Patent Application
20150285793
The present invention generally relates to a diagnostic device and assay for the detection of a target analyte in a fluid test sample. More specifically, the present invention relates to a downward or vertical flow diagnostic device having the reagents used in the assay contained within the device.
Point of Care Tests have become increasingly common over the past several years. These tests are simple to perform, require little or no extra equipment, and typically produce results in a relatively short period of time compared to traditional laboratory tests. There are primarily two technological platforms available in the Point of Care test market: lateral flow and flow-through, each of which can be used for many applications.
While both lateral flow and flow-through test devices utilize the same biological principles, namely detection of target analytes such as antibodies or antigens in a bodily fluid, they differ in their methods for detecting antibody-antigen complexes.
For example, Eisinger et al., U.S. Pat. No. 4,943,522, describes a lateral flow diagnostic device in which the test sample is applied to one end of the device and then transferred laterally across a membrane to be visualized in one or more indicator zones.
A common problem with most lateral flow devices is that the amount of time required to transfer the test sample laterally from the sample application zone to the indicator zones may not be ideal for certain time sensitive applications, such as emergency departments, labour and delivery settings and needle stick injuries.
Accordingly, flow-through devices offer advantages over lateral flow devices in that they are often more compact and can provide more rapid results. Examples of such flow-through devices can be found in Brown et al., U.S. Pat. No. 5,160,701 and Chan, U.S. Pat. No. 7,531,362.
Brown et al., USP '701 describe a flow-through device in which the fluid test sample is applied to a reaction matrix containing a capture reagent bound to the matrix that is capable of binding to a target analyte in the test sample. The remaining portion of the test sample flows through the reaction matrix into an absorbent pad. After several rounds of washing, the presence or absence of the target analyte in the fluid test sample is visually determined following the manual addition of a liquid indicator reagent. The Brown et al., device requires the manual addition of several buffers and reagents in order to achieve a result. These manual steps not only increase the amount of time required to complete an assay, but also increase the potential for error in the assay. In addition, some of reagents required for use with the Brown et al., device require refrigeration or have to be prepared immediately prior to use in the assay, thus limiting the environments in which the device can be used.
Chan, USP '362, provides a flow-through device that eliminates several of the washing steps required by Brown et al., thus decreasing the overall time required to successfully complete an assay. Furthermore, Chan provides a post-filter unit containing a dried indicator reagent that is applied to the test device after the test sample has permeated through the reaction matrix. A buffer is applied to the post-filter unit to resolubilize the indicator reagent. The resolubilized indicator reagent is then able to interact with the two-membered complex formed in the reaction zone between the target analyte and the capture reagent. By providing the indicator reagent in a dried format in a post-filter unit, the assay requires only a single buffer, fewer washing steps, and can be stored at room temperature.
Although the Chan device efficiently provides results in a timely manner, it would be desirable to have a rapid diagnostic device that improves the efficiency of the Chan device by further reducing the required number of manual additions of reagents and buffers than the traditional downward or vertical flow through devices.
It is an object of the present invention to provide a downward or vertical flow diagnostic device that requires less manual addition of reagents and buffers during operation and thus overcomes the limitations of the prior art.
It is a further object of the present invention to provide an assay that requires less reliance on the manual delivery of reagents and buffers to the assay, thus minimizing the potential for error.
According to an aspect of the present invention, there is provided a downward or vertical flow through test device for determining the presence or absence of a target analyte in a fluid test sample. The test device comprising a test area and a reagent storage area. The test area comprises: a reaction zone to which the fluid test sample is applied so that the fluid test sample flows into, then downwardly or vertically through the reaction zone, the reaction zone comprising an immobilized capture reagent that binds the target analyte in the deposited fluid test sample, and an absorbent zone in vertical communication with the reaction zone, the absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone and into the absorbent zone. The reagent storage area comprising: at least one cartridge housing a liquid reagent positioned above the test area and connected by a channel to the test area, whereby liberating the liquid reagent from the cartridge causes the reagent to flow vertically or downwardly through the channel onto the test area.
In one embodiment, a matrix having dried indicator reagent embedded therein is provided. The matrix being positioned between the cartridge and the test area, whereby the liberated liquid reagent flows through the matrix to reconstitute the indicator reagent.
In another embodiment, an actuator for breaking the cartridge is provided, wherein activating the actuator causes the release of the liquid reagent from the cartridge.
In a further embodiment, a liquid permeable member is positioned to support the cartridge allowing the liberated liquid reagent to flow downward or vertically through the liquid permeable member.
In yet a further embodiment, a second liquid permeable member positioned between the matrix and the test area. The second liquid permeable member can be positioned to support the matrix and functions to control the rate of flow of the liquid reagent containing the reconstituted indicator reagent from the matrix to the channel.
In an embodiment, two or more reagent storage areas are provided and each reagent storage area is connected to the test area via a dedicated channel. In another embodiment, the two or more reagent storage areas are provided and the reagent storage areas are connected to the test area via one or more common channels.
In a further embodiment, the reagent storage area is provided in a housing positioned on the test device. Alternatively, the reagent storage area is provided in a housing confined within the test device
In another embodiment, more than one immobilized capture reagent is provided in the reaction zone.
A flexible membrane that allows for compression of the cartridge can also be provided.
In an embodiment, a barrier surrounding the matrix to limit the lateral movement of the matrix within the reagent storage area.
In yet a further embodiment, the cartridge is pressure fitted into a space defined by the housing to prevent lateral movement of the cartridge within the housing.
