US20080269707A1 - Lateral Flow Device for Attachment to an Absorbent Article - Google Patents

Lateral Flow Device for Attachment to an Absorbent Article Download PDF

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Publication number
US20080269707A1
US20080269707A1 US11/741,943 US74194307A US2008269707A1 US 20080269707 A1 US20080269707 A1 US 20080269707A1 US 74194307 A US74194307 A US 74194307A US 2008269707 A1 US2008269707 A1 US 2008269707A1
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Prior art keywords
absorbent article
lateral flow
flow device
insert
absorbent
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Abandoned
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US11/741,943
Inventor
Xuedong Song
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Kimberly Clark Worldwide Inc
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Kimberly Clark Worldwide Inc
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Priority to US11/741,943 priority Critical patent/US20080269707A1/en
Assigned to KIMBERLY-CLARK WORLDWIDE, INC. reassignment KIMBERLY-CLARK WORLDWIDE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SONG, XUEDONG
Publication of US20080269707A1 publication Critical patent/US20080269707A1/en
Application status is Abandoned legal-status Critical

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Detecting, measuring or recording for diagnostic purposes; Identification of persons
    • A61B5/20Detecting, measuring or recording for diagnostic purposes; Identification of persons for measuring urological functions restricted to the evaluation of the urinary system
    • A61B5/207Sensing devices adapted to collect urine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0045Devices for taking samples of body liquids
    • A61B10/007Devices for taking samples of body liquids for taking urine samples
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00Bandages or dressings; Absorbent pads
    • A61F13/15Absorbent pads, e.g. sanitary towels, swabs or tampons for external or internal application to the body; Supporting or fastening means therefor; Tampon applicators
    • A61F13/42Absorbent pads, e.g. sanitary towels, swabs or tampons for external or internal application to the body; Supporting or fastening means therefor; Tampon applicators with wetness indicator or alarm
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306Solid-phase reaction mechanisms
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B2010/0003Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements including means for analysis by an unskilled person
    • A61B2010/0006Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements including means for analysis by an unskilled person involving a colour change
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00Bandages or dressings; Absorbent pads
    • A61F13/15Absorbent pads, e.g. sanitary towels, swabs or tampons for external or internal application to the body; Supporting or fastening means therefor; Tampon applicators
    • A61F13/84Accessories, not otherwise provided for, for absorbent pads
    • A61F2013/8473Accessories, not otherwise provided for, for absorbent pads for diagnostic purposes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00Bandages or dressings; Absorbent pads
    • A61F13/15Absorbent pads, e.g. sanitary towels, swabs or tampons for external or internal application to the body; Supporting or fastening means therefor; Tampon applicators
    • A61F13/84Accessories, not otherwise provided for, for absorbent pads
    • A61F2013/8488Accessories, not otherwise provided for, for absorbent pads including testing apparatus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/34Genitourinary disorders
    • G01N2800/348Urinary tract infections

Abstract

A lateral flow device for testing a bodily fluid, such as urine, blood, mucous, saliva, etc., is provided. The lateral flow device is configured to be attached to an absorbent article via an attachment mechanism located on the lateral flow device or the absorbent article. The lateral flow device includes a chromatographic medium (e.g., porous membrane) that defines a detection zone that provides a signal indicative of the presence or absence of the analyte. The device may also include a control zone that provides a signal indicative of whether a sufficient amount of bodily fluid has been provided and tested. Regardless of its specific configuration, the lateral flow device is configured to be attached onto the absorbent article to provide a user or caregiver with rapid information about a health condition. For example, the device may be attached onto a diaper to provide information about the presence of enzymes or other compounds often encountered with a patient having a urinary tract infection. This information may provide an early warning system to allow the user or caregiver to seek additional testing and/or treatment. Alternatively, semi-quantitative or quantitative results may be derived from the test.

Description

    BACKGROUND OF THE INVENTION
  • Multiple tests have been developed for detecting components in urine. Such tests can provide information about overall health as well as provide an indication of a health problem. When timely administered, such tests may also be able to provide an early indication of a health problem, which may be very advantageous for effective treatment. By way of examples, urine testing can be used to detect urinary tract infections, diabetes (including diabetic ketoacidosis), parasites, dehydration, dietary defects, cancer, high blood pressure, kidney disease, asthma, severe emphysema, alcoholism, systemic lupus erythematosus (SLE), glomerulonephritis, and leukemia.
