ASSAY
FIELDThe present invention relates to an assay apparatus, assay method, assay card and an assay card controller.
BACKGROUNDAssays are known. Such assays typically perform a diagnostic test to provide an indication of whether a particular target agent is present in a test sample. The complexity and functionality of these diagnostic devices varies considerably. For example, highly sophisticated diagnostic devices are typically provided in hospitals or laboratories for high- throughput diagnostic testing but these are typically only used in such environments because of their relatively high maintenance cost and complicated operation procedure.
There are less complicated devices also provided, in a hospital laboratory or in a physician’s clinic, which receive typically a blood or urine sample taken from the patient by a trained healthcare professional. The rest of the procedure is carried out automatically and the result of the diagnostic test is typically displayed on a liquid crystal display (LCD) screen or on paper using a printer. Other devices have dried or immobilised or lyophilized reagent inside. When a liquid test sample is mixed, the reagent is rehydrated. In some arrangements, a number of reagents are provided in the reaction chamber or bottle. Typically, each of these reagents is individually provided into the reaction chamber so that the reagents are introduced in the correct order. For example, a reagent may be required to pretreat any sensor reagent to active it from the storage condition, while other reagents may be required to bind to the test sample to make a detectable signal by, for example, optical or electrochemical means. After the reaction, washing reagents may be provided to wash out the unbound reagents. Also, some substrate reagent may be provided to convert or amplify any signal further. These devices are sometimes called point-of-care (POC) devices since they provide a handy means due to their small size, automatic sample and reagent handling, rapid result and low maintenance cost.
Many have replaceable cartridges that contain all disposable parts like reagents, sensor and sampling channel. Changing the cartridge makes the maintenance simple and easy.
Other POC devices exist such as the cellulose membrane based rapid lateral flow kit, often referred to as a ‘rapid test kit’. The rapid test kit has been so successful that it has become a de facto standard in the POC market since it has most of the desirable features of a more complex POC device such as low cost, disposability, quick response and simplicity.
Although each of these diagnostic devices provides many benefits, they each have their own shortcomings. Accordingly, it is desired to provide an improved diagnostic device or assay.
SUMMARYAccording to a first aspect, there is provided an assay apparatus, comprising: an assay card comprising a substrate having a plurality of compressible reagent reservoirs thereon, each of the plurality of compressible reagent reservoirs containing an associated reagent and each of the plurality of compressible reagent reservoirs being coupled with a reaction chamber operable to receive a test sample; and an assay card controller comprising a compression mechanism operable to compress each of the plurality of compressible reagent reservoirs to cause each associated reagent to be injected in a predetermined order into the reaction chamber to react with the test sample.
The first aspect recognises that a problem with rapid test kits is that although they are very simple, they lack the ability to perform sequential reagent delivery which makes it difficult to provide a simple device which is sensitive and reproducible. The first aspect also recognises that although other POC devices enable sequential reagent delivery, a problem with these devices is that they are complicated which makes them unnecessarily expensive, difficult to manufacture and may affect their reliability. Similarly, the first aspect recognises that although the highly sophisticated devices provided in hospital laboratories enable sequential reagent delivery, they are highly expensive, have high maintenance costs, and have complex operating procedures which requires highly skilled operators.
Accordingly, an assay apparatus is provided comprising an assay card and assay card controller. The assay card may comprise a substrate and may have a number of compressible, squeezable or squashable reagent reservoirs. Each of the reagent reservoirs may contain an associated reagent and each reservoir may be coupled with a reaction or test chamber into which a test sample may be placed. The assay card controller may comprise a compression mechanism which may compress each of the reagent reservoirs to cause the reagents contained therein to be displaced therefrom and provided to the reaction chamber in a predetermined order to react with the test sample. For example, such ordering may enable one reagent to be provided prior to another, one reagent to be provided whilst another is already being provided or more than one reagent to be provided substantially simultaneously. It will be appreciated that such an arrangement provides a sequential compression of the reagent reservoirs to enable sequential injection of reagent liquids into the reaction chamber in a pre- programmed manner using a very simple and reliable squeezing mechanism. This simple arrangement enables complex reagent reactions to occur which can be carefully controlled using a simple, low cost and reliable device which is easy to operate with a low level of skill and provides a sensitive and reproducible assay.
In one embodiment, each of the plurality of compressible reagent reservoirs is located at predetermined locations along a predetermined actuation path along the assay card to be followed by the compression mechanism. Accordingly, the location of each reservoir on the assay card may be carefully determined in order to ensure that each reagent is delivered in the correct order and at the correct time. The location of the reservoirs may be determined based on the knowledge of the location of the actuation path to be followed by the compression mechanism. Of course, the location of the reservoirs may be dependent on the particular arrangement of the compression mechanism and the path which that compression mechanism may follow. This provides for a simple, convenient and reliable arrangement which supports potentially complicated, multi-staged and time-critical reactions.
In one embodiment, the actuation path is linear. Accordingly, for a very simple compression mechanism which follows a linear actuation path, the reservoirs may be placed at appropriate locations at different points along the length of this linear actuation path.
