The present application claims the benefit of priority to Patent Application Serial No. 1908/CHE/2010 filed in India on July 5, 2010.

TECHNICAL FIELD [0001] The invention relates generally to low cost diagnostic devices and more specifically to diagnostic devices made of interweaving of two or more hydrophilic threads.

BACKGROUND

[0002] The detection of analytes including proteins, DNA/RNA and metabolites from body fluids and other samples of biological origin is essential for a variety of applications including medical testing, toxin detection and forensic analysis. Improved, point-of-care testing of such analytes is an urgent worldwide requirement (Yager, P.; Domingo, G. J.; Gerdes, J., Point-of-care diagnostics for global health. Annu Rev Biomed Eng 2008, 10, 107- 44). Most current diagnostic devices are inaccessible to developing and least developed economies. Further, typical assays require large and expensive laboratory instruments that are operated by trained personnel. At present, the only readily available assay format is the so-called "dipstick" technologies, which has increased the accessibility of many assays. However, the reliability and accuracy of these assays are still a concern in the form of false positives or false negatives. There is therefore a large unmet need for the development of systems that are low-cost, portable, convenient to handle and show high efficiency towards detection. These systems should also be capable of rapidly identifying a broad range of analytes from samples of biological origin.

[0003] Paper-based lateral flow immunoassays (LFIs) have been hugely successful in the market place with a variety of rapid tests such as home pregnancy tests being widely available. Visual readouts in the form of a color change are used for detection while sample flow occurs automatically through capillary action. Further, mature manufacturing processes are already available for such devices. However, LFIs come with a set of disadvantages too. They are not very reliable and do not provide for the ability to perform multiplex tests. One of the reasons for this is the lack of an ability to define a 'flow-path' in a paper based device (Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M., Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew Chem Int Ed Engl 2007, 46 (8), 1318-20). This study advanced such technology by patterning paper into selectively hydrophilic and hydrophobic portions. Thus, a patterned flow field can therefore be defined. However, paper-based devices still have some problems like the lack of mechanical stability and the absence of low-cost manufacturing methods that can deposit multiple reagents without heat treatment or exposure to high stress.

[0004] In a more recent development, cotton thread has been explored as a medium for microfluidic chip fabrication (Li, X.; Tian, J.; Shen, W., Thread as a Versatile Material for Low-Cost Microfluidic Diagnostics. ACS Applied Materials & Interfaces 2009, 2 (1), 1-6). Experiments were performed on single cotton threads or cotton threads that have been sewed onto a plastic substrate and color change based readouts were used to detect the presence of a reagent. Safavieh also provides cotton based microfluidic chips to be used as diagnostic devices (Roozbeh Safavieh, Maryam Mirzaei, Mohammad A. Qasaimeh, and David Juncker, Yarn Based Microfluidic s: From Basic Elements To Complex Circuits, Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences November 1 - 5, 2009, Jeju, Korea). Reiches et al have also attempted using cotton thread for diagnostic devices (RECHES ET AL: "Thread as a Matrix for Biomedical Assays", ACS APPLIED MATERIALS AND INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 2, no. 6, 1 January 2010 (2010-01-01), pages 1722-1728). In that study, Reiches et al attempted a variety of threads to be used for this purpose, including rayon, wool, nylon, polyester, hemp, acrylic, 50% acrylic-50% cotton, natural silk, before and after treatment, and concluded that cotton is the best option. In WO2009121043 (hereinafter Ό43), a diagnostic device comprising a hydrophilic thread is described, wherein the hydrophilic thread comprises a detection reagent. The hydrophilic thread used extensively in Ό43 was almost exclusively cotton. However, the use of nylon and polyester threads was also described therein, wherein it was observed that "these threads could be useful in devices that require faster wicking and do not entail the encapsulation of a thread within tape."

[0005] PCT/IB2010/053974 and PCT/IB2010/053976 describe a diagnostic device made of a hydrophilic thread comprising a diagnostic reagent that is woven into a hydrophobic matrix made of a hydrophobic fiber. The pattern of the weaving of the hydrophilic thread in the hydrophobic matrix defines a flow path for the sample. Thus, a simple method of fabricating diagnostic devices using well-known techniques of textile manufacturing was provided. The method and the device were demonstrated using a modified silk as the hydrophilic material and a coated silk as the hydrophobic matrix.

[0006] The field of point of care diagnostics requires continued improvements and further enhancements of the techniques presently available especially from the view of manufacturibility, costs and ease of use.

