WO2024226412A1

WO2024226412A1 - Test strip devices containing low volumes of colorimetric reagents and methods of producing and using same

Info

Publication number: WO2024226412A1
Application number: PCT/US2024/025624
Authority: WO
Inventors: Aaron Kauffmann
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2023-04-27
Filing date: 2024-04-22
Publication date: 2024-10-31

Abstract

A test strip device for detecting the presence of a plurality of target analytes in a biological sample is disclosed, along with methods of producing and using the test strip device. The test strip device includes a plurality of reagent pads secured to the substrate, and at least two of the plurality of reagent pads are formed of a separate material. The test strip device also includes a plurality of colorimetric reagents deposited on the reagent pads at spatially separate locations. Each of the colorimetric reagents is deposited on the reagent pad at a volume of less than 1 microliter.

Description

TEST STRIP DEVICES CONTAINING LOW VOLUMES OF COLORIMETRIC REAGENTS AND METHODS OF PRODUCING AND USING SAME

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63/498,711 , filed April 27, 2023. The entire contents of the abovereferenced patent application are hereby expressly incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] Numerous devices and methods exist for detecting analytes that may be present in a patient’s fluid sample, including, for instance, a patient’s urine sample. Such devices have been proven to be effective in diagnostic assays that detect the presence (or non-presence) as well as the quantity of certain analytes indicative of a patient’s health and biological profile, including, but not limited to, analytes and conditions associated with a patient’s urine sample. However, these devices, kits, and methods are limited in their configuration in that current configurations do not easily allow for the testing of a small volume of a patient’s fluid (i.e., urine) sample. As a result of this configuration, it is difficult to obtain an accurate analysis of a patient’s urine sample when a patient only produces a small volume of a liquid test sample (less than about 5 milliliters). In current test strip configurations, when the volume of a sample is low, the receptacle containing the sample must be manipulated (either manually or via machine) to facilitate the interaction between the analyte(s) of interest and the respective reagent(s) contained on the analyte testing pad(s). This can result in inaccurate and/or incomplete results (due to incomplete wetting of the analyte testing regions by the liquid sample), as well as spillage of the sample from the sample receptacle.

[0004] In addition, current configurations include sizable analyte testing pads along the total (or substantially total) length of the urine test strip (which typically have a length of about 11 centimeters), resulting in the need for increased amounts of reagents to be incorporated on each analyte testing pad. Current urinalysis test strips use approximately 15-20 pl of each reagent to produce 0.2” x 0.2” colorimetric pads. These pads are prepared by running paper rolls through troughs of colorimetric reagent, which can result in a substantial loss from dead volume.

[0005] In addition, the current configurations for urinalysis test strips contain at least 12 reagents that require at least four to five qualified different substrates for the testing pads to function as formulated, due to various different requirements of the reagents, such as, but not limited to, hydrophilic/hydrophobic requirements, surface area requirements, pH requirements, and the like.

[0006] Accordingly, a need exists for new and improved devices, kits, and methods that allow for the production of target analyte test strip devices as well as use thereof that can be used with a patient’s liquid test sample for detection of a plurality of analytes of interest.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0007] FIG. 1 is a perspective view of one non-limiting embodiment of a test strip device constructed in accordance with the present disclosure.

[0008] FIG. 2 is a perspective view of another non-limiting embodiment of a test strip device constructed in accordance with the present disclosure.

[0009] FIG. 3 is a perspective view of yet another non-limiting embodiment of a test strip device constructed in accordance with the present disclosure.

[0010] FIG. 4 is a perspective view of one non-limiting embodiment of production of test strip devices in accordance with the present disclosure.

[0011] FIG. 5 is a perspective view of another non-limiting embodiment of production of test strip devices in accordance with the present disclosure.

[0012] FIG. 6 is a photographic view of reagents stamped on a substrate (AMERSHAM™ nitrocellulose, Cytiva, Little Chalfont, UK) to produce a non-limiting embodiment of a test strip device in accordance with the present disclosure.