  • Such tests may be performed by having a patient voluntarily collect and provide a sample. However, patient collected urine samples may not be readily available with certain test subjects such as children, elderly adults, and injured or non-ambulatory patients. Additionally, it may be preferable to collect and test urine samples from these subjects at certain times or conditions where the patient is not necessarily in the presence of medical or otherwise specially trained personnel. Frequently, such subjects may be provided with a diaper or other absorbent article to collect urine. Then, the soiled article is provided to the medical or otherwise specially trained personnel for testing.
  • As such, a need exists for a device capable of testing bodily fluid that can be attached to an absorbent article such that the testing occurs at the time of the insult of the absorbent article.
  • SUMMARY OF THE INVENTION
  • In general, an absorbent article for testing a bodily fluid suspected of containing an analyte is disclosed. The absorbent article comprises an outer cover, a bodyside liner, and an absorbent core positioned between the outer cover and the bodyside liner. A lateral flow device is attached to the bodyside liner of the absorbent article. The lateral flow device includes a chromatographic medium that defines a detection zone. The detection zone is configured for exhibiting a signal indicative of the presence or absence of the analyte in the bodily fluid.
  • In another embodiment, the present invention is generally directed to an insert for testing a bodily fluid suspected of containing analyte. The insert includes a cover encasing a lateral flow device. Also, the insert includes an attachment mechanism configured to attach the insert to a bodyside liner of an absorbent article. In one embodiment, the insert can also include an absorbent material encased within the cover.
  • In yet another embodiment, a method for detecting the presence or absence of an analyte in urine is disclosed. The method includes attaching a lateral flow device to a bodyside liner of an absorbent article.
  • Other features and aspects of the present invention are described in more detail below.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, which makes reference to the appended figures in which:
  • FIG. 1 is a perspective view of an exemplary lateral flow device in accordance with one embodiment of the present invention;
  • FIG. 2 is a perspective view of an exemplary attachment mechanism of the lateral flow device in accordance with one embodiment of the present invention;
  • FIG. 3 is a perspective view of another exemplary attachment mechanism of the lateral flow device in accordance with one embodiment of the present invention;
  • FIG. 4 is a perspective view of an exemplary use of the lateral flow device in conjunction with an absorbent article in accordance with one embodiment of the present invention;
  • FIG. 5 is a perspective exploded view of an exemplary absorbent insert containing a lateral flow device for use with an absorbent article in accordance with one embodiment of the present invention;
  • FIG. 6 is a perspective view of an absorbent insert containing a lateral flow device attached to an absorbent article in accordance with one embodiment of the present invention; and
  • FIG. 7 is a perspective, exploded view of an absorbent article for attaching to a lateral flow device in accordance with one embodiment of the present invention.
  • Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.
  • Detailed Description of Representative Embodiments
  • Reference now will be made in detail to various embodiments of the invention, one or more examples of which are set forth below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, may be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations.
  • Generally speaking, the present invention is directed to a lateral flow device for testing a bodily fluid (e.g., urine, blood, mucous, saliva, etc.) that can be attached to an absorbent article. Since lateral flow devices are utilized to detect the presence or absence of compounds in a bodily fluid, the manufacturing and packaging environment of the device is controlled to prevent contamination of the device prior to its use. Controlling the manufacturing environment adds costs to the manufacture of the device. If the device were to be integrated within the construction of the absorbent article (e.g., positioned within the absorbent article's construction), then the manufacturing environment of the entire absorbent article would have to be controlled in order to prevent contamination of the device prior to its use, adding significant manufacturing and packaging costs to the absorbent article. However, according to the present invention, the lateral flow device can be separately manufactured under a controlled environment, and then attached to an absorbent article by the user or caregiver. Thus, only the manufacturing environment of the lateral flow device is subject to regulation, saving the cost of regulating the manufacturing environment of the absorbent article.
