g. by means of high volume or by means of an additive that allows passage past the block. Typically the function of the block 28 at the wick end of the system is controlled by volume and/or time. Note the volume of reagent added is sufficient to fill the excess volume sink to allow flow through the system. The non-bound or non-reacted sample is washed through the test zone by the reagent. The reagent may include a detecting agent and, if so, will allow the signal to be visualised or detected.

Example 3.2 - Unidirectional Sample Flow Assays Other embodiments can be envisaged in which sample applied to the assay device essentially flows in a single direction. These embodiments are illustrated schematically in Figures 4-6. Like components have been allocated common reference numerals with the components of the embodiments illustrated in Figures 2 and 3.

The following comments apply to the invention in general and especially to any one of the

described examples.

Flow of liquids within the assay device can be monitored by the use of a visible indicator (e.g. simple dyes), which can be impregnated into the device, dried down, or otherwise provided (e.g. in a buffer or otherliquid applied to the device). The assay devices may preferably be contained within an impermeable housing (especially a synthetic plastics material housing), but this is not essential.

Claims

Claims 1. An assay device having a liquid flow path, flow of liquid along

the flow path being initially impeded by a flow block comprising a substance which is suspendable or soluble in the liquid, such that contact of the flow block with the liquid suspends or dissolves at least part of the flow block over a period of time, so as subsequently to permit the liquid to flow along the flow path with less impedance.
2. A device according to claim 1, wherein the flow block comprises or consists of a substance which is dissolved in the liquid.
3. A device according to claim 1 or 2, wherein the flow block comprises or consists of a hydrophilic substance.
4. A device according to any one of the preceding claims, wherein the flow block comprises a peptide, polypeptide or a saccharide.
5. A device according to any one of the preceding claims wherein the flow block comprises sucrose and/or an albumen.
6. A lateral flow assay device according to any one of the preceding claims.
7. A device according to any one of the preceding claims comprising a plurality of flow blocks.
8. A device according to claim 7, wherein the plurality of flow blocks is disposed along a single flow path.
9. A device according to claim 7 comprising a plurality of flow paths and wherein at least one flow block is provided in each of the plurality of flow paths.
10. A method of performing an assay, the method comprising use of an assay device in accordance with anyone of the preceding claims.
11. An assay device as hereinbefore described and with reference to the accompanying drawings.