BRIEF DESCRIPTION

[0007] In one aspect, the invention provides a diagnostic device comprising: a hydrophilic matrix comprising at least one detection reagent; a hydrophobic matrix; wherein the hydrophilic matrix and hydrophobic matrix are woven together, wherein the hydrophilic matrix is characterized by having at least one thread derived from degummed silk and at least one thread made of a hydrophilic material that are inter- woven.

[0008] In another aspect, the invention provides a diagnostic device comprising: a hydrophobic matrix; a hydrophilic matrix comprising: a sample inlet zone, a flow zone, a testing zone, and a sample absorption zone; wherein the hydrophilic matrix is contained within the hydrophobic matrix, and wherein the hydrophilic matrix is characterized by having at least one thread derived from degummed silk and at least one thread made of a hydrophilic material that are interwoven.

[0009] In yet another aspect, the invention provides a method of making a diagnostic device, wherein the diagnostic device comprises a hydrophobic matrix and a hydrophilic matrix, wherein the method comprises: providing at least one strand of a degummed silk and at least one strand of a hydrophilic material; weaving together the at least one strand of the degummed silk and the at least one strand of the hydrophilic material to provide a hydrophilic matrix; providing at least one strand of a hydrophobic material; and weaving together the at least one strand of the hydrophobic material with the hydrophilic matrix to provide the diagnostic device.

DRAWINGS

[0010] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0011] FIG. 1 shows an exemplary embodiment of the hydrophilic matrix;

[0012] FIG. 2 shows an alternate embodiment of a configuration of the hydrophilic matrix;

[0013] FIG. 3 shows yet another alternate embodiment of a configuration of the hydrophilic matrix;

[0014] FIG. 4 shows yet another alternate embodiment of the hydrophilic matrix;

[0015] FIG. 5 shows a zoomed portion of the hydrophilic matrix showing the detail of the thread

[0016] FIG. 6 shows one embodiment of the arrangement for the diagnostic device of the invention; and

[0017] FIG. 7 shows one exemplary configuration of the diagnostic device of the invention. DETAILED DESCRIPTION

[0018] As used herein and in the claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0019] "Strand" as used herein refers to a single element (as a yarn) of a woven or plaited material. It may be used interchangeably with "yarn". "Thread" as used herein means at least two strands that have been bundled together appropriately to form a fiber. Bundling together of strands may be achieved using various standard techniques known in the art, and may include, twisting, braiding, holding multiple strands together tightly, and the like, and combinations thereof. Fiber as used herein may refer to a single strand of a material or a bundle of strands that are bundled together. [0020] Analyte, as used herein, refers to a substance or chemical constituent that is determined in an analytical procedure. For instance, in an immunoassay, the analyte may be a protein ligand or a binder, while in blood glucose testing, the analyte is glucose. In one instance, the analyte could be a gene that is a marker for the Hepatitis-B virus. In another exemplary instance, analyte may include a drug to be detected, such as cocaine from a blood analysis. The analytical procedure may include, for instance, fluorescence, mass- spectrometry, colorimetry, radio-imaging, electrochemical detection and the like, and combinations thereof. In some instances, analytes may refer to antibodies. In other instances, analytes may refer to antigens.

[0021] Antibody as used herein refers to protein that is used in the identification of specific antigen. The specific antigen is typically a marker of a disease or certain types of diseases. Sometimes, antibodies may also be referred to as immunoglobulins. Antibody may be primary or secondary antibody. Primary antibodies are antibodies raised against a specific antigen and are generally unlabelled. Primary antibodies may also be referred to as capture antibodies. Secondary antibody is an antibody that binds to primary antibody or fragments contained within the primary or capture antibody. Secondary antibody comprise label that render them useful for detection. Typical labels include fluorescence moiety, radio-active compounds, enzyme-linked labels, magnetically active particles, nanoparticles, quantum dots, latex particle labels, and the like, and combinations thereof. Depending on the label, the method used to detect, identify and quantitate may include fluorescence spectroscopy, radio- imaging, ELISA test and the like.

[0022] Antigen, as used herein, refers to a molecule that is recognized by an immune system of a living organism. Antigen also refers to molecular fragments that may be recognized by the immune system. It is generally known that a given antigen shows specificity to an antibody, and this property of an antigen is used in a variety of applications.

[0023] As noted herein, in one aspect the invention provides a diagnostic device comprising a hydrophilic matrix comprising at least one detection reagent and a hydrophobic matrix. The hydrophilic matrix and hydrophobic matrix are woven together to form the diagnostic device of the invention.

