FIELD OF THE INVENTION

The present invention relates to apparati and methods for lateral flow determination of analyte presence and concentration in fluid samples.

BACKGROUND OF THE INVENTION

The determination of the quantity of an analyte in a sample is of commercial importance in a broad array of compounds including but not limited to mycotoxins, toxins, drugs, proteins, peptides, oligonucleotides, inorganic compounds, food additives, nutritive compounds or components in bodily fluids such as blood and urine. One method of determination of the qualitative or quantitative presence of these analytes is through the use of immobilized ligands where the ligands include, but are not limited to, antibodies, enzymes and oligonucleotides. A particular application involves the use of lateral flow devices where the ligand is immobilized on a strip and the sample exposed to the immobilized ligand as a result of the lateral flow of the sample solution over the strip and through the area containing the immobilized ligand. To our knowledge, to date strip based tests have not previously incorporated a platform that enables the analysis of multiple concurrent strips all within the same uniform apparatus. All existing commercially available strip tests are based on a stand alone strip format, with the test strip either being encased in a single strip holding device, or simply as a free test strip.

There is a need for an apparatus capable of analysing multiple test strips in a single platform thereby providing high throughput analysis of a single analytes in a sample solution, or different analytes within one or more sample solutions. Using cellulose as a wicking material provides clear performance and cost advantages over other approaches known in the art. The use of cellulose as a wicking material in lateral flow devices, however, has not been successfully enabled for use with DNA based ligands. All previous disclosures in this area have involved the step of conjugating or associating DNA ligands with immobilized proteins on nitrocellulose membranes. Others trained in the art have discussed potential means of using aptamers in diagnostic assays but no one else has previously reduced it to practice. By far the majority of attempts to base diagnostic tests on DNA or RNA based ligands have involved the use of gold nano-particles (Tai-Chia Chiu et al., Sensors, 9 10356-10388). These applications require the aptamer to flow through a strip and thus do not enable the concentration of the analyte by an immobilized aptamer. J. Liu et al., (Angew Chem Int Ed Engl. 2006 Dec 4;45(47):7955-9) discuss immobilized aptamers as diagnostics, however the authors limit their overview to the conjugation of an aptamer to immobilized streptavidin through the use of a biotin label on the aptamer. The complex between the aptamer and the protein has the potential to reduce the binding affinity of the aptamer as well as increase the cost and complexity of the system. Protein immobilizations are performed on nitrocellulose surfaces rather than cellulosic ones.

Boese and Breaker (Nucleic Acids Res. 2007 October; 35(19): 6378-6388) have identified aptamers that bind apparently specifically to cellulose. They successfully developed a hybrid aptamer between their cellulose aptamer and an aptamer for adenosine triphosphate (ATP). They limited their demonstration of this technology to applications involving allosteric modifications to the aptamer as a function of binding to ATP in terms of its' ability to bind to cellulose. This demonstration does not envision the immobilization of such a hybrid aptamer to a cellulose surface in order to concentrate the analyte. Nor do the authors contemplate a lateral flow application.

Thus, there is also a need for an apparatus capable of analysis of analytes using cellulose as a wicking material.

There is also a need for a high throughput, lateral flow analysis apparatus capable of being analysed by existing analytical instruments currently used for reading signals from existing microtitre plates, such as microtitre plate fluorometers.

SUMMARY OF THE INVENTION

In one embodiment the present invention provides for an apparatus for determining the presence or the concentration of an analyte of interest in a sample solution characterized in that said apparatus comprises: a row of sample wells, each well configured for holding an concentration of the sample solution, and a platform supporting a plurality of lateral flow strips and absorbent pads, each one of said lateral flow strips having a receiving end for contacting the sample solution in one sample well, another end connected to an absorbent pad, and a capture Iigand immobilized at a specific location on the lateral flow strips, said capture Iigand being capable of binding the analyte of interest in the sample solution, wherein said binding is used for determining the presence or the concentration of the analyte in the sample.

In one aspect of the present invention, the apparatus for detection of analytes in sample solutions is configured to have the dimensions of a standard microtitre plate or dimensions substantially similar to those of standard microtitre plate. In further aspects of the present invention, the specific locations in each lateral flow strip corresponds to defined well positions within a standard microtitre plate format.

In another embodiment the present invention provides for a method for determining the presence or concentration of an analyte of interest in o ne or more sample solutions, characterized in that the method comprises the following steps: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone having a capture Iigand immobilized in a specific location within the capture zone, said capture Iigand being capable of binding the analyte of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; (c) allowing the one or more sample solutions to flow through the immobilized capture Iigand in the plurality of lateral flow strips, thereby concentrating and purifying the analyte of interest; (d) obtaining a measurable response from the specific location as a result of the capture Iigand binding to the analyte of interest, wherein said measurable response can be correlated to the presence or concentration of analyte of interest in the one or more samples.