DETAILED DESCRIPTION

[0013] Before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components or steps or methodologies set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting in any way.

[0014] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0016] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0017] All of the articles, compositions, kits, and/or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, compositions, kits, and/or methods have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles, compositions, kits, and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims. [0018] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: [0019] The use of the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As such, the terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a compound” may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term “plurality” refers to “two or more.”

[0020] The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0021] The use of the term “or” in the claims is used to mean an inclusive “and/or” unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition “A or B” is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0022] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase “in some embodiments” or “one example” in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims. [0023] Throughout this application, the terms “about” and “approximately” are used to indicate that a value includes the inherent variation of error for a com position/apparatus/ device, the method being employed to determine the value, or the variation that exists among the study subjects. That is, the terms “about” and “approximately” and variations thereof are intended to include not only the exact value qualified by the term, but to also include some slight deviations therefrom, such as deviations caused by measuring error, manufacturing tolerances, wear and tear on components or structures, settling or precipitation of cells or particles out of suspension or solution, chemical or biological degradation of solutions over time, stress exerted on structures, and combinations thereof, for example. In particular, for example, but not by way of limitation, when the term “about” is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0024] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, a composition, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherently present therein.

[0025] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. [0026] As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term “substantially” means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term “substantially adjacent” may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0027] As used herein, the phrases “associated with” and “coupled to” include both direct association/binding of two moieties to one another as well as indirect association/binding of two moieties to one another. Non-limiting examples of associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.

[0028] The term “sample” as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e. , plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), synovial fluid, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0029] The present disclosure is directed to test strip devices for detecting the presence of a plurality of target analytes in a fluidic biological sample, along with the production and use of these test strip devices in the detection of target analytes. The test strip devices comprise a plurality of paper reagent pads attached to a substrate, along with a plurality of colorimetric reagents deposited on the reagent pads in nanoliter volumes. At least one of the reagent pads is compatible for use with two or more colorimetric reagents such that the two or more colorimetric reagents can be spatially nano-deposited thereon for detection at separate locations on the single reagent pad. In addition, the sub-microliter volumes of colorimetric reagents will prevent the reagents from bleeding, diffusing, or otherwise touching one another and thereby preserve the integrity of each analyte assay being performed on the test strip device. Also, given the sub-microliter volumes of colorimetric reagents present, the test strip device is structured and configured for positioning within a diagnostic instrument for optical interrogation of each of the colorimetric reagents.

[0030] Turning now to the various non-limiting embodiments of the present disclosure, FIG. 1 depicts one non-limiting embodiment of a test strip device 10 constructed in accordance with the present disclosure. The test strip device 10 is used for detecting the presence of a plurality of target analytes in a biological sample. The test strip device 10 includes a lower substrate layer 12 that provides the test strip device with a first side 14, a second side 16, a first end 18, a second end 20, an upper surface 22, and a lower surface (not shown). In certain non-limiting embodiments, each of the first side 14 and the second side 16 comprises a length that is substantially longer than a length of each of the first end 18 and the second end 20. By way of example and not by way of limitation, the length of the first side 14 and the second side 16 may be as high as 20 times, 15 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, or 2 times the length of the first end 18 and the second end 20. In addition, while FIG. 1 depicts the device 10 as being substantially rectangular in shape, it should be understood to a person having ordinary skill in the art that the device 10 can be provided with any shape that will allow the device 10 to function in accordance with the present disclosure. The test strip device 10 is structured and configured for positioning within a diagnostic instrument for optical interrogation of a plurality of colorimetric reagents (described herein after) that are present on the test strip device 10.