[0024] The diagnostic device of the invention, as noted herein, comprises a hydrophobic matrix which is made from at least one strand of a hydrophobic-fiber composition. Hydrophobic as used herein refers to materials that are generally characterized by water-repellent property. Water repellence may be measured by techniques such as contact angle made by a drop of water on the surface of the material. Hydrophobic-fiber compositions are made from hydrophobic materials. Useful hydrophobic materials include polyamides such as, Nylon-66, Nylon-6, and other Nylons, aromatic polyamides such as Kevlar®; polyesters such as poly(ethylene terephthalate), poly(butylene terephthalate); poly(phenylene oxide); hydrophobic-coated silk, and the like, and combinations thereof. In one specific embodiment, the hydrophobic-fiber composition is a hydrophobic-coated silk. Typical hydrophobic coating useful in the invention include gold coating, tin coating, nickel coating, brass coating, copper coating, and the like.

[0025] The hydrophilic matrix is characterized by having at least one thread derived from degummed silk and at least one thread made of a hydrophilic material. Degummed silk is known in the art. Briefly, silk is a fiber obtained from silkworms, more specifically from the larvae of mulberry silkworms. The silk most useful in the invention are those that can be woven into textiles, such as that obtained from the silkworm Bombyx Mori, however, other forms of silk that may be synthetically made or produced from other sources may also be used for this invention. Chemically, silk fiber comprises a chain of amino acids, which possesses functional groups that may be further used for binding useful moieties. As used herein, functional groups are reactive chemical moieties that can interact with other reactive species to form physical or chemical bonds. Silk obtained from natural sources like Bombyx Mori may be treated with a suitable alkaline solution to remove extraneous material present on the surface. This process may be referred to in the art as degumming and the product obtained is referred to as degummed silk. For example, US2004/224406 describes a cleaned up silk fiber with the sericin extracted therefrom to provide a hydrophilic silk. The degummed silk may further be treated with other kinds of treatment solutions such as a surfactant and blocking agents to impart further useful properties to the silk material, and modified with appropriate reactive functional groups. For example, Davarpanah et el "Environmentally friendly surface modification of silk fiber: Chitosan grafting and dyeing" Applied Surface Science, vol. 255, no. 7, 15 January 2009 (2009-01-15), pages 4171-4176. PCT/IB2010/053976 describes methods of degumming silk and treating the degummed silk with solutions and blocking agents.

[0026] As used herein, hydrophilic material is a material that is capable of being wet with water. Hydrophilicity of a material is generally understood by the physical phenomena of wicking, as measured by wicking rate. Hydrophilicity may also be understood by the contact angle made by the water droplet on the surface of the material. The hydrophilic materials useful in the invention must also have the ability to form fibers. Useful hydrophilic materials for the diagnostic device include, but not limited to, poly(vinyl alcohol), cotton, rayon, cellulosic fibers, derivatives thereof, and blends therefrom. One skilled in the art will readily appreciate the effect of molecular weight, extent of crosslinking, presence of plasticizers and other additives, as well as other factors, that affect the fiber-forming abilities and fiber properties. The choice of material will become obvious to one skilled in the art without undue experimentation once the requirements of the diagnostic device and the conditions of use of the diagnostic device are known. In one particular embodiment, the hydrophilic material useful in the invention is cotton thread. [0027] Diagnostic reagent as used herein may be at least one of an antigen or an antibody. In some instances, the diagnostic reagent is an antigen. Antigens useful in the invention include, for example p24, gpl20, gp41, HIVII - gpl05, gp36, Hepatitis C - NS3, NS5, core antigen, ,β-Hcg (pregnancy), TSH (thyroid), FSH (female hormone), Troponin-T (cardiac), CpkMb, BNP, Myoglobin, HblAc, PSA, AFP, CEA, CA125, CA19.9, Progestorone, Testosterone, Estradiol. In yet other instances, the diagnostic reagent is a primary antibody. Useful primary antibodies include, for example, Anti HBs (IgG), Anti HBc (IgM), Anti-hcG, Anti-HIV- p24, Anti-HIV-gpl20, Anti-FSH, Anti-TSH, Anti Troponin, and Anti Plasmodium Falciparum, Toxoplasma IgM, Rubella IgM, Herpex Simplex Virus 2 IgM, Cytomegalovirus IgM, Typhidot IgM, Typhidot IgG, Dengue IgM, Dengue IgG, Leptospira IgM,. In situations wherein more than one diagnostic reagent is present, the second diagnostic reagent may be another primary antibody, or it may be a secondary antibody. In the case of the diagnostic device having a primary antibody as the first diagnostic reagent and a secondary antibody as the second diagnostic reagent, it must be understood that the secondary antibody must be present upstream to the primary antibody taking into account the flow of fluids along the final diagnostic device. As used herein, the terms "upstream" and "downstream" are used to indicate the relative position with respect to the flow of a solution on the diagnostic device. Thus, a spot on the diagnostic device that comes into contact with a flowing solution first is considered upstream to a position on the diagnostic device that comes into contact later. [0028] Immobilization may be achieved by any number of techniques known to those skilled in the art. This may include, for example, immersing, covalent bonding, coating, dipping, stamping, or combinations thereof. Depending on the choice of the hydrophilic polymer, there may be included a step for the preparation of the polymer to receive the first and/or second reagent. For example, as already stated herein, silk may be degummed to facilitate the immobilization of the reagents, whereas a fiber made from poly(vinyl alcohol) may be amenable to immobilization with reagents as such. Thus, the exact region of immobilization may be on the degummed silk, or on the hydrophilic material, or on both.