In another embodiment the present invention provides for a method for determining the presence or amount of two or more analytes of interest in one or more sample solutions, characterized in that said method comprises, (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone having two or more capture ligands immobilized in a specific location within the capture zone, each of the two or more capture ligands being capable of binding one of the two or more analytes of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; (c) allowing the one or more sample solutions to flow through the immobilized capture ligands, thereby concentrating and purifying the two or more analytes of interest; and (d) obtaining a measurable response from the specific location as a result of the two or more capture ligands binding to the two or more analytes of interest, wherein said measurable response can be correlated to the presence or amount of the two or more analytes of interest in the one or more samples. In another embodiment the present invention provides for a method for determining the concentration of one or more analytes of interest in one or more sample solutions, characterized in that said method comprises: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, a test zone having at least one test capture iigand capable of binding to the one or more analytes of interest, and a control zone having a control capture Iigand capable of binding a control compound; (b) adding a known amount of the control compound to the one or more sample solutions; (c) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions, thereby concentrating and purifying the one or more analytes of interest; (d) allowing the one or more sample solutions to flow through the reading zone and the control zone; and (e) obtaining a measurable response from the test zone and the control zone as a result of the test capture Iigand and the control capture Iigand binding to the one or more analytes of interest and the control compound respectively, wherein said measurable response in the test zone can be correlated to the measurable response in the control zone to determine the concentration of the one or more target analytes in the one or more sample solutions.

In another embodiment the present invention provides for a method for concentrating and purifying an analyte of interest characterized in that said method comprises: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone a capture Iigand immobilized in a specific location within the capture zone, the capture ligands being capable of binding to analyte of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; and (c) allowing the one or more sample solutions to flow through the immobilized capture ligands, wherein upon binding of said analyte of interest to said capture ligand, said analyte of interest is concentrated and purified at the specific location.

BRIEF DESCRIPTION OF DRAWINGS

A brief description of one or more embodiments is provided herein by way of example only and with reference to the following drawings, in which: Figure 1 (a) illustrates a side view of an apparatus for determining the presence or the amount of an analyte in a sample solution according to one embodiment of the present invention.

Figure 1 (b) illustrates a top view of an apparatus for determining the presence or the amount of an analyte in a sample solution according to one embodiment of the present invention.

Figure 2 illustrates a lateral flow strip assembly comprising a carrier strip and an absorbent pad in accordance with one aspect of the present invention.

Figure 3 illustrates a schematic side view of an apparatus for determining the presence or the amount of an analyte in a sample solution made from a 384 microwell backplate according to one embodiment of the present invention.

Figure 4 illustrates a top view of a substrate supporting a plurality of lateral flow strip assemblied in accordance with one embodiment of the present invention.

Figure 5 (a) illustrates a top view of a housing comprising a row of sample wells and a platform for receiving a substrate supporting a plurality of lateral flow strip assemblies in accordance with one embodiment of the present invention.

Figure 5 (b) illustrates a cross section corresponding to the dashed line B-B shown in Figure 5 (a) detailing the sample well region of the housing of Figure 5 (a). Figure 6 (a) illustrates a lateral flow strip having microtitre plate well definitions (A-E) in accordance with one embodiment of the present invention.

Figure 6 (b) illustrates a lateral flow strip having a 96 well microtitre plate definition. Reference 60 represents the positioning of DNA ligand in a specific location on the lateral flow strip corresponding to well C of a 96 well microtitre plate in accordance with one embodiment of the present invention.

Figure 7 is a mapping of applied DNA ligand on a lateral flow strip in accordance with one embodiment of the present invention.

Figure 8 are graphs illustrating ochratoxin A concentrations in a buffer solution using the multistrip, lateral flow apparatus in accordance with one embodiment of the present invention. Figure 8 (a) illustrates the regression of relative fluorescence units measured against known ochratoxin A levels. Figure 8{b) is a bar graph illustrating predicted ochratoxin A concentration measured (parts per billion, "ppb") and known ochratoxin A concentration (ppb). Figure 9 illustrates is a bar graph showing the predicted ochratoxin A concentration (ppb) in wine using an apparatus in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

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. Also, unless indicated otherwise, except within the claims, the use of "or" includes "and" and vice-versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (for example "including", "having" and "comprising" typically indicate "including without limitation"). Singular forms including in the claims such as "a", "an" and "the" include the plural reference unless expressly stated otherwise.