[0031 ] The test strip device 10 also includes a plurality of reagent pads attached or otherwise secured to the upper surface 22 of the substrate 12. The test strip device 10 of FIG. 1 is illustrated as having five reagent pads (represented by reference numerals 30, 32, 34, 36, and 38) attached thereto; however, it will be understood that the presence of five reagents pads is for the purposes of illustration only, and two to four or more than five reagent pads may be present on test strip devices that also fall within the scope of the present disclosure. The only requirement is that at least two reagent pads must be present on the test strip devices of the present disclosure. [0032] Also, in certain particular (but non-limiting) embodiments, at least two of the reagent pads are formed of a different, separate material from one another; that is, reagent pad 30 may be formed of a different material than reagent pad 32 (and/or one or more of reagent pads 34, 36, and/or 38). However, two or more reagent pads may be present that are formed of the same type of material (for example, but not by way of limitation, reagent pad 32 may be formed of the same type of material as reagent pad 34, 36, and/or 38), so long as at least two reagent pads are present that are formed of different materials.

[0033] In addition, the various reagent pads may abut one or more other reagent pad(s) and/or may be spatially separated from one or more other reagent pad(s) on the test strip device 10.

[0034] Each of the plurality of reagent pads has at least one colorimetric reagent dot/spot dispensed thereon. For example (but not by way of limitation), as illustrated in FIG. 1 , reagent pad 30 has colorimetric reagents 50, 52, 54, and 56 deposited thereon. Each of the colorimetric reagents 50, 52, 54, and 56 is deposited on the reagent pad 30 at a location that is spatially separate from the other three colorimetric reagents, and this allows for detection of a separate target analyte at each of the four spatially separate locations 50, 52, 54, and 56. In addition, each of the colorimetric reagents 50, 52, 54, and 56 is dispensed on the reagent pad 30 at a sub-microliter (i.e. , less than 1 microliter) volume. In certain non-limiting embodiments, the disposal of sub-microliter volumes of reagents is achieved by dispensing or stamping the small volume of reagents onto the reagent pad, and this allows for control of a uniform diameter for each reagent spot and thereby reduces or eliminates wicking away of reagent from the surface deposit.

[0035] Similarly, reagent pad 32 contains colorimetric reagents 58, 60, 62, and 64; each of these reagents 58, 60, 62, and 64 is dispensed on the reagent pad 32 in a sub-microliter volume and at a location that is spatially separate from the other three colorimetric reagents, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 58, 60, 62, and 64. Likewise, reagent pad 34 contains colorimetric reagents 66 and 68 that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other colorimetric reagent, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 66 and 68. Reagent pad 36 and reagent pad 38 are both illustrated as having only one colorimetric reagent (70 for reagent pad 36 and 72 for reagent pad 38) dispensed thereon in a sub-microliter volume. Each of colorimetric reagents 70 and 72 is spatially disposed on the reagent pad 36 or 38 at a location that allows for detection of a separate target analyte at each of the locations 70 and 72.

[0036] While the reagent pads 30, 32, 34, 36, and 38 are shown as having 1-4 colorimetric reagents disposed thereon, any of the test strip devices of the present disclosure may be provided with any number of sample testing dots/spots that allow for detection of a desired number of target analytes thereon. In certain particular (but non-limiting) embodiments, each reagent pad of the test strip devices constructed in accordance with the present disclosure contain (for example, but not by way of limitation), at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve colorimetric reagent sample testing dots/spots.

[0037] Currently available configurations of urine test strips include sizable analyte testing pads that require substantial amounts of colorimetric reagents to be incorporated into each testing pad, and only one colorimetric reagent is disposed on each large testing pad (i.e., each testing pad on a urine test strip only detects a single analyte). In addition, the various testing pads must be formed of different materials due to various different requirements of the reagents, such as, but not limited to, hydrophilic/hydrophobic requirements, surface area requirements, pH requirements, and the like. For example (but not by way of limitation), the currently available configurations for urinalysis test strips contain 12 testing pads that each contain a different reagent, and these testing pads include at least four to five qualified different substrates to allow the reagents to function as formulated.