In one embodiment, the actuation path extends along an axis of the assay card.
Accordingly, should the compression mechanism be arranged to travel along a path which is generally aligned with one of the axes of the assay card, then the reservoirs may be placed at predetermined locations along that axis of the assay card to ensure that they are actuated in the correct sequence and at the correct time. Also, any edges of the assay card which are aligned with that axis may be utilised to help guide the assay card when moving relative to the compression mechanism.
In one embodiment, the reaction chamber is located along the actuation path and those of the plurality of compressible reagent reservoirs located furthest away from the reaction chamber along the actuation path are compressible to enable injection of associated reagents prior to those of the plurality of compressible reagent reservoirs located closest to the reaction chamber. Accordingly, the reaction chamber may typically be located furthest away from that reagent reservoir which is intended to be the first which is activated by the compression mechanism, with the remaining reservoirs being actuated in the order which they lie along the actuation path between the first reagent reservoir and the reaction chamber.
In one embodiment, at least two of the plurality of compressible reagent reservoirs are located at the same predetermined location along the actuation path to enable simultaneous injection of associated reagents into the reaction chamber. Accordingly, more than one separate reagent reservoirs may be provided at the same distance along the actuation path in order to ensure that they are substantially simultaneously compressed to release their respective associated reagents. This helps to support increasingly complex reactions where multiple reagents may be required which cannot be stored in the same reservoir.
In one embodiment, each reservoir chamber comprises a reagent sack operable to receive the associated reagent. The reagent sack or balloon may be composed of a thin plastic bag filled with reagent liquid. The sack may be ruptured by the pressure derived from the roller movement. The ruptured sack may then release the reagent liquid into the reservoir. The sack may also play important role in protecting the reagent liquid from degradation due to the presence of humidity, oxygen and/or light.
In one embodiment, the assay card comprises: a waste chamber operable to receive excess from the reaction chamber. Accordingly, a waste chamber may be provided which may receive excess fluid displaced from the reaction chamber. Such excess fluid may typically occur as a result of the actuation of each reservoir. The size of the waste chamber may readily be calculated based on the size of the reagent reservoirs and the reaction chamber to ensure that no fluids are inadvertently emitted by the assay card during operation.
In one embodiment, the waste chamber comprises: material operable to retain the excess. Accordingly, material may be provided to retain excess fluid within the waste chamber and reduce the likelihood of these reagents inadvertently re-entering the reaction chamber.
Also, the provision of the material helps to retain the excess in the event of the rupture of the assay card.
In one embodiment, the assay card comprises: an inlet port coupled with the reaction chamber and operable to receive the test sample. Accordingly, the inlet port may be arranged to receive the test sample to be conveyed to the reaction chamber.
In one embodiment, the inlet port is sealable. By sealing the inlet port, the test sample 5 may be retained within the assay card and the inadvertent release of fluid during processing of the assay card may be prevented.
In one embodiment, the assay card comprises: at least one microfluidic channel operable to couple the inlet port with the reaction chamber. The provision of the microfluidic channel may conveniently enable the test sample to be conveyed to the reaction chamber under capillary action.
In one embodiment, the assay card comprises: a plurality of microfluidic channels operable to couple at each of the plurality of compressible reagent reservoirs with the reaction chamber. The provision of microfluidic channels between each of the reservoirs and the reaction chamber may help to minimise any inadvertent premixing or reagents prior to the assay card being used. Also, the use of microfluidic channels may help to minimise the amount of reagent which needs to be provided since very little volume is wasted in these channels and may maximise the rate at which any reagent is injected into the reaction chamber.
In one embodiment, the reaction chamber comprises: an indicator operable to provide an indication of a presence of a target agent within the test sample. Accordingly, the reaction chamber may be provided with an indicator providing any indication of the presence of a particular agent within the test sample. Such an indicator will typically be designed to be compatible with any detector to be utilised.
In one embodiment, the indicator comprises: a window through which the presence of the target agent may be detected.
In one embodiment, the window is transparent to enable optical detection of the presence of the target agent.
In one embodiment, the indicator comprises: an electrochemical detector operable to electrochemically detect the presence of the target agent, the electrochemical detector being coupled with a metallic coupling formed on the assay card to provide an electrical connection between the electrochemical detector and the assay card controller. Hence, an appropriate electrochemical detector may be provided on the assay card and coupled with the assay card controller.
In one embodiment, the reaction chamber comprises: a predetermined reagent.
Accordingly, the reaction chamber may be preconfigured to contain a particular reagent to be utilised to detect the presence of the target agent in the test sample. This ensures the reagent is already present within the reaction chamber and reduces the need to provide a separate reservoir containing that reagent.
In one embodiment, the predetermined reagent is provided on substrate within the reaction chamber. Accordingly, the reagent may be provided within a substrate within the reaction chamber in order to retain the reagent within the reaction chamber during the injection of other reagents during the processing of the assay card.