[0029] The hydrophilic matrix of the diagnostic device of the invention is advantageously made by the inter-weaving the at least one strand of the degummed silk and the at least one strand of the hydrophilic material. The method of inter-weaving is generally known among textile manufacturers (See for example http://www.britannica.com/EBchecked/topic/638448/weaving). A useful method of interweaving includes the use of a double warp double weft technique. Other methods of introducing warp and weft are known in the art. Warp means the lengthwise yarns while weft means the yarn that is threaded through the warp. The angle between the warp and the weft can influence the flow properties within the diagnostic device, and hence provides greater control over the use of the diagnostic device of the invention, which step is also included in the method of the invention. Currently, machines exist that are used extensively in the textile manufacturing industry for the production of finished textile goods using the double warp double weft technique along with other techniques.

[0030] A loom is an exemplary device used for weaving, and may be advantageously used for the production of the diagnostic device of the invention. Several types of looms are readily commercially available for use. Exemplary looms useful in the invention include, but not limited to, jacquard loom, dobby loom, treadle loom, power loom, and the like.

[0031] Fig. 1 shows an exemplary embodiment of a section of the hydrophilic matrix represented by numeral 10. Here, the weft 12 is shown to be made of silk 14 while the warp 16 is predominantly silk 18 but having two strands of hydrophilic material 20. As stated herein, a particularly useful hydrophilic material in the invention is cotton thread.

[0032] Such patterns in a hydrophilic matrix using two different materials give rise to differential flow in different regions. This leads to more effective design of diagnostic devices with clearer flow paths. As already noted herein, defining flow channels in diagnostic device is an important feature, which have been the main focus of several studies (see for example Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M., Patterned paper as a platform for inexpensive, low- volume, portable bioassays. Angew Chem Int Ed Engl 2007, 46 (8), 1318-20). The use of cotton thread especially allows for substantial reduction in costs of the diagnostic device. The use of silk allows for a protein based fiber, also available at a reasonable price, that can be used in a very cost efficient process such as weaving to fabricate the device.

[0033] Fig. 2 shows an alternate embodiment of a configuration of a section of the hydrophilic matrix 22. In this instance, the weft 24 is shown to be made of cotton 26 while the warp 28 is predominantly silk 30 with two strands of hydrophilic material 32.

[0034] Fig. 3 shows yet another alternate embodiment of a configuration of a section of the hydrophilic matrix 34. In this instance, the warp 80 is made of silk 36 while the weft 38 is predominantly made of silk 40 with two strands of hydrophilic material 42. One of ordinary skill in the art will understand that the two strands shown herein is an exemplary number used for illustration purposes only, and the number of strands may be anything, including, one, three, four, five, seven, ten, and so on.

[0035] Fig. 4 shows yet another alternate embodiment of a section of the hydrophilic matrix 44. In this instance, the warp 46 is made of silk, while the weft 48 is made of fiber 50, wherein the fiber is a bundle made of silk and cotton that have been braided together. Methods of braiding strands to make threads are known in the art. Fig. 5 shows a zoomed portion of the hydrophilic matrix showing the detail of the thread used in this exemplary embodiment.