[0038] In contrast, the scope of the present disclosure includes test strip devices 10 that contain multiple reagent pads that each have multiple colorimetric reagents dispensed thereon (for example, but not by way of limitation, colorimetric reagents 50, 52, 54, and 56 on reagent pad 30; colorimetric reagents 58, 60, 62, and 64 on reagent pad 32; colorimetric reagents 66 and 68 on reagent pad 34; etc.). In this manner, colorimetric reagents that can be dispensed on the same type of material can be clustered together in a spatially separate manner and at much reduced volumes that will allow for detection of multiple colorimetric reagents on a single reagent pad.

[0039] In certain non-limiting embodiments, any of the test strip devices of the present disclosure detect the presence of three or more target analytes, such as, but not limited to, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve analytes. Nonlimiting examples of analytes that may be detected by the colorimetric reagents include glucose, bilirubin, ketones, blood, proteins, urobilinogen, nitrites, leukocytes, albumin, creatinine, ascorbic acid, specific gravity, pH, and the like.

[0040] The various analytes are detected using one or more colorimetric reagents that are micro-deposited on the compatible paper reagent pad at spatially separate locations. Non-limiting examples of colorimetric reagents that may be utilized in accordance with the present disclosure include glucose oxidase, peroxidase, potassium iodide, 2,4-dichloroaniline diazonium salt, sodium nitroprusside, bromothymol blue, methyl vinyl ether, maleic anhydride, sodium hydroxide, diisopropylbenzene dihydroperoxide, 3, 3’, 5, 5’ -tetramethylbenzidine, methyl red, tetrabromophenol blue, p-diethylamino-benzaldehyde, p-arsanilic acid, 1 , 2, 3, 4- tetrahydrobenzo(h) quinolin-3-ol, derivatized pyrrole amino acid ester, bis (3’, 3”- diiodo-4’,4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetrabromosulfonepthalein, copper sulfate, diazonium salt, and the like, as well as any combinations thereof.

[0041 ] Each of the colorimetric reagents is dispensed on its respective reagent pad at a volume that allows the colorimetric reagent to function in accordance with the present disclosure; in particular, the volume should be sufficiently low so as to prevent the reagent from bleeding/diffusing through the reagent pad and contacting another colorimetric reagent, or diffusing out of the reagent pad and into the fluidic biological sample upon contact therewith. Non-limiting examples of volumes that may be utilized in accordance with the present disclosure include about 1 nl, about 5 nl, about 10 nl, about 25 nl, about 50 nl, about 75 nl, about 100 nl, about 150 nl, about 200 nl, about 250 nl, about 300 nl, about 350 nl, about 400 nl, about 450 nl, about 500 nl, about 550 nl, about 600 nl, about 650 nl, about 700 nl, about 750 nl, about 800 nl, about 850 nl, about 900 nl, about 950 nl, about 975 nl, and the like, as well as a range formed of two of the above values (i.e. , a range of from about 1 nl to about 900 nl, a range of from about 10 nl to about 800 nl, etc.) and/or a range formed of two values that each fall between two of the above values (i.e., a range of from about 3 nl to about 970 nl, a range of from about 35 nl to about 875 nl, etc.).

[0042] In a particular (but non-limiting) embodiment, colorimetric reagents 50, 52, 54, and 56 detect (in no particular order) leukocyte, occult blood, creatinine, and glucose; colorimetric reagents 58, 60, 62, and 64 detect (in no particular order) ketone, specific gravity, pH, and protein; colorimetric reagents 66 and 68 detect (in no particular order) bilirubin and whole blood cells; colorimetric reagent 70 detects urobilinogen; and colorimetric reagent 72 detects nitrite.

[0043] The test strip devices of the present disclosure may be provided with any dimensions that allow the test strip devices to function as described herein. In certain non-limiting embodiments, the device 10 has a width of less than or equal to about 1 cm, such as (but not limited to) about 0.9 cm, about 0.8 cm, about 0.7 cm, about 0.6 cm, about 0.5 cm, about 0.4 cm, about 0.3 cm, about 0.2 cm, about 0.1 cm, and the like, as well as a range formed of two of the above values (i.e., a range of from about 0.3 cm to about 1 cm, etc.).