In one embodiment, the assay card is extruded and thermoformed with the aid of a vacuum. Accordingly, the assay card may be simply manufactured using extrusion techniques.
This arrangement provides an assay card having no moving parts as such which reduces its complexity and improves reliability.
In one embodiment, the assay card comprises: a plurality of sheets arranged to form a laminate, at least one of the sheets being a thermoplastic having the plurality of compressible reagent reservoirs, the reaction chamber and the microfluidic channels formed thereon. Hence, a number of sheets may be provided which together form the assay card. One of the sheets may be a thermoplastic having the shape of a portion of the reservoirs of the microfluidic channels formed therein. Typically, the complete reservoirs and microfluidic channels may then be formed by attaching a further sheet to the extruded thermoplastic sheet.
In one embodiment, the compression mechanism comprises: a roller operable to move relative to the assay card, along the actuation path. A roller provides a particularly convenient, reliable and simple mechanism for compressing the reservoirs. The intended direction of travel of the roller may define the actuation path. It will be appreciate that embodiments may be provided in which the roller moves, with the assay card remaining static, or where the roller remains static and the assay card is moved, or a combination of both. As the roller moves relative to the assay card, the reservoirs it travels over may be compressed and the contents ejected.
In one embodiment, the compression mechanism comprises: a pair of rollers operable to receive the assay card therebetween and operable to move relative to the assay card, along the actuation path. By providing a pair of roller, it may be possible to grip the assay card between each roller to improve the controllability and reliability of the movement of the assay card relative to the rollers and to control the force applied during compression of the reservoirs.
In one embodiment, the compression mechanism comprises: gears operable to couple the pair of rollers. Accordingly, gears may be provided between the rollers to ensure that they are simultaneously activated and to ensure a fixed relationship between the rotation of the two rollers. For example, the gears may provide a unitary gear ratio to ensure that the two rollers operate at the same speed of rotation to avoid any slippage on the assay card.
In one embodiment, the assay card controller comprises: a speed controller operable to vary speed of movement the compression mechanism along the actuation path. Accordingly, the speed of the relative motion of the compression mechanism and assay card may be varied to suit the reaction times of the reagents. It will be appreciated that this enables, for example, reservoirs to be located at fixed positions and the speed of the compression mechanism varied which provides for a more compact assay card than would be possible if the compression mechanism was moved at a constant speed at the locations of the reservoirs needed to be varied to ensure the correct amount of time between delivery of the different reagents.
In one embodiment, the speed controller is operable to change direction of movement of the compression mechanism the along the actuation path. Accordingly, the direction of movement may be changed in order that, for example, only an initial portion of a reagent is delivered due to a change in direction of the movement during such delivery which causes some of the reagent to remain in the reservoir. Similarly, a change in direction may occur after delivery of a reagent to extend the time until another reagent is delivered. Likewise, such change in direction may enable the initial compression to occur at some point along the card and then enable movement of the compression mechanism away from that starting position towards two different edges of the assay card.
In one embodiment, the assay card controller comprises: a detector operable to detect the presence of the target agent. Such a detector may be arranged to detect a predetermined characteristic of the reaction with the test sample.
In one embodiment, the detector comprises: an optical detector operable to optically detect the presence of the target agent.
In one embodiment, the detector comprises: an amplifier operable amplify a signal provided from the electrochemical detector.
In one embodiment, the assay card controller comprises: an indicator operable to indicate the presence of the test agent in response to an indication provided by the detector.
Accordingly, the indicator may indicate whether the detector sufficiently indicates the presence of the target agent in the test sample. Such an indication may be based on a simple threshold amount to provide either a positive or negative result or may provide a quantitative indication of the amount or concentration of the target agent in the test sample.
According to a second aspect, there is provided an assay method, comprising the steps of: providing an assay card comprising a substrate having a plurality of compressible reagent reservoirs, each of the plurality of compressible reagent reservoirs containing an associated reagent and each of the plurality of compressible reagent reservoirs being coupled with a reaction chamber operable to receive a test sample; and compressing each of the plurality of compressible reagent reservoirs to cause each associated reagent to be injected in a predetermined order into the reaction chamber to react with the test sample.
In one embodiment, each of the plurality of compressible reagent reservoirs is located at predetermined locations along a predetermined actuation path to be compressed along the assay card.
In one embodiment, the actuation path is linear.
In one embodiment, the actuation path extends along an axis of the assay card.
In one embodiment, the reaction chamber is located along the actuation path and the step of compressing comprises: compressing those of the plurality of compressible reagent reservoirs located furthest away from the reaction chamber along the actuation path to enable injection of associated reagents prior to those of the plurality of compressible reagent reservoirs located closest to the reaction chamber.
In one embodiment, at least two of the plurality of compressible reagent reservoirs are located at the same predetermined location along the actuation path and the step of compressing comprises: simultaneously compressing the at least two of the plurality of compressible reagent reservoirs to simultaneously inject associated reagents into the reaction chamber.
In one embodiment, each reservoir chamber comprises a reagent sack operable to receive the associated reagent.