In one embodiment, one or more vents positioned between the barrier and the housing to facilitate the movement of air within the housing.
In an embodiment, the liquid reagent is an indicator reagent capable of binding to a two-membered complex formed between the capture reagent and the target analyte.
In one embodiment, the reagent storage area is located directly above the test area such that reagent liberated from the reagent storage area flows downward through an aperture onto the test area, flooding the entire test area.
In another embodiment, a separation zone is provided that is adapted to receive and separate particulate matter from a fluid portion of a test sample, such as whole blood. The separation zone being in fluid communication with the reaction zone. A channel can be provided to connect the separation zone to the reaction zone.
According to another aspect of the present invention, there is provided a method of determining the presence or absence of a target analyte in a fluid test sample. The method comprising the steps of: applying the fluid test sample to a reaction zone of a test unit, whereby the fluid test sample flows into and then downwardly or vertically through the reaction zone, the reaction zone comprising an immobilized capture reagent that binds the target analyte in the deposited fluid test sample to form a two-membered complex of a specific binding interaction; allowing the fluid test sample to flow into and then downwardly or vertically through the reaction zone into an absorbent zone in vertical communication with the reaction zone, the absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone so as to concentrate the two-membered complex in the reaction zone; activating a cartridge positioned above the test area to liberate a liquid reagent contained therein, transporting the liquid reagent through a channel to the reaction zone of the test area; allowing the liquid reagent to flow downwardly or vertically through the reaction zone and into the absorbent zone to bind with the two-membered complex concentrated in the reaction zone, with any unbound reactants being washed from the reaction zone; and observing a test result depicted by a presence or absence of a visually detectable signal on the reaction zone.
In one embodiment, the method further comprises the step of allowing the liberated reagent to flow through a liquid permeable member prior to being transported through the channel.
In another embodiment, the method further comprises the step of allowing the liberated reagent to flow through a matrix comprising a dried indicator reagent to resolubilize the indicator reagent prior to being transported through the channel.
In a further embodiment, the liquid reagent is an indicator reagent capable of binding to the two-membered complex.
According to another aspect of the present invention, there is provided a downward or vertical flow through test device for determining the presence or absence of a target analyte in a fluid test sample. The test device comprising a test area and a reagent storage area. The test area comprising: a reaction zone to which the fluid test sample is applied so that the fluid test sample flows into and then downwardly or vertically through the reaction zone, the reaction zone containing an immobilized capture reagent that binds the target analyte in the deposited fluid test sample, and an absorbent zone in vertical communication with the reaction zone, the absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone and into the absorbent zone. The reagent storage area comprises: at least one cartridge housing a liquid reagent, and a liquid permeable member positioned between the cartridge and the test area; and a channel connecting the reagent storage area to the test area for facilitating transfer of the liquid reagent from the cartridge to the reaction matrix; wherein the liquid permeable member prevents solid material from the cartridge from entering the channel.
In one embodiment, a matrix is provided having dried indicator reagent embedded therein, the matrix being positioned between the liquid permeable member and the test area, whereby the liberated liquid reagent flows through the matrix to reconstitute the indicator reagent.
In another embodiment, an actuator is provided for breaking the cartridge, wherein activating the actuator causes the cartridge to break and release the liquid reagent therefrom.
In a further embodiment, the liquid permeable member is positioned to support the cartridge to allow the liberated liquid reagent to flow downward or vertically through the liquid permeable member.
In yet a further embodiment, a second liquid permeable member is positioned between the matrix and the test area.
In a still further embodiment, the second liquid permeable member is positioned to support the matrix and wherein the second liquid permeable member controls the rate of flow of the liquid reagent containing the reconstituted indicator reagent from the matrix to the channel.
In an embodiment, the channel is angled from the reagent storage area towards the test area to control the rate of flow of the liberated liquid reagent to the test area.
In another embodiment, two or more reagent storage areas are provided and each reagent storage area is connected to the test area via a dedicated channel. Alternatively, each reagent storage area is connected to the test area via one or more common channels.
In a further embodiment, the liquid permeable member controls the rate of flow of the liquid reagent from the cartridge to the channel once the cartridge has been activated and the liquid reagent liberated therefrom.
In some embodiments, the reagent storage area is provided in a housing positioned on the test device. In others, more than one immobilized capture reagent is provided in the reaction zone.
According to another aspect of the invention, there is provided an apparatus for determining the presence or absence of at least one target analyte in a fluid test sample. The apparatus comprises a reagent storage area and a test device. The reagent storage are comprising: at least one cartridge housing a liquid reagent positioned above the test area, and a channel connecting the cartridge to a test area. Liberating the liquid reagent from the cartridge causes the reagent to flow vertically or downwardly through the channel onto the test area. The test device housed in the apparatus comprises: a test area and a matrix. The test area comprising a reaction zone and an absorbent zone. The fluid test sample is applied to the reaction zone so that the fluid test sample flows into and then downwardly or vertically through the reaction zone. The reaction zone comprises an immobilized capture reagent that binds the target analyte in the deposited fluid test sample, and an absorbent zone in vertical communication with the reaction zone. The absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone and into the absorbent zone. The matrix is positioned between the cartridge and the test area, as has a dried indicator reagent embedded therein. Liquid reagent liberated from the cartridge flows through the matrix to reconstitute the indicator reagent. The indicator reagent comprises a fluorescent material. The apparatus also has a light source for exciting the fluorescent material when the indicator reagent is bound to the target analyte and a detector for detecting the emission of fluorescence from the bound indicator reagent.