  • The lateral flow assay device generally includes a chromatographic medium (e.g., porous membrane) that defines a detection zone for providing a signal indicative of the presence or absence of an analyte. The device may also include a control zone that provides a signal indicative of whether a sufficient amount of bodily fluid has been provided and tested. Regardless of its specific configuration, the lateral flow assay device is configured to be attached to the absorbent article to provide a user or caregiver with rapid information about a health condition. For example, the device may be attached to a diaper to provide information about the presence of enzymes or other compounds often encountered with a patient having a urinary tract infection. This information may provide an early warning system to allow the user or caregiver to seek additional testing and/or treatment. Alternatively, semi-quantitative or quantitative results may be derived from the test.
  • I. Lateral Flow Assay Device
  • Generally speaking, a lateral flow assay device is employed in the present invention to perform a heterogeneous assay. A heterogeneous assay is one in which a species is separated from another species prior to detection. Separation may be carried out by physical separation, e.g., by transferring one of the species to another reaction vessel, filtration, centrifugation, chromatography, solid phase capture, magnetic separation, and so forth. The separation may also be nonphysical in that no transfer of one or both of the species is conducted, but the species are separated from one another in situ. In some embodiments, for example, a heterogeneous immunoassay is performed that utilizes mechanisms of the immune systems, wherein antibodies are produced in response to the presence of antigens that are pathogenic or foreign to the organisms. These antibodies and antigens, i.e., immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism that may be used to determine the presence or concentration of that particular antigen in a fluid test sample. In other embodiments, however, the heterogeneous assay may employ non-specific chemical reactions to achieve the desired separation.
  • In any event, the use of a lateral flow assay device provides a variety of benefits, including a more uniform flow of the bodily fluid and reagents during testing. This may enhance the accuracy of the test and minimize the need for external control mechanisms. Referring to FIG. 1, for example, one embodiment of a lateral flow assay device 10 will now be described in more detail. As shown, the device 10 contains a chromatographic medium 14 optionally supported by a rigid support 12. The chromatographic medium 14 may be made from any of a variety of materials through which the test sample is capable of passing. For example, the chromatographic medium 14 may be a porous membrane formed from synthetic or naturally occurring materials, such as polysaccharides (e.g., cellulose materials such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose); polyether sulfone; polyethylene; nylon; polyvinylidene fluoride (PVDF); polyester; polypropylene; silica; inorganic materials, such as deactivated alumina, diatomaceous earth, MgSO4, or other inorganic finely divided material uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon or rayon); porous gels, such as silica gel, agarose, dextran, and gelatin; polymeric films, such as polyacrylamide; and so forth. In one particular embodiment, the chromatographic medium 14 is formed from nitrocellulose and/or polyether sulfone materials. It should be understood that the term “nitrocellulose” refers to nitric acid esters of cellulose, which may be nitrocellulose alone, or a mixed ester of nitric acid and other acids, such as aliphatic carboxylic acids having from 1 to 7 carbon atoms.
  • The size and shape of the chromatographic medium 14 may generally vary as is readily recognized by those skilled in the art. For instance, a porous membrane strip may have a length of from about 10 to about 100 millimeters, in some embodiments from about 20 to about 80 millimeters, and in some embodiments, from about 40 to about 60 millimeters. The width of the membrane strip may also range from about 0.5 to about 20 millimeters, in some embodiments from about 1 to about 15 millimeters, and in some embodiments, from about 2 to about 10 millimeters. Likewise, the thickness of the membrane strip is generally small enough to allow transmission-based detection. For example, the membrane strip may have a thickness less than about 500 micrometers, in some embodiments less than about 250 micrometers, and in some embodiments, less than about 150 micrometers.
  • As stated above, the support 12 carries the chromatographic medium 14. For example, the support 12 may be positioned directly adjacent to the chromatographic medium 14 as shown in FIG. 1, or one or more intervening layers may be positioned between the chromatographic medium 14 and the support 12. Regardless, the support 12 may generally be formed from any material able to carry the chromatographic medium 14. The support 12 may be formed from a material that is transmissive to light, such as transparent or optically diffuse (e.g., transluscent) materials. Also, it is generally desired that the support 12 is liquid-impermeable so that fluid flowing through the medium 14 does not leak through the support 12. Examples of suitable materials for the support include, but are not limited to, glass; polymeric materials, such as polystyrene, polypropylene, polyester (e.g., Mylar® film), polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxides, methacrylates, and polymelamine; and so forth. To provide a sufficient structural backing for the chromatographic medium 14, the support 12 is generally selected to have a certain minimum thickness. Likewise, the thickness of the support 12 is typically not so large as to adversely affect its optical properties. Thus, for example, the support 12 may have a thickness that ranges from about 100 to about 5,000 micrometers, in some embodiments from about 150 to about 2,000 micrometers, and in some embodiments, from about 250 to about 1,000 micrometers. For instance, one suitable membrane strip having a thickness of about 125 micrometers may be obtained from Millipore Corp. of Bedford, Mass. under the name “SHF180UB25.”