In one embodiment, the assay card comprises a waste chamber operable to receive excess from the reaction chamber.
In one embodiment, the method comprises the step of retaining excess within material provided within the waste chamber.
In one embodiment, the method comprises the step of: receiving the test sample at an inlet port of the assay card.
In one embodiment, the method comprises the step of: sealing the inlet port.
In one embodiment, the assay card comprises at least one microfluidic channel operable to couple the inlet port with the reaction chamber.
In one embodiment, the assay card comprises a plurality of microfluidic channels operable to couple at each of the plurality of compressible reagent reservoirs with the reaction chamber.
In one embodiment, the reaction chamber comprises an indicator and the method comprises the step of: providing an indication of a presence of a target agent within the test sample.
In one embodiment, the indicator comprises a window and the step of providing an indication comprises: detecting the presence of the target agent through the window.
In one embodiment, the window is transparent and the step of detecting comprises: optically detecting the presence of the target agent.
In one embodiment, the indicator comprises an electrochemical detector operable to electrochemically detect the presence of the target agent, the electrochemical detector being coupled with a metallic coupling formed on the assay card to provide an electrical connection between the electrochemical detector and the assay card controller.
In one embodiment, the reaction chamber comprises a predetermined reagent.
In one embodiment, the predetermined reagent is provided on substrate within the reaction chamber.
In one embodiment, the assay card is extruded and thermoformed with the aid of a vacuum.
In one embodiment, the assay card comprises a plurality of sheets arranged to form a laminate, at least one of the sheets being a thermoplastic having the plurality of compressible reagent reservoirs, the reaction chamber and the microfluidic channels formed thereon.
In one embodiment, the step of compressing comprises: moving a roller relative to the assay card, along the actuation path.
In one embodiment, the step of compressing comprises: moving a pair of rollers operable to receive the assay card therebetween relative to the assay card, along the actuation path.
In one embodiment, the pair of rollers are coupled by gears.
In one embodiment, the step of compressing comprises: varying a speed of the compressing each of the plurality of compressible reagent reservoirs to cause each associated reagent to be injected into the reaction chamber to react with the test sample with predetermined timings.
In one embodiment, the method comprises the step of: detecting the presence of the target agent.
In one embodiment, the step of detecting comprises: optically detecting the presence of the target agent.
In one embodiment, the step of detecting comprises: amplifying a signal provided from the electrochemical detector to electrochemically detect the presence of the target agent.
In one embodiment, the method comprises the step of: indicating the presence of the test agent in response to an indication provided by the step of detecting.
According to a third aspect, there is provided an assay card, comprising: a substrate having a plurality of compressible reagent reservoirs, each of the plurality of compressible reagent reservoirs containing an associated reagent and each of the plurality of compressible reagent reservoirs being coupled with a reaction chamber operable to receive a test sample.
In one embodiment, each of the plurality of compressible reagent reservoirs is located at predetermined locations along a predetermined actuation path along the assay card.
In one embodiment, the actuation path is linear.
In one embodiment, the actuation path extends along an axis of the assay card.
In one embodiment, the reaction chamber is located along the actuation path and those of the plurality of compressible reagent reservoirs located furthest away from the reaction chamber along the actuation path are compressible to enable injection of associated reagents prior to those of the plurality of compressible reagent reservoirs located closest to the reaction chamber.
In one embodiment, at least two of the plurality of compressible reagent reservoirs are located at the same predetermined location along the actuation path to enable simultaneous injection of associated reagents into the reaction chamber.
In one embodiment, each reservoir chamber comprises a reagent sack which operable to receive said associated reagent.
In one embodiment, the assay card comprises: a waste chamber operable to receive excess from the reaction chamber.
In one embodiment, the waste chamber comprises: material operable to retain the excess.
In one embodiment, the assay card comprises: an inlet port coupled with the reaction chamber and operable to receive the test sample.
In one embodiment, the inlet port is sealable.
In one embodiment, the assay card comprises: at least one microfluidic channel operable to couple the inlet port with the reaction chamber.
In one embodiment, the assay card comprises: a plurality of microfluidic channels operable to couple at each of the plurality of compressible reagent reservoirs with the reaction chamber.
In one embodiment, the reaction chamber comprises: an indicator operable to provide anindication of a presence of a target agent within the test sample.
In one embodiment, the indicator comprises: a window through which the presence of the target agent may be detected.
In one embodiment, the window is transparent to enable optical detection of the presence of the target agent.
In one embodiment, the reaction chamber comprises: a predetermined reagent.
In one embodiment, the predetermined reagent is provided on substrate within the reaction chamber.
In one embodiment, the assay card comprises: an electrochemical detector operable to electrochemically detect the presence of the target agent, said electrochemical detector being coupled with a metallic coupling to provide an electrical connection between the electrochemical detector and the assay card controller.
In one embodiment, the assay card is extruded and thermoformed with the aid of a vacuum.