Definitions

"Lateral flow strip assembly" refers to a lateral flow strip physically connected at one end to an absorbent pad, as illustrated in Figure 2. "Microplate readers" refers to instruments designed to detect biological, chemical or physical events of samples in microtitre plates. Common microplate readers include fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization microplate readers. Overview

The innovation disclosed herein provides for apparati whereby multiple solid carrier strips may be held by a single platform. The apparati according to different embodiments of the present invention enable high throughput analytical methods whereby samples may be added to a plurality of sample wells in the apparati, each sample well being configured for receiving an end of one individual solid carrier strip. The sample solutions in each well may be allowed to flow through the strips simultaneously and all strips may then be analyzed for the presence and/or concentration of one or more analytes present in each sample concurrently. Alternatively, a subset of the strips down to one strip at a time may be processed in the same apparatus. It would be clear to one trained in the art that this approach provides higher throughput capacity for analysis, while at the same time decreasing experimental error. It would also be clear to one trained in the art that the apparati and methods of the present invention would be broadly applicable to all analytes/ligand interactions. Apparatus

Referring to Figure 1 , an apparatus 1 is described in accordance with one embodiment of the present invention. The apparatus 1 may comprises of a platform 10 having a supporting side 11 that may support a plurality of solid carrier strips 25 for lateral flow analysis of fluid samples 35 that may be held in sample wells 30. The apparatus 1 may be square or rectangular and having a front side 12, a back side 14 and two lateral sides 3. A line or row 32 of sample wells 30 capable of holding the fluid samples 35 may be aligned along the front side 12 of the apparatus 1. The sides 12, 13, 14 of the apparatus 1 may be raised above the supporting side 11 of the platform 10. A plurality of lateral flow strips 25, such as chromatography paper strips, may be disposed on the supporting side 11 of the platform 10 from the front side 12 to the back side 14 of the apparatus 1. One end of the lateral flow strips (the receiving end) 26 may extend into and be immersed in one sample weli 30 holding a sample 35 to be analyzed, with the other end of the strip (the back or opposite end) 27 in physical contact with an absorbent pad 28 or other means that may be used to accelerate the flow of the sample through the lateral flow strips 25. A capture ligand 29 may be immobilized in a specific location or capture zone on the lateral flow strips 25. The capture ligand should be capable of binding the analyte of interest in the sample thereby forming a ligand/analyte complex. In one aspect of the present invention, one or more capture ligands may be immobilized in the specific location such that each capture ligand is capable of binding a different analyte of interest. Figure 2 illustrates a lateral flow strip assembly in accordance with one embodiment of the present invention. Referring to Figure 2, a lateral flow assembly comprises a lateral flow strip 25c connected to an absorbent pad 28c. The lines shown on the lateral flow strip 25c marked as 17 and 17b refer to points or lines where the strip 25c may be folded at a 90° angle. Point 17b corresponds to a fold downwards to enter the sample well, and fold 17 refers to a fold back onto a plane parallel to that of the majority of the lateral flow strip 25c to conform to the bottom of the sample well. The portion of the lateral flow strip that extends across the bottom of the well is referred to as the toe.

The lateral flow strips that may be used with the apparatus of the present invention include cellulose, nitrocellulose and/or nylon lateral flow strips. In one aspect of the present invention, the lateral flow strip is a chromatography paper strip. However, other lateral flow strips known in the art may also be used with the apparatus of the present invention. Whatman #4 Chromatography Paper may be suitable for the lateral flow strips. These strips may be cut to a width of 6 mm and a length of 4.4cm for use in the apparati of the present invention.

To accelerate the flow of solutions across the lateral flow strips, accelerating means such as absorbent pads may be affixed to the stop end of the lateral flow strip. An enablement of this invention may involve the use of a Cellulose Fibre Pad (Millipore) as the absorbent pad. The absorbent pad may have a length of about 3.5 cm, a width of about 6 mm. As illustrated in Figure 2 one enablement of this invention may be to establish an overlap 26c between the lateral flow strip 25c and the absorbent pad 28c (for example an overlap of about 2 mm). The capture ligand may include oligonucleotides, antibodies, and/or enzymes and/or combinations of these chemical moieties. One enablement of this invention may include the use of immobilized capture ligands on the strip that are capable of binding to mycotoxins, toxins, drugs, proteins, peptides, oligonucleotides, inorganic compounds, food additives, nutritive compounds, and/or components of bodily fluids such as blood or urine.