According to another aspect of the invention, there is provided an apparatus for determining the presence or absence of at least one target analyte in a fluid test sample. The apparatus comprises a reagent storage area and a test device. The reagent storage are comprising: at least one cartridge housing a liquid reagent positioned above the test area, and a channel connecting the cartridge to a test area. Liberating the liquid reagent from the cartridge causes the reagent to flow vertically or downwardly through the channel onto the test area. The test device housed in the apparatus comprises: a test area and a matrix. The test area comprising a reaction zone and an absorbent zone. The fluid test sample is applied to the reaction zone so that the fluid test sample flows into and then downwardly or vertically through the reaction zone. The reaction zone comprises an immobilized capture reagent that binds the target analyte in the deposited fluid test sample, and an absorbent zone in vertical communication with the reaction zone. The absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone and into the absorbent zone. The matrix is positioned between the cartridge and the test area, as has a dried indicator reagent embedded therein. Liquid reagent liberated from the cartridge flows through the matrix to reconstitute the indicator reagent. The indicator reagent comprises a detectable material.
These and other features, aspects and advantages of the present invention will become better understood with regard to the following description and accompanying drawings wherein:
The following description is of one particular embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to an “antigen” or “antibody” is intended to include a plurality of antigen molecules or antibodies.
As employed throughout the description, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
Absorbent Zone—the term “absorbent zone” is intended to include one or more layers of a permeable (e.g. porous or fibrous) material, which layers can be the same or different, and are capable of drawing or wicking fluid by capillary action. The absorbent zone should also be capable of absorbing a substantial volume of fluid that is equivalent to or greater than the total volume capacity of the material itself, and thus have a high absorbent capacity.
Analyte (or target analyte)—the compound or composition of interest to be detected in a biologically derived fluid test sample. Examples of analytes may include drugs, pharmaceutical metabolites, hormones, polypeptides, proteins including immunoglobulins, polysaccharides, nucleic acids, and combinations thereof.
Antibody—an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
Antibodies useful in conducting the immunoassays of the present invention include those specifically reactive with various analytes the detection of which in biological fluids is desired. Such antibodies are preferably IgG or IgM antibodies or mixtures thereof, which are essentially free of association with antibodies capable of binding with non-analyte molecules. The antibodies may be polyclonal or monoclonal and are commercially available or may be obtained by mouse ascites, tissue culture or other techniques known to the art. A typical description of hybridoma procedure for the production of monoclonal antibodies may be found in Wands, J. R., and V. R. Zurawski, Gastroenterology 80:225 (1981); Marshak-Rothstein, A., et al.; J. Immunol. 122:2491 (1979); Oi, V. Y. and L. A. Herzenberg, “Immunoglobulin Producing Hybrid”, Mishell B. B. and S. M. Shiigi (eds) Selected Methods in Cellular Immunology, San Francisco: W. H. Freeman Publishing, 1979; and U.S. Pat. No. 4,515,893 issued to Kung, et al. The use of mixtures of monoclonal antibodies of differing antigenic specificities or of monoclonal antibodies and polyclonal antibodies may be desired. It is further contemplated that fragments of antibody molecules may be used as specific binding reagents according to the invention including half antibody molecules and Fab, Fab′ or F(ab′)2 fragments known in the art. Regardless of the particular source or type of antibodies, however, it is preferred that they be generally free of impurities. The antibodies may be purified by column chromatographic or other conventional means but are preferably purified according to known affinity purification techniques. Antibodies materials may also be labeled with colloidal particles, fluorescent markers or chemi-luminescent molecules according to the invention and used in sandwich type assays for the detection of antigen analytes or in competition assays for the detection of antibody analytes.
Antigen—antigens and haptens useful in carrying out the immunoassays of the present invention include those materials, whether natural or synthesized, which present antigenic determinants for which the analyte antibodies are specifically reactive when used according to the present invention. Synthesized antigens include those which are constructed according to conventional chemical syntheses as well as those constructed according to recombinant DNA techniques. Antigen materials may also be labeled with colloidal particles, fluorescent markers or chemi-luminescent particles according to the invention and used in sandwich type assays for the detection of antibody analytes or in competition assays for the detection of antigen analytes.
Capture Reagent—any compound or composition capable of recognizing a particular spatial and/or chemical structure of an analyte. In the case of an analyte which is a specific immunoglobulin species, the capture reagent may be the specific protein or eptitope recognized by the immunoglobulin. Other types of capture reagents include naturally occurring or synthetically produced receptors, antibodies, antigens, enzymes, Fab fragments, lectins, nucleic acids, avidin, protein A, and the like.
Fluid test sample—the fluid test sample is assayed to form a detectable reaction product on the reaction membrane of the test unit. In preferred assay embodiments, the fluid test sample is biologically derived (e.g. whole blood, plasma, serum, urine, saliva, etc.) and is suspected to include as the target analyte, typically an antigen, antibody, or hapten capable of being bound by the capture reagent immobilized on the reaction membrane. In some cases, prior to application to the device the fluid may be mixed with a buffer.
Indicator Reagent—a conjugate comprised of a specific binding member to the target analyte and a label or enzyme conjugated to the specific binding member. Additionally, the indicator reagent can be comprised of a general marker protein, e.g. Protein A, Protein G, or anti-IgG conjugated to a label. For example, in an assay for detecting antibody as a target analyte, a preferred indicator reagent would be protein A labeled with colloidal gold. Other indicator reagents may also include a labeled anti-human antibody directed to the antibody of interest, e.g. goat anti-human IgG labeled with colloidal gold for the detection of human antibody in a fluid test sample. Alternatively, the specific binding member or the general marker protein may be conjugated to an enzyme, such as horseradish peroxidase.