In one embodiment, the assay card comprises: a plurality of sheets arranged to form a laminate, at least one of the sheets being a thermoplastic having the plurality of compressible reagent reservoirs, the reaction chamber and the microfluidic channels formed thereon.
According to a fourth embodiment, there is provided an assay card controller comprising: a compression mechanism operable to compress each of a plurality of compressible reagent reservoirs containing an associated reagent provided on an assay card to cause each associated reagent to be injected in a predetermined order into a reaction chamber provided on the assay card to react with a test sample.
In one embodiment, the compression mechanism is operable to follow a predetermined actuation path along the assay card to compress each of the plurality of compressible reagent reservoirs.
In one embodiment, the compression mechanism comprises: a roller operable to move relative to the assay card, along the actuation path.
In one embodiment, the compression mechanism comprises: a pair of rollers operable to receive the assay card therebetween and operable to move relative to the assay card, along the actuation path.
In one embodiment, the assay card controller comprises: gears operable to couple the pair of rollers.
In one embodiment, the assay card controller comprises: a speed controller operable to vary speed of movement the compression mechanism along the actuation path.
In one embodiment, the speed controller is operable to change direction of movement of the compression mechanism the along the actuation path.
In one embodiment, the assay card controller comprises: a detector operable to detect the presence of the target agent.
In one embodiment, the detector comprises: an optical detector operable to optically detect the presence of the target agent.
In one embodiment, the detector comprises: an amplifier operable to amplify a signal provided from the electrochemical detector.
In one embodiment, the assay card controller comprises: an indicator operable to indicate the presence of the test agent in response to an indication provided by the detector.
Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art.
Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements;
Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,
N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential
Techniques, John Wiley & Sons; J. M. Polak and James O’D. McGee, 1990, In Situ
Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984,
Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of
DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual :
Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring
Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed
Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0- 87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala,
Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and
Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench,
Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0- 87969-630-3. Each of these general texts is herein incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURESEmbodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
Figure 1 illustrates components of the assay apparatus according to one embodiment;
Figure 2 illustrates an assay card according to one embodiment;
Figure 3 illustrates a section through the assay card,
Figure 4 illustrates an example process for performing a multi-stage reaction according to one embodiment;
Figures 5A to 5C illustrate an example operation for drawing a test sample according to one embodiment;
Figure 6 illustrates the assay card being presented to the assay apparatus;
Figures 7A to 7D illustrate relative movement of the assay card with respect to rollers of the assay apparatus along a compression path;
Figure 8 illustrates optical changes in reaction material due to varying concentrations of a target agent;
Figure 9 illustrates a fluid diagram showing the equivalent function of the assay card;
Figures 10 and 11 illustrate desired example reactions to be performed by reagents within the reaction chamber;
Figure 12 illustrates a sensor disk utilised in the reaction chamber in one embodiment; and
Figure 13 illustrates processing steps for the preparation of the sensor disk.
DETAILED DESCRIPTIONASSAY APPARATUS
Figure 1 illustrates an arrangement of an assay apparatus, generally 10, according to one embodiment. The assay apparatus 10 provides a simple, reliable and effective arrangement for determining the presence of a target agent in a test sample. The main components of the apparatus are shown with its cover removed to help improve clarity. The apparatus comprises an assay card 20 which, as will be explained in more detail below, contains a number of reagent reservoirs, each of which contains a reagent which reacts with the test sample introduced into the assay card 20 via an inlet port 180 in order to determine the presence of the target agent, as will also be described in more detail below. The assay card 20 includes an aperture 30 in its reaction chamber to enable a detection of the characteristics of the reaction occurring within the reaction chamber to be made in order to determine the presence of the target agent.
In this example, the apparatus comprises an optical source 40 and an optical detector 50, although other types of source and detector may be provided dependent upon the characteristics of the particular reactions that are intended to occur. Accordingly, the optical properties of the fluid resulting from the reaction occurring within the reaction chamber can be determined when the aperture 30 is aligned with the optical source 40 and the optical detector 50. Typically, the concentration of the target agent can be determined from these optical characteristics and an indication of the presence of the target agent can be provided on the display 60. However, it will be appreciated that other characteristics could be measured to determine the presence of a target agent. For example, an electrochemical detector may be provided which couples with metallic strips on the assay card. The electrochemical detector then measures electrochemical characteristics of the fluid in the reaction chamber, the metallic strips convey any signal to an amplifier which amplifies these characteristics, if required, and an indication of the presence of the target agent can be provided on the display 60.
In overview, the operation of the assay apparatus 10 is as follows. The assay card 20 having a test sample provided therein is introduced into the apparatus between a pair of polymer-coated rollers 70. A switch 80 is activated which sends a signal to a roller controller 90 to drive the pair of rollers 70 using a motor within the roller controller 90 via the gear train 100. The motor may be a stepper motor or may be provided with a position indicator to enable the roller controller 90 to provide accurate control. The presence of the gear train 100 causes each roller within the pair 70 to counter rotate and slowly draw the assay card between the rollers in the direction A. As will be explained in more detail below, this causes the reservoirs on the assay card to be compressed sequentially to cause reagents therein to be released into the reaction chamber simultaneously and/or in series. In this example, the reagents are selected to cause a change in the optical properties of the resultant composition in the reaction chamber, with the optical properties varying in dependence on the concentration of target agent within the test sample. When the aperture 30 is aligned with the optical source 40 and the optical detector 50, the optical properties of the composition can be detected and processed to provide an indication of the presence of the target agent using the display 60.