  • The chromatographic medium 14 may be cast onto the support 12, wherein the resulting laminate may be die-cut to the desired size and shape. Alternatively, the chromatographic medium 14 may simply be laminated to the support 12 with, for example, an adhesive. In some embodiments, a nitrocellulose or nylon porous membrane is adhered to a Mylar® film. An adhesive is used to bind the porous membrane to the Mylar® film, such as a pressure-sensitive adhesive. Laminate structures of this type are believed to be commercially available from Millipore Corp. of Bedford, Mass. Still other examples of suitable laminate device structures are described in U.S. Pat. No. 5,075,077 to Durley, III, et al., which is incorporated herein in its entirety by reference thereto for all purposes.
  • The device 10 may also contain an absorbent material 18 that is positioned adjacent to the medium 14. The absorbent material 18 can help promote capillary action and fluid flow through the medium 14. In addition, the absorbent material 18 receives fluid that has migrated through the entire chromatographic medium 14 and thus draws any unreacted components away from the detection region to help reduce the likelihood of “false positives.” Some suitable absorbent materials that may be used in the present invention include, but are not limited to, nitrocellulose, cellulosic materials, porous polyethylene pads, glass fiber filter paper, and so forth. The absorbent material may be wet or dry prior to being incorporated into the device. Pre-wetting may facilitate capillary flow for some fluids, but is not typically required. Also, as is well known in the art, the absorbent material may be treated with a surfactant to assist the wicking process.
  • To initiate the detection of an analyte, the bodily fluid (e.g., urine) may be applied to a portion of the chromatographic medium 14 through which it may then travel in the direction illustrated by arrow “L” in FIG. 1. Alternatively, the fluid may first contact a sample application zone 16 that is in fluid communication with the chromatographic medium 14. The sample application zone 16 may be defined by a separate pad or material as shown in FIG. 1, or simply defined by the chromatographic medium 14. In the illustrated embodiment, the fluid may travel from the sample application zone 16 to a conjugate pad (not shown) that is placed in communication with one end of the sample pad. The conjugate pad may contain one or more diffusively immobilized reagents, and be formed from a material through which a fluid is capable of passing (e.g., glass fibers). Some suitable materials that may be used to form the absorbent material 18 and/or sample pad include, but are not limited to, nitrocellulose, cellulose, porous polyethylene pads, and glass fiber filter paper. If desired, the sample pad may also contain one or more assay pretreatment reagents, either diffusively or non-diffusively attached thereto.
  • Regardless of the particular manner in which it is formed, the lateral flow assay device of the present invention employs one or more zones for providing an indicator of the presence of an analyte. More specifically, such zone(s) typically contain a chemical or biological reagent that interacts with the analyte and/or other reagents to generate a signal (e.g., visual signal). Referring again to FIG. 1, for example, the lateral flow assay device 10 includes a detection zone 20 within which a capture reagent is disposed. Typically, the capture reagent is applied in a manner so that it does not substantially diffuse through the matrix of the chromatographic medium 14 (i.e., non-diffusively immobilized). This enables a user to readily detect the change in color that occurs upon reaction of the capture reagent with other compounds. The capture reagent may, for example, form an ionic and/or covalent bond with functional groups present on the surface of the chromatographic medium 14 so that it remains immobilized thereon. For instance, particles, such as described below, may facilitate the immobilization of the reagent at the detection zone 20. Namely, the reagent may be coated onto particles, which are then immobilized on the chromatographic medium 14 of the device 10. In this manner, the reagent is able to readily contact compounds flowing through the medium 14.