Label—a label may be any molecule or particle bound or conjugated to a specific binding member, or general marker protein which can produce a signal. A label can also include a substrate capable of producing a visually detectable signal when reacted with an enzyme conjugated to the general marker protein. In one embodiment, the label is preferably a “direct” label which is capable of spontaneously producing a detectible signal without the addition of ancillary reagents and will be easily detected by visual means without the aid of instruments. For example, colloidal gold particles can be used as the label. Other suitable labels may include other types of colloidal metal particles, minute colored particles, such as dye sols, and coloured latex particles. Many such substances will be well known to those skilled in the art.
Matrix—The term “matrix” is intended to include a porous material which is impregnated with a dried indicator reagent that can be readily resolubilized upon addition of a buffer reagent thereto.
Reaction Zone—the term “reaction zone” is intended to include a porous material to which the capture reagent(s) and other molecules employed in the analytical assay are bound as well as additional porous supporting material, if any, that forms the lower surface of the reaction zone. The capture reagent(s) immobilized or bound in the reaction zone may be bound in one, two, or more discernable and separate areas so that multiple target analytes in a single test sample can be analyzed simultaneously. Optionally, the reaction may further comprise an immobilized control molecule or reagent capable of non-specifically capturing material from the fluid test sample in a discernable and separate area.
Specific Binding Member—this describes two or more complementary members of a specific binding interaction which have binding affinity for one another. The specific binding members may be naturally derived or synthetically produced. One member of the specific binding interaction has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and/or chemical structure of the other complementary member. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin/streptavidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate, and the like.
The downward or vertical flow through test device of the present invention is useful for determining the presence or absence of a target analyte in a fluid test sample, such as a bodily fluid. For example, the test sample could be serum, plasma, whole blood, saliva, urine, etc and the target analyte may be an antigen, antibody, natural or synthetic steroid, hormone, pharmaceutical metabolite and the like.
As shown in
Depending on the detection limits the user wishes to impose on the diagnostic assay, the capture reagent can be deposited singly or in various combinations in the reaction zone. For example, a panel of two or more different specific binding members selected as the capture reagent for the diagnostic assay may be applied to different regions of the same reaction membrane so that the presence of multiple analytes in a single fluid test sample may be simultaneously analyzed, e.g. for the detection of HIV and HCV. Preferably, the capture reagent is deposited in a discrete test zone having an area substantially smaller than that of the entire surface area of the porous material used in the reaction zone. Various patterns that are convenient for the distribution of the capture reagent may include, but are not limited to, numerals, letters, dots, lines and symbols, or the like, which display the detectable signal upon completion of the assay. It is preferred that the pattern of the discrete test zone be in the form of a single line to enhance the visability of the test result.
Since the present apparatus is designed to be used in a method for detecting a target analyte in a fluid test sample, a capture reagent must be provided which will recognize and be capable of specifically binding to the target analyte. One of ordinary skill in the art will appreciate that the term “specific binding” refers to the interaction that will occur between two or more complementary non-identical components to form a complex. Examples of such binding pairs include antigens and antibodies, hormones (and other intracellular messengers) and cell receptors, sugars and lectins. Either member of the specific binding pair can be immobilized to the reaction zone with the other member being the analyte being detected in the test sample. Exemplary, but not exclusive of the present technology, is the specific binding interaction that occurs as a result of antibody-antigen interactions. However, it should be realized that the use of terms such as antigen and antibody are not mutually exclusive since antibodies can act as antigens for other antibodies.
In addition to the capture reagent, a defined area of the exposed reaction zone may also contain a control molecule. In this regard, color development at the test site may be compared with the color of one or more standards controls to determine whether the reagents are stable and the test is performing properly. In general, when testing for the presence of target analyte, the diagnostic device will have a built-in control of an antibody directed to human immunoglobulin G (IgG), IgM, IgE, or IgA. Thus when a fluid test sample is added to the diagnostic device, immunoglobulin will bind to the control region regardless of whether or not target analyte happens to be present in the sample. For example, a suitable control may be established by using Protein A which is disclosed in U.S. Pat. No. 5,541,059 (Chu). Other suitable controls are well known in the art.
As noted above, the capture reagent, and the optional use of controls, are typically applied only to defined regions of the exposed surface of the reaction zone. The capture reagent will often be applied to a region within the center of the reaction zone such that the perimeter of the exposed surface of the reaction zone will not have any capture reagent bound thereto. On the other hand, in some situations, it may be appropriate to cover the entire exposed surface of the reaction zone with the capture reagent. If, however, capture reagent is immobilized onto a limited region of the exposed surface of the reaction zone, the porous material or membrane from which the zone is made can be treated with a blocking composition that prevents the target analyte and other components of the sample from non-specifically binding to the reaction zone. For assays where non-specific binding is not problematic, a blocking step will be unnecessary. Also, the use of a good quality paper-backed nitrocellulose may make a blocking step unnecessary in some assays. However, if a blocking step is needed, common blocking solutions comprising bovine serum albumin (BSA) or other proteins which do not interfere with, or cross-react with, reagent materials of the assay can be used.