[0036] One skilled in the art will also understand that the techniques described herein may be effectively used to provide threads made of strands having two different compositions, or three different compositions, or four different compositions, or more, wherein at least one of the composition is degummed silk. Thus, in one embodiment, the thread is made from at least one strand of degummed silk, at least one strand of cotton, and at least one strand of polyester fiber. In another exemplary embodiment, the thread is made from at least one strand of degummed silk, and at least one strand of nylon fiber. In yet another exemplary embodiment, the thread is made using at least one strand of degummed silk, at least one strand of polyester fiber, at least one strand of cotton, at least one strand of nylon and at least one strand of nylon fiber. The exact properties desired for the hydrophilic matrix in terms of a defined flow path, stiffness, tensile properties, and other factors, will determine the number and the exact composition of the strands used to form the thread.

[0037] Further, the techniques of weaving may be precisely controlled to obtain threads having multiple strands of varying compositions in the weft region only, or in the warp region only, or in both. Also, specific portions of the weft region or warp region or both may be made to comprise specific compositions as described herein. In one embodiment, the weft region of the hydrophilic matrix comprises threads having strands of multiple compositions, such as two different compositions, or three different compositions, and so on, while the warp region comprises threads made of strands from degummed silk only. The weaving technique allows for precision control, accuracy, reproducibility, and scalability. Other variations and possibilities may become obvious to one skilled in the art, and is contemplated to be within the scope of the invention.

[0038] Fig. 6 shows one embodiment of the arrangement for the diagnostic device of the invention 52 comprising a hydrophilic matrix 54 contained within a hydrophobic matrix 56. The hydrophobic matrix is shown to comprise sections of woven silk 82, along with sections of silk and hydrophilic material 84 interspersed between each other in this exemplary configuration. In this exemplary embodiment, the shape of the matrices are as shown, however, other shapes and arrangements are contemplated within the scope of the invention. Also, multiple hydrophilic matrices within the hydrophobic matrix are also contemplated.

[0039] In one exemplary configuration of the diagnostic device of the invention shown in Fig. 7 and represented by numeral 58, it comprises the hydrophobic matrix 60 that contains the hydrophilic matrix 62 that defines a sample inlet zone 64, a flow zone 66, a testing zone 68 and a sample absorption zone 70. The sample inlet zone 64 is the portion of the device wherein a sample to be tested is introduced into the device. Typical samples that may be used with the diagnostic device 58 includes bodily fluids such as blood, saliva, sputum, urine, and the like, and combinations thereof. Methods of introducing the sample are known in the art, and may include, pipetting, spotting, and the like. The flow zone 66 defines the path of flow of the sample. The testing zone typically includes an antigen or an antibody corresponding to the test being conducted. The sample may comprise a corresponding antigen or antibody which interacts with the counterpart in the testing zone and gives rise to a signal. The interactions with the interacting reagents and methods of giving rise to a signal are well known in the art. The signal may then be used to provide a qualitative determination of the presence or absence, such as in a pregnancy test, or a quantitative measurement, such as amount of blood glucose level. After the testing zone, the flow path leads to the sample absorption zone 70, wherein the liquid is absorbed. One skilled in the art would be able to understand that the sample absorption zone may be made of a more absorptive material. [0040] The blend of silk and hydrophilic material may form a part of any portion of the hydrophilic matrix or the entire hydrophilic matrix, as the situation demands. In one exemplary embodiment, as stated herein, the interwoven mix of silk and hydrophilic material may be along the flow zone 66. In another embodiment, the interwoven mix of silk and hydrophilic material may be in the sample absorption zone. In this instance, cotton thread as the hydrophilic material would be a low-cost greater absorbing material, and hence provide a great advantage in its use in the interwoven mix. In yet another embodiment, the interwoven mix of silk and hydrophilic material may be used in the sample inlet zone. In a further embodiment, the interwoven mix of silk and hydrophilic material is only on the testing zone.

[0041] Weaving as a method of the invention is particularly attractive as it is conducive for scale-up for manufacturing a large number of diagnostic devices within a given period of time. The method is also amenable to introducing multiplexed diagnostic devices. Weaving also uses skill and equipment that already exist. Further, the technique along with the materials used provides for economic feasibility of the fabricated devices to be provided at a low-cost for developing and least developed economies. [0042] Thus, in another aspect, the invention provides a method for making diagnostic devices. The method involves weaving together at least one strand of degummed silk with at least one strand of the hydrophilic material to provide the hydrophilic matrix, wherein the hydrophilic material is as defined herein. Subsequently, the hydrophilic matrix is then interwoven with at least one strand of a hydrophobic material. [0043] While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.