In one embodiment of the present invention, a plurality of lateral flow assemblies may be attached directly to the platform of the apparatus. In one aspect of the present invention an adhesive film that has adhesive on both sides may be used to attach the lateral flow strip assemblies to a backing card, which may be composed of plastic. The lateral flow strips extend beyond the card and may be folded to fit the conformation of the loading wells. The backing may be attached directly to the platform of the apparatus through the use of two-sided tape, or double sided adhesive film, or through the use of a liquid adhesive. One enablement of this apparatus may be based on existing microtitre dishes, such as those including 96, 384 and 1536 well formats. Figure 3 illustrates a schematic drawing of a multiple lateral flow strip apparatus made from a 384-microwell back plate 32. A channel between the wells A to H within the microtitre plate 32 may be created by lowering the wall between the wells A to H such that a strip 25b may be placed over the wells but below the top surface 34 of the microtitre dish 32. Also shown in Figure 3 are sample solution 35 capture ligand 50b and absorbing pad 40b. It is also possible to lay the strips on top of the plate without reducing the height of the intravening walls.

In another embodiment, illustrated in Figures 4 and 5, the apparatus for lateral flow analysis of the present invention may comprise an assembly comprising a backing or substrate 40 for supporting a plurality of lateral flow strip assemblies and a housing 50. The backing 40 may be comprised of a supporting side 42, a front end portion 44 having a front edge 48, and a back end portion 46. A plurality of lateral flow solid strip carriers 41 and absorbent pads 47 may be disposed on the supporting side 42 in a substantially parallel arrangement therein. Each lateral flow strip carrier 41 may be comprised a front or sample receiving end 43 and a back or opposite end 45. The sample receiving end 43 may be disposed in the proximity of the front edge 48 of the substrate 40. In one aspect of the present invention illustrated in Figure 4, the sample receiving end 43 may extend beyond the front edge 48 of the substrate 40.

Absorbent pads 47 or other means for accelerating the flow of the sample solution along the strip carrier 41 may be connected to the back end 45 of the plurality of solid strip carriers 41. The absorbent pads 47 may be disposed on the supporting side 42. In one aspect of the present invention, the lateral flow carrier strips 41 and the absorbent pads 47 may overlap 39.

In one aspect of the present invention, the supporting side of the substrate may be adhesive. The lateral flow strips and absorbent pads may held in place by affixing them to the adhesive supporting side of the substrate. One enablement for such an adhesive substrate would involve the use of super white polystyrene (0.01" thick) laminated on one side with GL-187™ acrylic pressure sensitive adhesive. Multiple strips may then be affixed to one adhesive substrate as shown in Figure 4.

In one aspect of the present invention, the receiving end 43 of the lateral flow strips may be allowed to extend beyond the front edge 48 of the substrate 40, for example the receiving end may extend by about 10-20 mm beyond the front edge 48. This receiving end portion 43 of the lateral flow strip may be folded, for example at about a 90° angle twice thereby creating folds 49a, 49b.

With reference to Figures 4 and 5, in one aspect of the present invention, the substrate may be disposed substantially within a housing and retained within the housing. Figure 5 illustrates a housing 50 which may comprise of a platform 52 and a row of sample wells 54 which may be aligned at one end of the platform 52 The platform 52 may be configured for receiving the substrate 40 supporting the plurality of lateral flow assemblies such that sample receiving end 43 of the each lateral flow carrier strip 41 may be in fluid contact with one loading well 54. The extension folds 49a, 49b of the lateral flow strips 41 previously mentioned, allow the lateral flow strips 41 to follow the contours of the loading well 54 The extensions 49a, 49b of the lateral flow strips 41 may be aligned down the side of the sample wells 54 adjacent to the platform 52, and along the floor of the loading well. This latter aspect is referred to as the toe of the strip. The presence of the toe increases the surface area of strip exposed to substrate while helping to maintain the position of the strip within the sample well.

One advantage of the apparati of the present invention may be that the lateral flow strip assemblies may be aligned with predefined microtitre plate well definitions and as such be readily read by a microtitre plate fluorescent reader or any other type of microplate reader.