The sensitivity of reaction-membrane type immunoassays (i.e. the ability to detect very low levels of target substance) can be increased if the sample is concentrated through the reaction zone 3. For example, concentration of the sample through a reaction zone 3 can be achieved by having an absorbent material, or pad, beneath the reaction zone 3 that draws the sample, which is added to the surface of the reaction zone, through to the absorbent material below. The absorbent zone 4 can be generated from any material capable of wicking fluid by way of capillary action, such as cotton or paper. Membrane-based immunoassays that utilize various absorbent materials to concentrate sample are exemplified in U.S. Pat. Nos. 5,185,127, 5,006,464, 4,818,677, 4,632,901, and 3,888,629.
An absorbent material of the absorbent zone 4 is situated underneath the lower surface of the reaction zone 3 so as to be in direct fluid communication with the reaction zone 3. Thus, the upper surface of the absorbent material is adjacent to at least a portion of the lower surface of the reaction zone 3. Fluid communication contact can involve direct physical contact of the absorbent material with the reaction zone or may optionally include the separation of a portion of the absorbent material from the reaction zone by an intervening spacer layer which has an opening therein. Accordingly, the spacer layer still permits direct contact between the reaction zone and the absorbent zone thereby enabling the assay reagents to flow uniformly from the upper surface down to the lower surface of the assay apparatus. Although not critical to the performance of the apparatus, the spacer layer also serves to hold the porous membrane of the reaction zone. The spacer layer may be made of any rigid or semi-rigid material that does not bind or interact with assay reagents used in conjunction with the invention. Exemplary of materials for the spacer layer are fiberglass, paper, hydrophilic polypropylene, or cellulose. The thickness of the spacer layer will generally be in the range of about 0.1 mm to 1 mm. In embodiments of the invention where ease of manufacture and reduced costs are desired, the upper surface of the absorbent material is typically placed immediately adjacent the lower surface of the reaction zone.
The selection of material for the absorbent zone 4 is not critical and a variety of fibrous filter materials can be used, including one or more layers of the same or different materials, providing that the material selected is compatible with the target analyte and the assay reagents. Any conventionally employed absorbent material that is capable of drawing or wicking fluid through a porous membrane, such as for example, by capillary action, can be used in the present invention. The absorbent material should be capable of absorbing a volume of fluid test sample that is equivalent or greater than the total volume capacity of the material itself. Useful known materials include cellulose acetate fibers, polyester, polyolefin or other such materials. The absorbent material provides a means to collect the sample by providing uniform “suction” to deliver the sample from the well, through the reaction zone, and down into the absorbent material. Thus, the absorbent body also acts as a reservoir to hold the sample, and various reagents that are used when the assay is performed. Accordingly, when used in assays where relatively large volumes of fluid are used, the absorbent material should have high absorbent capacity so as to prevent or minimize the possibility of back-flow of sample and reagents from the absorbent body back into the reaction membrane.
The reagent storage area 5 comprises a cartridge 6 and a liquid permeable member 7. The reagent storage 5 can be positioned either within the device 1 (as shown in
The cartridge 6 houses a liquid reagent for use in the assay. In the context of the application, the term reagent not only refers to a substance used in the detecting and measuring of a component in the assay, but also to any liquid used in the assay, such as buffers and wash solutions. The cartridge 6 can be made from a liquid impermeable, but pliable, material, such as, but not limited to, plastic. Alternatively, the cartridge 6 is manufactured from a material that can be broken or punctured to liberate the liquid contained therein. In some instances, it might be advantageous to manufacture the cartridge 6 from a material that is capable of protecting the liquid housed therein from light or changes in temperature. Alternatively, the cartridge 6 may be provided as a cavity within the housing 10 of the device or a cavity within an apparatus containing the device. In this arrangement, the liquid reagent is stored in the cavity or in a cartridge positioned in the cavity and pressure applied to the housing 10 or the cartridge 6 causes the liquid reagent to be liberated from the cavity or cartridge. A seal, one-way valve or similar means retains the liquid in the cavity until the cartridge is activated and the liquid reagent is liberated therefrom. Similarly, a one-way valve or similar means can be incorporated into the cartridge 6 so that when pressure is applied to the cartridge, the liquid reagent retained therein is liberated.
In one embodiment, the cartridge 6 is positioned directly and vertically above at least a portion of the test area 2 (
The liquid housed in the cartridge 6 can be released by putting sufficient manual pressure on the cartridge 6 to force the liquid through the one-way valve (if equipped), or to rupture, in the case of a breakable cartridge. For example, a button or flexible membrane 12 can be provided near or surrounding the cartridge 6 to force the liquid out of the cartridge 6 or to break the cartridge outright. When the button or flexible membrane 12 is depressed, the compressive force on the cartridge 6 causes it to rupture, or the liquid contained therein to be forced through the one-way valve, and thus release the liquid contained therein. Optionally, an actuator 11 can be provided near the cartridge 6 to assist in disrupting the structural integrity of the cartridge 6. The actuator 11 may be positioned below the cartridge 6 (not shown) such that depression of the flexible button 12 causes the cartridge 6 to contact the actuator positioned below it. Similar to the button or flexible membrane 12, the actuator 11 can rupture the cartridge 6 by compression or the actuator 11 can force the liquid out of the cartridge 6. In some instances, it may be advantageous to provide both an actuator 11 and a button 12 to collectively compress the cartridge 6. Alternatively, the flexible member 12 may consist of piercing members that are able to puncture or fracture the cartridge 6.