  • Another zone that may be employed in the lateral flow assay device 10 for improving detection accuracy is a control zone 22. The control zone 22 gives a signal to the user that the test is performing properly. More specifically, reagents may be employed that flow through the chromatographic medium 14 upon contact with a sufficient volume of the bodily fluid being tested. These reagents may then be observed, either visually or with an instrument, within the control zone 22. The control reagents generally contain a detectable substance, such as luminescent compounds (e.g., fluorescent, phosphorescent, etc.); radioactive compounds; visual compounds (e.g., colored dye or metallic substance, such as gold); liposomes or other vesicles containing signal-producing substances; enzymes and/or substrates, and so forth. Other suitable detectable substances may be described in U.S. Pat. Nos. 5,670,381 to Jou, et al. and 5,252,459 to Tarcha et al., which are incorporated herein in their entirety by reference thereto for all purposes.
  • If desired, one or more of the reagents employed in the assay device may be disposed on particles (sometimes referred to as “beads” or “microbeads”). Among other things, the particles enhance the ability of the reagent to travel through a chromatographic medium. For instance, naturally occurring particles, such as nuclei, mycoplasma, plasmids, plastids, mammalian cells (e.g., erythrocyte ghosts), unicellular microorganisms (e.g., bacteria), polysaccharides (e.g., agarose), etc., may be used. Further, synthetic particles may also be utilized. For example, in one embodiment, latex microparticles that are labeled with a fluorescent or colored dye are utilized. Although any synthetic particle may be used in the present invention, the particles are typically formed from polystyrene, butadiene styrenes, styreneacrylic-vinyl terpolymer, polymethylmethacrylate, polyethylmethacrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinylpyridine, polydivinylbenzene, polybutyleneterephthalate, acrylonitrile, vinylchloride-acrylates, and so forth, or an aldehyde, carboxyl, amino, hydroxyl, or hydrazide derivative thereof. When utilized, the shape of the particles may generally vary. In one particular embodiment, for instance, the particles are spherical in shape. However, it should be understood that other shapes are also contemplated by the present invention, such as plates, rods, discs, bars, tubes, irregular shapes, etc. In addition, the size of the particles may also vary. For instance, the average size (e.g., diameter) of the particles may range from about 0.1 nanometers to about 1,000 microns, in some embodiments, from about 0.1 nanometers to about 100 microns, and in some embodiments, from about 1 nanometer to about 10 microns. Commercially available examples of suitable particles include fluorescent carboxylated microspheres sold by Molecular Probes, Inc. under the trade names “FluoSphere” (Red 580/605) and “TransfluoSphere” (543/620), as well as “Texas Red” and 5- and 6-carboxytetramethylrhodamine, which are also sold by Molecular Probes, Inc. In addition, commercially available examples of suitable colored, latex microparticles include carboxylated latex beads sold by Bang's Laboratory, Inc.
  • The location of the detection zone 20 and control zone 22 may vary based on the nature of the test being performed. In the illustrated embodiment, for example, the control zone 22 is defined by the chromatographic medium 14 and positioned downstream from the detection zone 20. In such embodiments, the control zone 22 may contain a material that is non-diffusively immobilized in the manner described above and forms a chemical and/or physical bond with the control reagents. When the control reagents contain latex particles, for instance, the control zone 22 may include a polyelectrolyte that binds to the particles. Various polyelectrolytic binding systems are described, for instance, in U.S. Patent App. Publication No. 2003/0124739 to Song, et al., which is incorporated herein in it entirety by reference thereto for all purposes. In alternative embodiments, however, the control zone 22 may simply be defined by a region of the absorbent material 18 to which the control reagents flow after traversing through the chromatographic medium 14.
  • Regardless of the particular control technique selected, the application of a sufficient volume of the test sample to the device 10 will cause a signal to form within the control zone 22, whether or not the enzyme or other analyte of interest is present. Among the benefits provided by such a control zone is that the user or other personnel are informed that a sufficient volume of test sample has been added without requiring careful measurement or calculation. This provides the ability to use the lateral flow assay device 10 without the need for externally controlling the reaction time, test sample volume, etc. In the case of the elderly, children, or patients unable to communicate clearly, control zone 22 provides an indication that a sample was discharged, collected, and successfully tested.