In operation, a fluid or liquid sample may be loaded in each loading well of an apparatus of the present invention and contacting the receiving end of the lateral flow strip carriers disposed on the platform. Through capillary action, the fluid samples may wick through the solid strips towards the opposite stop end. Thus, the fluid samples may be brought into contact with the capture ligand that is immobilized in a specific location on the lateral flow strips. The presence or amount of an analyte in the samples may be determined in each sample concurrently. Alternatively, a subset of the strips down to one strip at a time may be processed in the same apparatus. The presence or amount of the analyte of interest in the fluid samples may be measured using instruments capable of providing a measurable response as a result of the capture ligand binding to the analyte of interest at the specific location where the capture ligand is immobilized. The measurable response may then be correlated to the presence or amount of analyte of interest in the samples. If necessary, a signalling means, such as a colorimetric or fluorescent dye, may be added to the strip at the point where the capture ligand may be immobilized. The colorimetric or fluorescent intensity of the dye may be then evaluated with an appropriate instrument such as a fluorometer. The concentration of the analyte of interest in the samples may be determined by correlating the measured response to the amount of analyte in the sample fluid.

In one aspect of the present invention, the specific location may correspond to a determined position within a standard microtitre plate well definition. Having the specific location correspond to a position within a standard microtitre plate well definition enables the use of mircoplate readers as the instruments to measure the capture ligand binding to the analyte. In one embodiment of the present invention, the amount of one or more analytes of interest in multiple sample solutions may be determined by comparing a measurable response of the analyte of interest to the measurable response of a known control included in the solutions. This embodiment has the advantage of facilitating analysis of control compounds inherent to an individual sample solution within the same sample solution for which the target analyte is being analyzed.

The Capture Ligand

It would be clear to one trained in the art that a number of suitable capture ligands may be used with the multistrip platform of the present invention, including antibodies and DNA ligands.

In one aspect of the present invention the capture ligand may be a DNA ligand. One enablement of this invention may be to apply a DNA ligand to a specific location on the lateral flow strip. The lateral flow strips of the apparati of the present invention may be defined to correspond to standard microtitre plate well definitions, as illustrated in Figure 6(a). The DNA ligand, or any other suitable capture ligand, may be placed, for example, in the centre of a position 60 that would correspond to the row of wells known as C in a microtitre plate format, as illustrated in Figure 6 (b). It would be clear to one trained in the art that the amount of DNA ligand used may vary depending on the nature of that ligands' binding capacity to the target molecule or analyte and the range of concentrations that are desirable to measure. One enablement of the invention may be to apply about 120 pmoles of DNA ligand in a volume of about 200 nl at this point.

The position of the DNA ligand on the lateral flow strip may be verified through the addition of a signalling means. In one embodiment of the present invention a fluorescent lanthalide like terbium may be added to the strip at the specific location where the DNA ligand was placed and the fluorescence of this lanthalide may then be measured. For example if the lanthalide is terbium then the excitation of 280 nm, and an emission of 545 nm may be used. At these setting the light energy may be exciting the DNA directly and emitted as fluorescence by the terbium. With the use of certain microplate reading fluorometers such as the B G Omega, it may be possible to measure the fluorescence of the DNA ligand on the strip, for example using time resolved fluorescence with a lag time of about 70 isec, and a total integration time of about 1 ,000 to 2,500 psec. It may also be possible to map a 30 x 30 grid of spots within the area in which the DNA ligand has been loaded and thereby derive a three-dimensional map of the distribution of the DNA ligand on the spot in question (see Figure 7).

It would be clear to one trained in the art that this method of measuring the application of the DNA ligand on the capture zone would be useful as a means of assessing the consistency of ligand application to the strips. It would also be clear to one trained in the art that Figure 7 illustrates that the DNA ligand applied to the strip in this manner taught herein is distributed in a regularly shaped peak.

In general it has been found that DNA ligands do not bind with as high a level of affinity for their target analytes as comparative antibodies do. As such the incorporation of DNA ligands in a lateral flow device as provided in this invention is of particular value as the apparati of this invention allow for the concentration of analyte at the detection point. A significant advantage of this invention is that the DNA ligand may be applied in a small surface area within a specific location of the lateral flow strip, leading to a high level of concentration of the ligand/analyte interaction. This may enable an increase in the signal to noise ratio and the high levels of sensitivity observed in the examples provided herein. The focusing of the ligand/analyte binding area is also enabled through the use of focused fluorescence measurement on the spot in question. The ability of existing fluorometers to map fluorscent signals within a circle area adds to the sensitivity observed herein.

Increasing DNA Ligand Binding Activity

The inventors have demonstrated that DNA ligands may be immobilized onto paper (cellulose) surfaces without a need for conjugation. It is presumed that the charge interaction between the DNA ligand and the glucose molecules within the paper may be sufficient to immobilize the DNA ligand on the lateral flow strip. Given that this process of immobilization would largely be a random process and as such the region of a portion of any given DNA ligand that may be useful for binding to a corresponding target could be involved in binding to cellulose the inventors have developed two preferred enablements. One enablement of the present invention may be to increase the probability of maintaining binding activity of the DNA ligand by increasing the copy number of binding sites or binding domains within the DNA ligand. The inventors discovered the binding activity of the DNA ligand for its target analyte may be increased by simply repeating the DNA ligand sequence in a contiguous copy.