A liquid permeable member 7 can be positioned between the cartridge 6 and the test area 2. When used, the liquid permeable member 7 prevents solid material from the cartridge 6 from entering the test area 2 and contaminating the assay. In addition, the liquid permeable member 7 can also be designed to control the flow rate of the liquid reagent from the cartridge 6 to a channel 8; or directly to the test area, once the cartridge 6 has been activated and the liquid reagent liberated therefrom. Moreover, the liquid permeable member 7 can function alone or in combination with an actuator 11 to assist in the breaking or rupturing of the cartridge 6. In this case, it would be preferable, but not necessary, for the liquid permeable member 7 to be sharp, rough or jagged on the surface that comes into contact with the cartridge 6.
In one embodiment, the liquid permeable member 7 is positioned to support the cartridge 6 to allow the liberated liquid reagent to flow downward or vertically through the liquid permeable member 7. In other embodiments, the liquid permeable member 7 can be positioned along the channel 8 to prevent pieces of the cartridge 6 from entering the test area. In this case, the cartridge 6 is supported by either the device housing 10 or a separate supporting member (not shown).
Typically, the liquid permeable member 7 is formed from a thermoplastic polymer, such as polyethylene, or a thermosetting polymer, such as vulcanized rubber. In these cases, one or more apertures of a prescribed diameter, or width, pass through the liquid permeable member 7 in order to prevent pieces of the cartridge 6 from entering the test area 2. In addition, the size and shape of the apertures can control the flow rate of the liquid exiting the liquid permeable member 7 as well as concentrate the stream of the liquid flowing thereform. Although inert materials, such as medical-grade plastics, may be most desirable for use in the present device, since they are less likely to contaminate the assay, other materials, such as metals and fibrous membranes may be used in certain circumstances.
The reagent storage area 5 to the test area 2 can be connected by a channel 8. The channel 8 gathers the liberated reagent from the cartridge 6 and transfers it to the test area 2. At the test area 2, the channel 8 terminates above the reaction zone 3 to allow the reagent to be deposited onto the reaction zone 3 where it can then flow downward or vertically through the zone 3. In one embodiment, the channel 8 is angled downward and away from the reaction storage area 5 toward the test area 2 to allow for the gravity-assisted movement of the reagent through the channel 8. In other embodiments, the channel 8 may be either angled against or perpendicular to the force of gravity. In the case where gravity is not assisting in the transfer of the reagent, the flow rate of the reagent through the channel will have to be sufficient to transfer the reagent from the reagent storage area 5 to the test area 2. This may be accomplished by providing an actuator 11 that exerts sufficient force on the cartridge 6 to force the liquid reagent to move against the force of gravity to the test area 2. Alternatively, the actuator 11 may be designed to not only liberate the liquid reagent from the cartridge, but also to apply pressure on the liquid to force it through the channel 8 into the test area 2. A separate plunger (not shown) may also be provided to assist in the transfer of the liquid reagent from the cartridge 6 to the test area 2.
Further control of the flow rate of the reagent entering the test area 2 can be achieved by adjusting the angle of the channel 8 between the reagent storage area 5 and the test area 2. In addition, altering the path of the channel 8, or the width of the channel 8, can also provide additional control of the flow rate of the reagent entering the test area 2. Finally, controlling pressure by a plunger may also be used to control of flow rates of reagent entering the test area 2.
In a further embodiment, as shown in
Further control of the flow rate of the reagent entering the test area 2 can be achieved by adjusting the shape of the walls of the channel 8. In addition, altering the path of the reaction area or the channel 8, can also provide additional control of the flow rate of the reagent entering the test area 2. In one embodiment, the walls of the channel 8 form a funnel that terminates in an aperture 17 (
As shown in
In an alternate embodiment a matrix 15 can be included in the device shown in
The selection of the material for the matrix 15 is not critical and can be any suitably absorbent, porous or capillary possessing material through which the reagent, in the form of a multifunctional buffer, and resolubilized indicator reagent may be transported by wicking action. The criteria of selection is that the material allow for the resolubilization and mixing of the dried indicator reagent upon addition of the multifunction buffer, as well as initiate the transfer of the buffer and freshly dissolved indicator reagent to the reaction zone 3 of the test area 2.
Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used for the matrix 15 including, but not limited to cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose, fiberglass, cloth, films of polyvinyl chloride, and the like. The material should be chosen for its ability to release the indicator reagent upon reconstituting with the multifunctional buffer. Moreover, the fluid flow through the matrix 15 should be laminar as opposed to turbulent flow characteristics, which adequately allows for initial mixing of the buffer with the indicator reagent.
In an embodiment shown in
The use of indicator reagents to detect the presence of a target analyte in a test sample is well known in the. Examples of such indicators are described in Chan U.S. Pat. No. 7,531,362, the contents of which is herein incorporated by reference. For example, but not limited to, the dried indicator reagent can be in the form of an antibody coupled (i.e. physically linked) to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include, Indo-1, Cascade Blue, AMCA, DAPI, Alexa 350, Hoeschst 33342, PacificBlue, MarinaBlue, eCFP, Cascade Yellow, Propidmm Iodide, Alexa 430, eGFP, FITC, Alexa 488, Phodamine 123, RPE, Acridine Orange, eYFP, PE, DsRed2, Ds-Red, PE-Texas Red, 7-AAD, Per-CP, PE-Cy5, DRAQ5, PE-Cy5.5, PE-Cy7, Alexa 633, To-Pro-3, APC, Cy5, Alexa 647, Alexa 660, Cy5.5, Alexa 680 and APC-Cy7; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
Similar to the cartridge 6, the matrix 15 may be supported by the housing 10 of the device 1, by a separate supporting member (not shown) or a second liquid permeable member 7′. The second liquid permeable member 7′ can have the same or different characteristics as the liquid permeable member 7. The ability to retard the passage of pieces of the cartridge 6 will be of less importance with the second liquid permeable member 7′. In some instances, the reagent entering the matrix 15 must have a certain flow rate to ensure proper resolubilization of the indicator reagent, this flow rate may be different from the flow rate required for proper saturation of the reaction zone 3. Accordingly, the second liquid permeable member 7′ may be provided to either retard or accelerate the flow rate of the reagent containing the dried indicator reagent.