ASSAY CARD
Figure 2 illustrates an assay card, generally 20°, according to one embodiment. The assay card 20° comprises a substrate 110 which is a laminate having an extruded thermoplastic layer into which reagent reservoirs 120 to 160, microfluidic channels 180, a reaction chamber 170 and a waste chamber 190 is provided, collectively referred to as an assay arrangement, as illustrated in more detail in Figure 3 below. The extruded thermoplastic may be thermoformed with the aid of a vacuum. These extruded components are compressible by the pair of rollers 70. In this example, there are provided five reagents reservoirs, each of which contains a different reagent. As can be seen, the reagent reservoirs 120 to 160 may be different sizes, dependent upon the volume of reagent to be contained therein, and are located at different locations on the surface of the assay card 20°. Typically, the reagent reservoirs 120 to 160 are dome-shaped or cylindrical. Each reservoir chamber may contain a reagent sack (not shown) which contains each associated reagent. The reagent sack provides protection for each reagent from humidity, light and/or oxygen. The reagent sack also enables convenient manufacture of the assay card since no loose fluids are present. Also, by retaining the reagents in the sack, inadvertent premixing is avoided. Each sack is small and thin. The sack is punctured upon the action of the rollers to enable injection of the reagent into the reaction chamber.
The position of the reagent reservoirs 120 to 160 on the assay card 20’ is determined by the order in which the reagents are intended to be delivered into the reaction chamber 170.
In this example, the assay card 20’ is intended to be drawn into the assay apparatus 10 in the direction marked by the arrow A. In other words, the assay card 20” moves relative to the rollers 70 in the direction A. Accordingly, the reagents are delivered into the reaction chamber 170 via the associated microfluidic channels 180 as the reagent reservoirs are compressed in order which pumps and seals at the same time, withstanding any backpressure. First, reagent reservoir 120 is compressed, followed by reagent reservoir 130, then reagent reservoir 140, then reagent reservoir 150 and finally reagent reservoir 160. If the assay card 20° is drawn into the assay apparatus at constant speed, then reagent reservoir 120 is compressed at time tl, reagent reservoir 130 at time t2, reagent reservoir 140 at time t3, reagent reservoir 150 at time t4, and reagent reservoir 160 at time tS. In that example, different time differences occur between the times at which each reaction reservoir is compressed. These delivery times can be varied by locating the reservoirs at different positions on the card in the direction A, based on the speed of the rollers 70. Alternatively, and typically in practice, it will be appreciated that the delivery times are varied by varying the speed of the rollers or even stopping the rollers for periods of time. Such an arrangement provides for a much more compact assay card. Also, when stopping the rollers provides a stationary hydrodynamic phase which is useful for providing time for biochemical reactions, particularly those which are slow such as antigen- antibody binding.
As will also be explained in more detail below, as each reagent is introduced into the reaction chamber 170 to react with the test sample and any surplus fluid is displaced into the waste chamber 190 where it is retained typically by an absorbent material such as a pulped material.
It will be appreciated that embodiments may provide for two or more reagent reservoirs being located at exactly the same location so that the associated reagents are simultaneously injected. Furthermore, it will be appreciated that the length of one of the reagent chambers may extend further towards the reaction chamber 170 than the other in order that one associated reagent is continued to be injected after the injection of another has completed.
The assay card 20° may be provided with an inlet port 180 and aperture 30 as shown in
Figure 1. In this embodiment, the aperture 30 enables the optical properties of the fluid within the reaction chamber to be measured. However, in another embodiment, a metallic coupling may be provided to enable an electrical connection between an electrochemical detector provided in the reaction chamber and an amplifier of the assay apparatus to be made.
The inlet port 180 is coupled with the reaction chamber 180 via a microfluidic channel 180 as shown in Figure SA. As also shown in Figure 5A, a narrow gate 210 is formed in the microfluidic channel 180, which may be sealed using a heating technique to decouple the reaction chamber 180 from the inlet port 180 once the test sample has been introduced.
Alternatively, a further roller or other compression device may be provided to squeeze the test sample into the reaction chamber and then retain its position to seal the inlet port 180.
Although the assay cards 20; 20° are shown as having one assay arrangement on one side of the substrate, it will be appreciated that more than one assay arrangement may be provided on each assay card and that these assay arrangements may be provided on more than one side of the assay card. This would enable, for example, a single assay card to perform multiple tests on the same test sample (assuming the inlet port was coupled was multiple reaction chambers) or the same test to be performed on multiple test samples, or a combination of both. Of course, it will be appreciated that the assay apparatus may need additional optical sources and the optical detectors.