Another enablement of the present invention may be to increase the probability of maintaining binding activity of the DNA ligand by providing a DNA ligand that includes a binding domain for the target analyte and a sufficient additional portion to enable a specific binding to the cellulose of the lateral flow strip. Methods of Using the Apparati of the Present Invention

The present invention also comprises methods of determining the presence or concentration of analytes of interest in sample solutions.

In one embodiment, a method is provided for determining the presence or concentration of analytes of interest in sample solutions. A method for determining the presence or concentration of an analyte of interest in sample solutions may comprise the steps of: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone having a capture ligand immobilized in a specific Iocation within the capture zone, said capture ligand being capable of binding the analyte of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; (c) allowing the one or more sample solutions to flow through the immobilized capture ligand in the plurality of lateral flow strips; (d) obtaining a measurable response from the specific iocation as a result of the capture ligand binding to the analyte of interest, wherein said measurable response can be correlated to the presence or concentration of analyte of interest in the one or more samples.

Any of the apparati of the present invention may be used in the methods provided herein. The specific Iocation may, for example, correspond to a determined position within a standard microtitre plate well definition. Having the specific location correspond to a position within a standard microtitre plate well definition enables the use of mircoplate readers as the instruments to measure the capture ligand binding to the analyte.

One or more sample solutions may be analysed simultaneously. The sample solutions may be obtained from one source or from different sources. In another embodiment of the methods of the present invention, the presence and concentration of two or more analytes of interest may be analysed. In this embodiment the method may comprise: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone having two or more capture ligands immobilized in a specific location within the capture zone, each of the two or more capture ligands being capable of binding one of the two or more analytes of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; (c) allowing the one or more sample solutions to flow through the immobilized capture ligands; and (d) obtaining a measurable response from the specific location as a result of the two or more capture ligands binding to the two or more analytes of interest, wherein said measurable response can be correlated to the presence or amount of the two or more analytes of interest in the one or more samples.

In another embodiment, the present invention may provide for a method for determining the concentration of one or more analytes of interest in one or more sample solutions, characterized in that said method comprises, (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, a test zone having at least one test capture ligand capable of binding to the one or more analytes of interest, and a control zone having a control capture ligand capable of binding a control compound; (b) adding a known amount of the control compound to the one or more sample solutions (c) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; (d) allowing the one or more sample solutions to flow through the reading zone and the control zone; and (e) obtaining a measurable response from the test zone and the control zone as a result of the test capture ligand and the control capture ligand binding to the one or more analytes of interest and the control compound respectively, wherein said measurable response in the test zone can be correlated to the measurable response in the control zone to determine the concentration of the one or more target analytes in the one or more sample solutions. This embodiment of the present invention has the advantage of facilitating analysis of control compounds inherent to an individual sample solution within the same sample solution for which the target analyte(s) is/are being analyzed.

Allowing the sample solutions to flow through the lateral flow strips may result in concentrating and purifying the analyte(s) of interest at the specific location where the capture ligand(s) is/are immobilized, thereby providing for a method to concentrate the analyte(s) of interest at a specific location for subsequent tests. In this embodiment of the invention the method for concentrating and purifying an analyte of interest may comprise: (a) providing an apparatus comprising a plurality of lateral flow strips, each lateral flow strip including a receiving end, a back end in contact with an absorbent pad, and a capture zone, said capture zone a capture ligand immobilized in a specific location within the capture zone, the capture ligands being capable of binding to analyte of interest; (b) contacting each receiving end of the plurality of lateral flow strips with one of the one or more sample solutions; and (c) allowing the one or more sample solutions to flow through the immobilized capture ligands, wherein upon binding of said analyte of interest to said capture ligand, said analyte of interest is concentrated and purified at the specific location.

Advantages of the Present Invention

The present invention provides several novel embodiments and aspects that are advantageous over known lateral flow detection devices available in the art, including:

(1) The use of cellulose as a wicking material has not been successfully enabled for use with DNA based ligands previously. All previous disclosures in this area have involved the step of conjugating or associating DNA ligands with immobilized proteins on nitrocellulose membranes. The direct immobilization of DNA onto cellulose provides clear performance and cost advantages over approaches known in the art.