In an embodiment, a single reagent storage area 5 is provided linked to the test area 2 via a channel 8. However, providing multiple reagent storage areas, each linked to the test area via a channel, may allow for greater flexibility in the types of assays that can be run on the device (
Alternatively multiple reagent storage areas as shown in
The flexibility and adaptability of the present device to house one or more reagents useful in a diagnostic assay, as well as the ability to control the rate in which the reagents are delivered to the test area, allows for various diagnostic tests and assays to be run in the device. For example, a single reagent storage area can be provided with the cartridge housing a universal multifunctional buffer, such as described in Chan U.S. Pat. No. 7,531,362. In this case, the matrix described herein above, having a dried indicator reagent, such as colloidal gold conjugated to an antibody capable of recognizing a two-membered complex, is provided.
In this exemplary method, the fluid test sample is applied to the test area, specifically to the reaction zone. The test sample passes downward or vertically through the reaction zone and the target analyte, if present, binds to the capture reagent embedded in the reaction zone. The absorbent zone draws the fluid through the reaction zone, so as to concentrate the two-membered complex formed between the capture reagent and the target analyte on the reaction zone. The multifunctional buffer housed in the cartridge is then liberated by way of the button or flexible membrane, or actuator, and allowed to flow through the liquid permeable member. The universal buffer then enters the matrix to resolubilize the indicator reagent. The universal buffer, containing the indicator reagent, flows through the channel to be deposited on the reaction zone. The universal buffer and indicator reagent flows downwardly or vertically through the reaction zone and into the absorbent zone. The indicator reagent binds to the two-membered complex concentrated in the reaction zone, with any unbound reactants being washed from the reaction zone. The test result is depicted by the presence or absence of a visually detectable signal on the reaction zone.
In an alternate embodiment, the universal buffer housed in the cartridge contains an indicator reagent, thus a separate matrix is not required to supply this component to the assay. In this case, the steps involved in running the assay would be the same as described above, however, the universal buffer would not have to pass through a matrix to introduce the indicator reagent to the reaction zone.
In some instances, it may be advantageous to include one or more washing steps in the assay. If this is the case, the wash solutions may be introduced to the test area manually in a dropwise manner. However, additional reagent storage areas may be provided with each cartridge housing a solution useful in the assay. Compared to the manual addition of the solutions, providing additional reagent storage areas to deliver the additional reagents improves the overall efficiency of the assay and operability of the device.
To facilitate the detection of a target analyte in a sample containing particulate matter, an alternate embodiment of the present invention provides a separation zone 24 capable of receiving and separating the fluid and particulate portions of a sample, such as a whole blood sample where serum is separated from the blood cells (RBC) and other cellular components (
Various methods for the separation of particulate matter from a liquid sample have been described. For example, blood cells can be selectively separated from the fluid portion of blood using separation coatings, erythrocyte aggregating and agglutinating agents, materials having asymmetric pore sizes, polymer-containing matrixes, and multilayer systems, to name a few, e.g. U.S. Pat. No. 3,768,978 to Grubb et al., U.S. Pat. No. 3,902,964 to Greenspan, U.S. Pat. No. 4,477,575 to Vogel et al., U.S. Pat. No. 4,594,372 to Zuk, U.S. Pat. No. 4,753,776 to Hillman et al., U.S. Pat. No. 4,816,224 to Vogel et al., U.S. Pat. No. 4,933,092 to Aunet et al., U.S. Pat. No. 5,055,195 to Trasch et al., U.S. Pat. No. 5,064,541 to Jeng et al., U.S. Pat. No. 5,076,925 to Roesink et al., U.S. Pat. No. 5,118,428 to Sand et al., U.S. Pat. No. 5,118,472 to Tanaka et al., U.S. Pat. No. 5,130,258 to Makino et al., U.S. Pat. No. 5,135,719 to Hillman et al., U.S. Pat. No. 5,209,904 to Forney et al., U.S. Pat. No. 5,212,060 to Maddox et al., U.S. Pat. No. 5,240,862 to Koenhen et al., U.S. Pat. No. 5,262,067 to Wilk et al., U.S. Pat. No. 5,306,623 to Kiser et al., U.S. Pat. No. 5,364,533 to Ogura et al., and U.S. Pat. No. 5,397,479 to Kass et al.
In a preferred embodiment, the separation zone 24 is an elongate or rectangular strip of porous material having intrinsic physical properties which enable it to preferentially and sufficiently entrap or retain the particulate matter in the sample within the separation zone 24. A first end of the separation zone 24, located a short lateral or vertical distance from the reaction zone 3, defines a region for receiving the sample during the first step of the assay protocol, and prior to introduction of the target analyte at the reaction zone 3. A second end of the separation zone 24, in fluid communication with the reaction zone 3, helps to promote the movement of the particulate-free fluid portion of the sample from the first end of the separation zone 24 to the reaction zone 3 for eventual analysis. The separation zone 24 and the reaction zone 3 can either contact one another in order to ensure optimal transfer of the sample from one zone to the other, or be separated by a channel 25, as described above. Accordingly, the materials selected for the separation zone 24 and the reaction zone 2 may overlap slightly with one another in order to ensure adequate migration of the particular-free portion of the whole sample.