The assay cards 20; 20° may be provided with an index hole (not shown) to mark a starting position of the assay card when entering the rollers 70. It will be appreciated that a starting position of the assay card may also be determined by the card being detected when entering the rollers 70 by activating a sensor on the assay apparatus 10. Likewise, the assay cards 20; 20° may be provided with a apertures or markings such as a linear encoding pattern
(not shown) which are sensed by a detector on the assay apparatus 10 and used to feedback position information to the roller controller 90.
Figure 3 illustrates a section through the assay card 20; 20°. The assay card 20; 20’ is a laminate comprising a substrate layer 200, to which is bonded a thermoplastic layer 210. The thermoplastic layer 210 is formed from a suitable thermoplastic material which is compressible, but not necessarily elastic such as, for example, polyvinyl chloride, polypropylene, polyethylene, polyethylene terephthalate, poly(methyl methacrylate), and the like. The thermoplastic layer 210 is formed using vacuum-assisted hot embossing to form parts of the reagent reservoirs 120 to 160, the reaction chamber 170, the microfluidic channels 180 and the waste chamber 190. A hole may be punched through the substrate 200 and the thermoplastic sheet 210 at the appropriate location to locate the aperture 30. Suitable windows may then be sealed into the resultant holes, optionally with a sensor disc located there between, as will be explained in more detail below.
Also, it will be appreciated that reservoirs could be provided which are compressed to form a negative pressure which enables fluid to be draw into a particular region.
EXAMPLE OPERATION
Figure 4 illustrates an example process for performing a multi-stage reaction that may be undertaken when processing the assay card 20 with the assay apparatus 10. At step S10, the test sample is drawn into the card, as illustrated with reference to Figures SA to 5C. As illustrated in Figures SA and 5B, the test sample 200 (such as a blood sample) is drawn into the inlet port 180 and flows to the aperture 30 in the reaction chamber 170 by capillary force.
Any excess of the test sample 200 goes to the waste chamber 190. After the test sample 200 is drawn, a gate 210 of microfluidic channel 180 is sealed off by means of hot wire sealer provided separately with sampling kit.
Returning to Figure 4, once the test sample 200 has been sealed into the assay card 20, then the assay card 20 is placed into the assay apparatus 10 as shown in Figures 6 and 7A. The reaction chamber 170 is preconfigured to contain an immobilized antibody, typically provided on a substrate as will be described in more detail below. The assay card 20 is drawn through the pair of rollers 70 in the direction A until, at step S20, pair to rollers 70 compress the reservoir 120 and the test sample 200 is washed by the reagent contained in the reservoir 120 being injected into the reaction chamber 170 to flush the test sample and any unbound target agent, such as an antigen, as illustrated in Figure 7B. At this stage, the speed of the pair of rollers 70 may reduce or movement stop completely to enable the washing to complete.
The assay card 20 continues to move relative to the rollers 70 until, at step S30, the enzyme labelled antibody is injected from the reagent reservoir 130, as illustrated in Figure 7C. The enzyme labelled antibody binds to the antigen which is already adhered to the immobilized antibody on the substrate creating a sandwich structure: antibody-antigen- antibody. Again, at this stage, the speed of the pair of rollers 70 may reduce or movement stop completely to enable the reaction to complete.
The assay card 20 continues to move relative to the pair of rollers 70 until, at step S40, the reservoir 140 releases its reagent to wash any unbound antibodies, as shown in Figure 7D.
Again, at this stage, the speed of the pair of rollers 70 may reduce or movement stop completely to enable the reaction to complete.
The assay card 20 continues to move relative to the pair of rollers 70 until, at step S50, a reagent is released from the reagent reservoir 150, as also illustrated in Figure 7D. Again, at this stage, the speed of the pair of rollers 70 may reduce or movement stop completely to enable the reaction to complete. The reagent molecules react with the enzyme to generate luminescence, fluorescence or a colour change.
Accordingly, it can be seen that the reagent reservoir 140 engages with the pair of rollers 70 slightly earlier than the reagent reservoir 150. However, it will be appreciated that there will be a period when the associated reagents will be simultaneously released into the reaction chamber 170.
The assay card 20 continues to move relative to the pair of rollers 70 until, at step S60, a washing reagent is released from the reagent reservoir 160. Again, at this stage, the speed of the pair of rollers 70 may reduce or movement stop completely to enable the reaction to complete.
The assay card 20 continues to move relative to the pair of rollers 70 until, at step S70, the window 30 aligns with the optical transmitter 40 and the optical detector SO whereupon a colour change in the reaction materials is detected to provide an indication of the concentration of the presence of the target agent within the test sample, as illustrated in more detail in Figure 8. Figure 8 shows an example antibody immobilised spot in the aperture 30 which is stained with, for example, silver. As the concentration of any target agent or antigen in the test sample increases, the grey level becomes darker. The grey level can be detected by the optical emitter 40 and detector 50. The emitter 40 may be a LED, the detector S50 may be a photodiode or may be a sensitive black and white charge-coupled device (CCD) camera. This grey level may be detected and a corresponding concentration of target agent displayed on the display 60.