[0044] Each of the layers of any of the test strip devices disclosed or otherwise contemplated herein may be formed of any material, so long as the test strip devices formed therefrom is capable of functioning as described herein. Non-limiting examples of materials from which the substrate may be formed include acrylic, polystyrene, styrene-acrylonitrile, polycarbonate, polyethylene terephthalate, and the like, as well as any combinations thereof.

[0045] In certain non-limiting embodiments, each of the reagent pads may be independently formed of a quantitative or qualitative natural or synthetic filter paper. Non-limiting examples of paper materials from which each of the reagent pads may be independently formed include cellulose, nitrocellulose, cotton, spun glass, silica glass, soda lime glass, glass microfiber, cellulose acetate, cellulose nitrate, nylon, polyester film, polyethylene terephthalate, polycarbonate, and polystyrene, and the like, as well as any combinations thereof.

[0046] Each of the layers (i.e., substrate and reagent pads) of any of the test strip devices disclosed or otherwise contemplated herein may be provided with any thickness, so long as the test strip devices formed therefrom are provided with a desired thickness that allows for the conductance of the plurality of analyte assays and measurement thereof within a diagnostic instrument. For example (but not by way of limitation), each of the layers may have a thickness independently selected from about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, and the like, as well as a thickness that falls within a range of two of the above values (i.e., a range of from about 0.1 mm to about 1 mm, etc.). In addition, the test strip devices of the present disclosure may be provided with any total thickness, so long as the test strip device is capable of performing the plurality of analyte assays. For example (but not by way of limitation), the test strip devices may be provided with a total thickness of about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1 .6 mm, about 1 .7 mm, about 1 .8 mm, about 1 .9 mm, about 2.0 mm, about 2.1 mm, about 2.2 mm, about 2.3 mm, about 2.4 mm, about 2.5 mm, about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm, about 3.0 mm, about 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, about 3.5 mm, about 3.6 mm, about 3.7 mm, about 3.8 mm, about 3.9 mm, about 4 mm, and the like, as well as a total thickness that falls within a range of two of the above values (i.e. , a range of from about 0.4 mm to about 4 mm, etc.).

[0047] The test strip devices of the present disclosure are configured for use with fluidic biological samples to enable detection of a plurality of target analytes within the fluidic biological sample. Any fluidic biological sample capable of flowing through the flow path of the test strip devices of the present disclosure may be utilized in the methods disclosed herein. Non-limiting examples of fluidic biological samples that may be utilized in accordance with the present disclosure include urine, whole blood or any portion thereof (i.e., plasma or serum), synovial fluid, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and the like, as well as any combinations thereof.

[0048] In certain particular (but non-limiting) embodiments, the substrate and reagent pads of the test strip devices of the present disclosure are formed of substantially non-chromatographic (i.e., non-lateral flow) materials to ensure that the colorimetric reagents are generally maintained at or near their dispensed location and do not bleed or diffuse sufficiently through or out of the reagent pad or substrate. In addition, the volumes of the dispensed and dried colorimetric reagents are small enough so as to not completely diffuse to the edges or second side of the reagent pads; because of this, when a pad is uniformly rehydrated (i.e., with fluidic biological sample), the colorimetric reagents do not favor further diffusion. As such, the biological sample must be directly dispensed (i.e., pipetted) on the reagent pads of the test strip devices, or the reagent pads must be submerged or otherwise adequately wetted with the fluidic biological sample to ensure contact between the sample and each colorimetric reagent. [0049] In certain particular (but non-limiting) embodiments, the test strip devices of the present disclosure include urinalysis test strip devices. In a particular (but nonlimiting) embodiment, the test strip devices comprise at least 12 colorimetric reagents for the detection of at least 12 target analytes in urine.