(2) The immobilization of the DNA ligand within an apparatus such that the point of immobilization is oriented within the existing reading matrix of existing microtitre plate fluorometers enables a high level of sensitivity without a need to develop dedicated equipment for this application. The focusing of detection within a small physical area that is oriented with the existing reading capacity of time resolved fluorometers would be realized by one trained in the art as a substantial advance in terms of reducing cost and increasing sensitivity over existing technology.

(3) The combination of a lateral flow strip, the apparatus of this invention, and time resolved fluorometery provides a basis for quantitative determination of anaiyte concentration at levels that were only previously achievable with the combination of immuno-affinity columns and fluorescence HPLC analysis. It would be clear to one trained in the art that this invention provides a substantial improvement over such existing technology.

(4) The combination of multiple strips within one format increases the reliability of analysis by decreasing potential for experimental error. It would be clear to one trained in the art that there would implicitly be lower variation among samples within a test kit, as the material used to assemble each kit would be derived from the same lot, whereas kits that provide individual tests have the potential to be derived from different lots of material. The multi-strip format of this invention provides a more valid basis for comparison between unknown samples and samples of known concentration (controls) as these comparisons are performed within the same experimental apparatus, simultaneously.

It may be possible to use the injection capacity of existing fluroremeters to provide the application of terbium solution required for fluorescence enhancement in certain of the enablements listed herein. The inclusion of such an application would provide more automation over the methods, decrease potential for operator error, and maintain more consistent timing between the application of terbium and the measurement of the sample.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES

Example 1 : Determination of Ochratoxin A (OTA) concentration in a sample

One enablement of the detection of a target molecule with the multistrip, lateral flow apparati described within this invention is the use of the DNA ligand specific for OTA for the determination of OTA concentrations in sample matrices. 120 pmoles of OTA 1.12.2 an OTA DNA ligand described in WO/2009/086621 , the contents of which are incorporated herein by reference, was immobilized at specific locations on the strips. 200 μΙ of solutions containing known concentrations O TA were loaded into the loading wells of the apparatus and allowed to wick across the lateral flow strips and pass through the immobilized DNA ligand. A replication of each OTA concentration was performed. The absorbent pads were removed and the strips dried with a blow dryer. Following drying a 0.5 μΙ aliquot of a 10 mM terbium chloride solution was added to the specific locations on the strips and the strips were immediately measured in a microtitre plate fluorescent reader. Fluorescence readings were performed with a lag time of 70 sec, an integration of 2,000 psec, with 25 flashes at an excitation wavelength of 370 nm and an emission of 540 nm. This was measured on a Tecan Infinity fluorometer (Figure 8 (a)). The regression of the observed differences in relative fluorescence units measured against the known OTA levels in the samples was significant (r ² = 0.9956). This regression was used with the individual experiments to determine predicted OTA concentrations and standard deviations of these predictions based on the use of the multistrip platform (Figure 8(b)). It would be clear to one trained in the art that this multistrip format can be used effectively to determine the concentration of an analyte in a sample solution with parts per billion (ppb) sensitivity. An alternative enablement involves the extension of this measurement to include well mapping in a 30 x 30 grid. This latter enablement allows for clarification of data that may be due to contaminants in the sample matrix through a variety of means described in more detail in subsequent examples.

Example 2: Use of the apparatus for the determination of OTA content in grain OTA was extracted from grain samples using a method recommended by the Canadian Grain Commission which is based on the method of Langseth et al. _t J. Chromatography, 478, (1989), 269-274.

A 30 g sample of grain is combined with 5 g of Celite, 25 ml of 0.1 M H3PO ₄ and 150 ml of chloroform. This was shaken on a flatbed shaker at 280 excursions per minute for 45 min. After this extraction step, the solutions were centrifuged at 40C at 3,000 rpm for 10 min. The organic phase of the biphasic solution recovered from the centrifugation was filtered a filter paper. 25 ml of this filtrate was collected and washed three times with an excess volume of chloroform. This extract was concentrated to near dryness on a rotary evaporator with the water bath maintained at 50°C, and the cooling bath at 5°C. The products were redissolved in 6 ml dichloromethane and added to Silica Sep-Pak columns. Sep-Pak columns were conditioned by exposure to 5ml hexane followed by 5 ml of dichloromethane. The samples were added to the Sep-Pak at a drop wise rate. The Sep-Pak columns were then rinsed with 0 ml of dichloromethane, then 10 ml of hexane, and finally with 10 ml of toluene. The OTA bound by the Sep-Pak column was eluted with the addition of 12 ml of a 9:1 mixture of toluene and acetic acid. Eluted samples were dried under nitrogen gas at 50°C. Dried samples were redissolved in 1 ml acetonitrile with vortexing for one min. At this point a further 1 ml of 0.5% acetic acid was added and the solutions were sonicated for 15 min. Samples were then centrifuged at 3000 rpm at 40C for 10 min. The supernatant was filtered.