A variety of materials can be used for the separation zone 24 such as glass fiber, glass fiber/cellulose mixtures, cellulose, or other proprietary materials, including synthetic materials, e.g., nylon. In some instances, a permeable glass fiber matrix is employed as the blood separation material to facilitate the separation of red blood cells from whole blood. A variety of grades of different thicknesses and absorbencies of glass fiber materials are commercially available to facilitate blood separation and include, for example, GF-24, GF-25, and #33, available from Schleicher & Schuell (Keene, N.H., U.S.A.); G143, G144, and G167, available from Ahistrom (Mount Holly Springs, Pa., U.S.A.); GFQA30VA, GF/P 30, GF/DE 30, GF/SE 30, GF/CM30VA, GF/CM 30, F 075-14, F487-09, GF DVA, GFVA 20, and GD-2, available from Whatman (Fairfield, N.J., U.S.A.).
Useful glass fiber/cellulose mixture materials include F255-07 90 glass/10 cellulose, F255-09 70 glass/30 cellulose, F255-11 50 glass/50 cellulose, and F255-12 50 glass/50 cellulose, available from Whatman.
Useful cellulose materials include 598, available from Schleicher & Schuell. Miscellaneous or other materials falling outside the above categories can also be used, including HemaSep V and Leukosorb; which article of manufacture according to the subject invention available from Pall BioSupport (Port Wash., N.Y., U.S.A.).
One useful nylon material is Nylon 6.6 Transfer Membrane, which is commercially available under the tradename Biodyne B (Pall Specialty Materials, Port Wash., N.Y.). In addition, the material known as “PlasmaSep”, available from Whatman, can be used.
Although the shape and dimensions of the separation zone 24 can be tailored to the particular sample being tested, one embodiment has a narrow rectangular form and dimensions suitable for allowing efficient removal of a substantial amount of red blood cells from the whole blood sample during migration of the fluid portion of the sample from the first end to the second end of the zone 24. Thus, in effect, while a narrow rectangular shape is preferred to channel fluid portion of the blood sample to the reaction zone 24, the dimensions may vary depending on the intrinsic properties (e.g. absorbency, migration rate, etc.) of the material selected for the separation zone 24. In one embodiment, the separation zone 24 is made using the glass fiber material F487-09, available from Whatman, having dimensions between approximately 4 to 7 mm in width, between approximately 10 and 15 mm in length, and between approximately 0.2 mm and 1.0 mm in thickness. More specifically, the separation material is about 7 mm in width by about 10 mm in length and about 0.5 mm in thickness. These dimensions are optimized to be capable of receiving and separating the total volume of a whole blood sample, e.g. two drops of blood.
The separation material typically has a rigid or semi-rigid carrier or backing affixed to its lower surface to provide support and reduce seepage of the particulate-free fluid portion of the sample while it migrates towards the reaction zone. Suitable materials for use as a carrier or backing include, for example, hydrophobic materials such as polycarbonate, polyethylene, Mylar, polypropylene, vinyl, cellophane and polystyrene, etc. as well as water-proofed or fluid-resistant cardboard or similar materials. The carrier or backing may be affixed either directly or indirectly to the separation material by means of a fluid-resistant adhesive. Alternatively, the separation material may be positioned within a cavity within the housing of the device. In this case, the use of a carrier or backing would be optional, as the housing itself would prevent seepage of the particulate-free fluid.
Suitable adhesives are well-known in the art. The carrier may be of any shape and of almost any size which may conveniently be handled. However, the carrier is preferably similar in shape and size to the separation material. Thus, the carrier is preferably formed as an elongate or rectangular strip having a length and width similar to or the same as the separation material.
As shown in
If the device 1 is contained within an apparatus 20, as described above, the reagent storage area 6 can be moved from the actual device 1 and included within structure of the apparatus 20. In this case, it is possible to replace the cartridge 6 without having to replace the whole device 1 or apparatus 20. Moreover, by including the reagent storage area 6 in the structure of the apparatus 20, for example within a well or cavity on the surface of the apparatus 20, allows for automated release of the liquid contained within the cartridge 6 through the use of a plunger or other form of device that would be capable of compressing or breaking the cartridge 6. If the reagent storage area 6 is included in the apparatus 20, instead of the device 1, a channel 21 should be provided to direct the liquid from the cartridge 6 (when liberated from the cartridge 6) to the matrix 15.
In one embodiment, the separation zone 24, as described above, could be moved to the surface of the apparatus 20 or within the apparatus 20, and the liquid portion of the sample transported via a channel 25 to the reaction zone 3.
It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.
This application is a Continuation-in-Part Application of U.S. patent application Ser. No. 13/124,758, filed Jul. 1, 2011, which is the national phase entry of PCT/CA2009/001468 filed Oct. 16, 2009, and claims priority of U.S. Provisional Patent Application No. 61/106,291 filed Oct. 17, 2008. The contents of all these documents are incorporated herein in their entireties for any and all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61106291 | Oct 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13124758 | Jul 2011 | US |
| Child | 14747495 | US |