  • The detection zone 20, control zone 22, or any other zone employed in the lateral flow assay device 10 may generally provide any number of distinct detection regions so that a user may better determine the concentration of the enzyme or other analyte within the test sample. Each region may contain the same or different materials. For example, the zones may include two or more distinct regions (e.g., lines, dots, etc.). The regions may be disposed in the form of lines in a direction that is substantially perpendicular to the flow of the test sample through the device 10. Likewise, in some embodiments, the regions may be disposed in the form of lines in a direction that is substantially parallel to the flow of the test sample through the device 10.
  • The specific reagents employed in the lateral flow assay device depend on the analyte of interest and the assay technique employed. In one particular embodiment, for example, it may be desirable to detect the presence of leukocytes in urine as an early diagnosis of urinary tract infection (“UTI”). Although leukocytes are normally present in the urine, it has been determined that the threshold limit for pathological levels is about 1×104 leukocytes per milliliter of uncentrifuged urine. When leukocytes are present in urine, leukocyte esterase is produced and may be used as a biomarker for the presence of leukocytes.
  • A variety of reagents may be used for detecting the presence of the leukocyte esterase enzyme. One such reagent is a substrate that is chemically acted upon or “cleaved” by the enzyme of interest to release a product. For example, the substrate may be an ester that is catalytically hydrolyzed in the presence of leukocyte esterase to yield an aromatic compound. The aromatic esters may include, for instance, indoxyl esters having the following general formula:
  • Figure US20080269707A1-20081030-C00001
  • wherein, R may be substituted or unsubstituted, and may be an alkyl group, an alkyoxy group, a hydroxyalkyl group, an alkylene group, a fatty acid group, and so forth. In addition, the aromatic rings may also be substituted or unsubstituted. Specific examples include, for instance, indoxyl acetate, indoxyl butyrate, indoxyl laureate, indoxyl stearate, indoxyl ester of a N-blocked amino acid or peptide and thioindoxyl analogs thereof, and N-Tosyl-L-alanine 3-indoxyl ester. Such indoxyl esters are hydrolyzed by the leukocyte esterase to form a benzopyrrole, such as indoxyl, which has the following structure:
  • Figure US20080269707A1-20081030-C00002
  • Lactate esters may also be used, such as described in U.S. Pat. Nos. 5,464,739 to Johnson, et al. and 5,663,044 to Noffsinger, et al., which are incorporated herein in their entirety by reference thereto for all purposes. Lactate esters are generally hydrolyzed by the leukocyte esterase to provide a hydroxy-pyrrole compound. Other suitable ester substrates include thiazole esters, pyrrole esters, thiophene esters, naphthyl esters, phenoxyl esters, quinolinyl esters, such as described in U.S. Pat. Nos. 5,750,359 to Huh et al.; 4,657,855 to Corey, et al.; and Japanese Publication No. 03210193 to Kawanishi, et al., which are incorporated herein in their entirety by reference thereto for all purposes.
  • Typically, the substrate is diffusively immobilized on the lateral flow assay device 10 prior to application of the urine or other bodily fluid. The substrate is preferably disposed downstream from the sample application zone 16. In this manner, the test sample is capable of mixing with the enzyme upon application. If desired, the pH may be maintained at a relatively neutral level to facilitate the desired enzyme-catalyzed reaction, such as described above. To accomplish the desired pH level, a buffer may be mixed with the substrate prior to application to the device 10. Alternatively, the buffer may be separately applied to the lateral flow assay device 10 so that it is capable of mixing with the reagents upon exposure the bodily fluid being tested.