[0050] FIGS. 2 and 3 illustrate test strip devices 10a and 10b, respectively, that are identical to the test strip device 10 described with reference to FIG. 1 , except that the test strip devices 10a and 10b have different configurations of reagent pads and colorimetric reagents. The test strip device 10a of FIG. 2 has eight reagent pads attached or otherwise secured thereto; these reagent pads are represented by reference numerals 100, 102, 104, 106, 108, 110, 112, and 114. Reagent pad 100 has two colorimetric reagents 120 and 122 that are each dispensed thereon in a submicroliter volume and at a location that is spatially separate from the other colorimetric reagent, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 120 and 122. Reagent pad 102 has colorimetric reagents 124, 126, and 128 that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other two colorimetric reagents, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 124, 126, and 128. Reagent pad 106 has colorimetric reagents 132 and 134 that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other colorimetric reagent, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 132 and 134. Then each of reagent pads 104, 108, 110, 112, and 114 is illustrated as having only one colorimetric reagent (indicated by reference numerals 130, 136, 138, 140, and 142, respectively) dispensed thereon in a sub-microliter volume. Each of colorimetric reagents 130, 136, 138, 140, and 142 is spatially disposed on their respectively reagent pad at a location that allows for detection of a separate target analyte at each of the locations 130, 136, 138, 140, and 142.

[0051] It will be understood that the test strip devices can be provided with any number of reagent pads, and that each of these reagent pads may be provided with any size and shape, so long as the reagent pad can function as described herein. Further, it will be understood that the reagent pads may be attached to the substrate in any configuration and in any spacing along the substrate, so long as the test strip device can function as described herein. For example, but not by way of limitation, note the different number, sizes, shapes, and placements of the reagent pads 30, 32, 34, 36, and 38 of test strip device 10 of FIG. 1 compared to the reagent pads 100, 102, 104, 106, 108, 110, 112, and 1 14 of test strip device 10a of FIG. 2. Therefore, any number, size, shape, and placement of reagent pads along the substrate to form the test strip device falls within the scope of the present disclosure.

[0052] FIG. 3 illustrates a test strip device 10b that is identical to the test strip device 10a of FIG. 2, except that some of the colorimetric reagents are placed at slightly different locations on the reagent pads. In particular, the test strip device 10b has eight reagent pads attached or otherwise secured thereto; these reagent pads are represented by reference numerals 100b, 102b, 104b, 106b, 108b, 110b, 112b, and 114b. Reagent pad 100b has two colorimetric reagents 120b and 122b that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other colorimetric reagent, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 120b and 122b. Reagent pad 102b has colorimetric reagents 124b, 126b, and 128b that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other two colorimetric reagents, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 124b, 126b, and 128b. Reagent pad 106b has colorimetric reagents 132b and 134b that are each dispensed thereon in a sub-microliter volume and at a location that is spatially separate from the other colorimetric reagent, thereby allowing for detection of a separate target analyte at each of the spatially separate reagent locations 132b and 134b. Then each of reagent pads 104b, 108b, 110b, 112b, and 114b is illustrated as having only one colorimetric reagent (indicated by reference numerals 130b, 136b, 138b, 140b, and 142b, respectively) dispensed thereon in a sub-microliter volume. Each of colorimetric reagents 130b, 136b, 138b, 140b, and 142b is spatially disposed on their respectively reagent pad at a location that allows for detection of a separate target analyte at each of the locations 130b, 136b, 138b, 140b, and 142b.

[0053] Note the slightly different placement of each of the colorimetric reagents 128b, 130b, 136b, 138b, 140b, and 142b on their respective reagent pad compared to their corresponding colorimetric reagent 128, 130, 136, 138, 140, and 142 in FIG. 2. This adjustment demonstrates that the colorimetric reagents may assume any spatial configurations on the reagent pads, so long as they are sufficiently spatially separated so as to allow individual detection thereof upon optical interrogation via a diagnostic instrument.