Hence, as can be seen, the processing operation is sequential. Although the timing is important, this can be easily controlled by carefully locating the reservoirs 120 to 160 at the appropriate distance along the assay card 20 in the direction A and by controlling the speed of the movement relative to the rollers 70 by changing the rotation speed or even switching the rollers 70 on and off. The flow rate may also affect performance and this can also be readily controlled by varying the rotation speed of the rollers 70, adjusting the size of the microfluidic channels 180 and/or the cross-sectional area of the reagent reservoirs 120 to 160 presented to the rollers 70.
In one embodiment the reagents are a washing solution, a gold nanoparticle labelled antibody solution, a silver enhancement solution A and solution B, a washing solution.
Figure 9 illustrates a fluid diagram showing the equivalent function of the assay card 20,20’ and shows the five equivalent pumps for pumping the associated reagents into the reaction chamber 170. The quantity of reagent is controlled by the volume of the associated reagent reservoir 120 to 160. The flow rate is a function of the cross-sectional area of the associated reagent reservoir 120 to 160 presented to the pair of rollers 70 and the speed at which the assay card 20 moves relative to the rollers 70. The operation time is a function of the location of each reaction reservoir along the actuation access A and the speed with which the assay card moves relative to the pair of rollers 70, which 1s controlled by the controller 90.
EXAMPLE REACTIONS
Figures 10 and 11 illustrate desired example reactions to be performed by reagents within the reaction chamber 170.
Figure 10 illustrates an antibody 300 immobilised on a sensor card 310 provided within the aperture 30 of the reaction charober 170. The target agent or antigen 330 is contained in the test sample 200. The enzyme labelled antibody 320 is supplied from a reagent
TESETVOIT,
As shown in Figure 11, embodiments may utilise a silver enhancement of gold in a nano-particles technique. Typically, gold nano—particles are used as an antibody label because of the gold nano-particle’s deep red colour and the ease of conjugation with the antibody [Immunochemistry, 8, 1081(1071)]. However, colour detection of gold nano-particles requires expensive optical filters and a sensitive detector. Also, the signal is often not sensitive enough for some applications. To amplify the signal, therefore, a silver enhancement technique is utilised (as described in US patent 6602669). Silver nitrate and silver acetate are good sources of silver ions, while hydroquinone, n-propyl galate, p-penylenediamine and formaldehyde are commonly used as reducing agents.
SENSOR DISK
As shown in Figure 12, an embodiment uses a separate sensor disk 250 made of a piece of indium-tin-oxide (ITO) film, which is a conductive and transparent film widely used in the touch-screen industry. The ITO film provides well defined and stable surface for antibody immobilization. The sensor disk 250 is inserted in to the reaction chamber 170 just before the aperture 30 is covered with a cover film.
As shown in Figure 13, an embodiment uses (3-glycidoxypropyl)trimethoxysilane (GPTES) 211 as a cross linker between the ITO surface hydroxyl group and the antibody’s amine group. The ITO film of the sensor disk 250 is cleaned and treated with acid to activate the surface hydroxyl group. Then, the cross linking agent is applied on the activated ITO surface by dipping the ITO film in 1% solution of GPTES for overnight at room temperature .
The antibody 213 solution is dropped onto the surface and incubated for overnight at 4°C.
Additional rinsing and blocking with bovine serum albumin (BSA) steps finalize the antibody deposition.
It will be appreciated that the sensor disk may be provided with a layer of biochemical agent such as an enzyme or an antibody that reacts specifically to certain molecules to be detected in the test sample.
Accordingly, it can be seen that embodiments provide a versatile diagnostic platform for detecting chemical or biological agents in biological samples like blood or urine, as well as water, soil and other environmental samples.
Embodiments provide a means of sequential injection of reagent liquids into a reaction chamber in a pre-programmed manner by means of very simple and reliable squeezing mechanism. In one embodiment, a name-card sized, thin diagnostic device is provided on which five reagent reservoirs and one waste chamber are extruded, while those reservoirs and chambers are interconnected with microfluidic channels. The location of each reservoir is carefully determined to make sure the contained liquid is squeezed out sequentially. For example, a typical sandwich immunoassay requires following steps: the antigen in a sample is bound with the antibody immobilized on the reaction zone. A washing solution is pumped to the reaction zone to flush the sample and unbound antigen. An enzyme labelled antibody solution is pumped to the reaction zone. The antibody is bound to the antigen which is already adhered to the immobilized antibody creating a sandwich structure: antibody-antigen- antibody. The washing solution is pumped to the reaction zone again to flush the unbound antibody. The last step is to pump the substrate solution to the reaction zone. The substrate molecules react with the enzyme generating luminescence, fluorescence or colour change. The light signal is detected by an appropriate electro-optical detector module.
REFERENCESEach of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents (“application cited documents”) and any manufacturer’s instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer’s instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.
Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.