  • Regardless, an aromatic compound is released through cleavage of the substrate that is capable of inducing a color change in the presence of certain reagents. The released aromatic compound is a nucleophile in that it contains a group that is electron rich (e.g., amine) and that may form bonds with electron deficient groups. For example, indoxyl esters are hydrolyzed by the leukocyte esterase to form indoxyl. Indoxyl contains an electron-rich, aromatic ring system that is capable of undergoing electrophilic attack by a diazonium ion having the generic formula:
  • Figure US20080269707A1-20081030-C00003
  • The diazonium ion may be zwitterionic in that the counterion of the diazonium moiety is covalently bound to the ring system. The ring system of the diazonium ion may be substituted or unsubstituted. The ion may be provided by a variety of suitable diazonium salts, such as diazonium chlorides, diazonium acid sulphates, diazonium alkyl sulphates, diazonium fluoborates, diazonium benzenesulphonates, diazonium acid 1,5-naphthalenedisulphonates, and so forth. Specific examples of diazonium salts are 1-diazo-2-naphthol-4-sulfonate; 1-diazophenyl-3-carbonate; 4-diazo-3-hydroxy-1-naphthylsulfonate (DNSA); 4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate (NDNSA); 4-diazo-3-hydroxy-1,7-naphthyldisulfonate; 2-methoxy-4-(N-morpholinyl) benzene diazonium chloride; 4-diazo-3-hydroxy-7-bromo-1-naphthylsulfonate; and 4-diazo-3-hydroxy-7-[1, oxopropyl]-1-naphthylsulfonate. One particularly desired diazonium salt is 5-chloro-2-methoxybenzenediazonium chloride, which has a yellow color and is classified under the name “Diazo Red RC” or “Fast Red RC.” More specifically, “Fast Red RC” has the following structure:
  • Figure US20080269707A1-20081030-C00004
  • Other suitable diazonium salts are classified by the common names “Fast Red B” and “Fast Blue B.” Still other suitable diazonium salts may be described in U.S. Pat. Nos. 4,637,979 to Skjold, et al.; 4,806,423 to Hugh, et al.; and 4,814,271 to Hugl, et al., which are incorporated herein in their entirety by reference thereto for all purposes.
  • As indicated above, the nucleophilic aromatic compounds released by the hydrolysis of the substrate are capable of undergoing electrophilic attack by a reagent (e.g., diazonium ion). This reaction is often referred to as “coupling” and results in the formation of another reagent having a different color. For example, diazonium ions may react with aromatic compounds to form an aromatic azo compound having the generic formula, R—N═N—R′, wherein “R” and “R′” are aryl groups. Without intending to be limited by theory, it is believed that this reaction induces either a shift of the absorption maxima towards the red end of the spectrum (“bathochromic shift”) or towards the blue end of the spectrum (“hypsochromic shift”). The type of absorption shift depends on the nature of the resulting azo molecule and whether it functions as an electron acceptor (oxidizing agent), in which a hypsochromic shift results, or whether it functions as an electron donor (reducing agent), in which a bathochromic shift results. The absorption shift provides a color difference that is detectable, either visually or through instrumentation, to indicate the presence of leukocyte esterase or other enzymes within the test sample. For example, prior to contact with an infected test sample, the diazonium ion may be colorless or it may possess a certain color. However, after contacting the test sample and reacting with an aromatic compound released by hydrolysis of the substrate, an aromatic azo compound will form that exhibits a color that is different than the initial color of the diazonium ion. Exemplary aromatic azo compounds that may be formed include dimethyldiazene, diphenydiazene, 1-naphthyl-2-naphthyl diazene, 3-chlorophenyl-4-chlorophenyl diazene, methylvinyl diazene, and 2-naphthylphenyl diazene. In one particular embodiment, for instance, “Fast Red RC” (yellow), a diazonium ion, may react with indoxyl to form an aromatic azo compound that is red and has the following general structure (may be substituted or unsubstituted):
  • Figure US20080269707A1-20081030-C00005
  • Normally, the above-described diazonium ion is immobilized within the detection zone 20 of the lateral flow assay device 10. The diazonium ion may be applied directly to the medium 14 or first formed into a solution prior to application. Various solvents may be utilized to form the solution, such as, but not limited to, acetonitrile, dimethylsulfoxide (DMSO), ethyl alcohol, dimethylformamide (DMF), and other polar organic solvents. For instance, the amount of a diazonium salt in the solution may range from about 0.001 to about 100 milligrams per milliliter of solvent, and in some embodiments, from about 0.1 to about 10 milligrams per milliliter of solvent. In one particular embodiment, the detection zone 20 is defined by the chromatographic medium 14 and formed by coating a solution thereon using well-known techniques and then dried. The diazonium ion concentration may be selectively controlled to provide the desired level of detection sensitivity.
  • Typically, the diazonium ion is applied in a manner so that it does not substantially diffuse through the matrix of the chromatographic medium 14 (i.e., non-diffusively immobilized), which enables a user to readily detect the change in color that occurs upon reaction of the diazonium ion with a nucleophilic aromatic compound. The diazonium ion may form an ionic and/or covalent bond with functional groups present on