Samples were split at this point for analysis on the apparatus described within this invention ("OTA Sense" in Table 1 ) and for analysis via fluorescence based high performance liquid chromatography (HPLC). HPLC analysis was performed with a C18 column at a temperature of 30°C, at a flow rate of 0.9 ml/min with a gradient from 30% acetonitrile to 60% acetonitrile versus water. The ochratoxin A peak is measured with an excitation of 310 nm, and an emission of 470 nm.

Multistrip analysis platforms were prepared as described in Example 1 (?) with 120 pmoles of DNA ligand OTA 1.12.2 loaded in the middle of what be the C row of wells in a microtitre plate. An aliquot of 100 μΙ of each sample was combined with 100 μΙ of Universal Grain Buffer (UGB) (15 mM Tris, 150 mM Hepes, 120 mM NaCI, 5 mM KCI, and 7 mM CaCI ₂ ). These solutions were combined in the loading well of the multistrip apparatuse, with the sample solution being added first. Wicking of solutions occurs immediately. The solutions were allowed to wick at room temperature across the strips and onto the absorbent pads until the wells were seen to be empty. At this point a further five minutes was allowed to ensure that the solution on the strips migrated to the absorbent pads. The absorbent pads were then removed with a pair of forceps, and the strips were dried with a blow dryer for five minutes.

Immediately before each strip was measured a 0.5 μΙ aliquot of a 5 mM TbCI solution was added onto the position where the aptamer was loaded. Fluorescence of the strips was then immediately measured with an excitation wavelength of 380 nm, and an emission wavelength of 545 nm on a Tecan Infinity fluorescent microplate reader. A lag period of 70 sec was imposed between excitation and emission, with a total integration time of 1 ,500 psec. 25 flashes were used for each measurement. These values were compared to values obtained with standard OTA concentrations of 0, 2 and 5 ppb. The results obtained with the multistrip platform were sufficiently similar to the results obtained by HPLC analysis to confirm the efficacy of this test method.

Table 1 : Comparison of performance of multistrip platform with HPLC analysis

OTA-

Sample HPLC Sense

1 5.4 5.63

2 2.8 3.02

3 <1.0 <1

4 <1.0 <1

5 <1 .0 <1

6 1.1 <1

7 <1.0 1.22

8 1.2 <1

9 <1.0 <1

10 2.3 1.54

1 1 1.8 2.07

12 4 2.49

13 1.1 0.97

14 <1.0 <1

15 <1.0 <1

16 <1.0 <1 It would be clear to one trained in the art that the use of the apparatus described in this invention has been demonstrated to perform with equal efficacy to accepted approved methods for the detection of the target in question.

Example 3: Use of the apparatus for the determination of ochratoxin A content in wine

The Liquor Control Board of Ontario confirmed that no Ontario wine available for sale has an ochratoxin concentration above 1 ppb. To this end we purchased a sample of French Cross wine, a red Ontario wine as an OTA standard with a concentration less than 1 ppb. We added OTA to a sample of this wine to achieve a final concentration of 5 ppb. An equal volume of this sample, and a sample that was not spiked with ochratoxin A were combined with an equal volume of Loading Buffer (Tris 165mM pH 7.5, NaCI 120mM, 500uM CaCI2). 200 μΐ of this combined solution was loaded in replicate in separate loading wells on the multistrip apparatus in replicate. These samples were processed as described in Example 2 except that 0.5 μΙ of a 10 mM terbium solution was added to the immobilized ligand for fluorescence detection.

Strips were excited at a wavelength of 280 nm and an emission of 545 on a BMG Fluostar Omega with 25 flashes, 70 sec lag time, 2,000 psec integration time, and a GAIN of 2,000. We!ls were mapped in a 30 x 30 grid over a 0.6 mm circle. The area under the peaks for each sample was determined and the averages regressed against the known OTA values. This regression was then used to estimate the predicted OTA concentrations in each replicate. The result is shown in Figure 9. The standard deviations obtained with this analysis were +/- 0.1 ppb for 0 ppb OTA, and +/- 0.5 for 5 ppb OTA. It would be clear to one trained in the art that this level of variation in estimates provides a sound basis for determining OTA concentration with confidence down to levels of 1 ppb.