[0054] In a particular (but non-limiting) embodiment, with reference to FIGS. 2-3, colorimetric reagents 120/120b and 122/122b detect (in no particular order) leukocyte and occult blood; colorimetric reagents 124/124b, 126/126, and 128/128b detect (in no particular order) ketone, specific gravity, and pH; colorimetric reagent 130/130b detects protein/albumin; colorimetric reagents 132/132b and 134/134b detect (in no particular order) bilirubin and whole blood cells; colorimetric reagent 136/136b detects urobilinogen; colorimetric reagent 138/138b detects nitrite; colorimetric reagent 140/140b detects creatinine; and colorimetric reagent 142/142b detects glucose. However, as described herein above, the placement of various colorimetric reagents on a particular reagent pad (or placement of various colorimetric reagents on two or more reagent pads) is shown in the Figures for purposes of example only. It will be understood that the disposal of one or more colorimetric reagents on the same or different reagent pads is determined based on several factors, such as (but not limited to) the compatibility of multiple colorimetric reagents for use with a particular reagent pad material, a diameter of each reagent spot/dot, available space on a single reagent pad, detection parameters of the diagnostic instrument with which the test strip devices are being used, and the like.

[0055] Certain non-limiting embodiments of the present disclosure are directed to methods of determining the presence of at least one target analyte in a fluidic biological sample. In the method, any of the test strip devices disclosed or otherwise contemplated herein is contacted with a fluidic biological sample whereby each of the reagent pads of the test strip device is directly wetted with the fluidic biological sample such that the fluidic biological sample interacts with each of the colorimetric reagents of the test strip device. As disclosed herein above, in certain particular (but nonlimiting) embodiments, the substrate and reagent pads of the test strip devices of the present disclosure are formed of substantially non-chromatographic (i.e. , non-lateral flow) materials to ensure that the colorimetric reagents are generally maintained at or near their dispensed location and do not bleed or diffuse out of the reagent pad or substrate; therefore, contacting the test strip device with the fluidic biological sample requires that the biological sample be directly dispensed (i.e., pipetted) on the reagent pads of the test strip devices, or the reagent pads be submerged or otherwise adequately wetted with the fluidic biological sample to ensure contact between the sample and each colorimetric reagent. The test strip device is then positioned within a diagnostic instrument, and the diagnostic instrument is actuated to optically interrogate each of the colorimetric reagents to determine if the at least one target analyte is present in the fluidic biological sample.

[0056] Certain non-limiting embodiments of the present disclosure are directed to methods of preparing a batch of any of the test strip devices disclosed or otherwise contemplated herein. FIG. 4 illustrates a system 200 for preparing the test strip devices, in which a roll 202 of substrate material is partially unrolled in a direction 204 to provide an unrolled section 206. A first ribbon 208 of filter paper is secured to the unrolled section 206 at a perpendicular angle to the direction of roll 204, and a second ribbon 210 of filter paper (that is different from the filter paper of the first ribbon 208) is secured to the unrolled section 206 at a perpendicular angle to the direction of roll 204 at a position that is spatially separated from the first ribbon 208. The first and second ribbons 208 and 210 may be secured to the unrolled section 206 by any means known in the art or otherwise contemplated herein; in one non-limiting embodiment, the first and/or second ribbons 208 and 210 have an adhesive applied to a lower surface thereof for securing the ribbon 208 and/or 210 to the unrolled section 206 of substrate material.

[0057] The unrolled section 206 of substrate material having the first and second ribbons 208 and 210 secured thereto forms an assembly 212. A first colorimetric reagent 214 is deposited in a row of spots on the first ribbon 208, and a second colorimetric reagent 216 is deposited in a row of spots on the second ribbon 210. Each of the colorimetric reagents 214 and 216 is deposited at each spot in a volume of less than 1 microliter, and each spot of colorimetric reagent is separated from the adjacent spot(s) of the same colorimetric reagent by a distance substantially equal to a width of a test strip device being produced.

[0058] Additional colorimetric reagents may also be deposited in a row of spots on the first or second ribbons 208 or 210 and in the same manner as the other colorimetric reagents 214 and 216; for example (but not by way of limitation), a third colorimetric reagent 218 is illustrated as being deposited in a row of spots on the first ribbon 208. The spots of the colorimetric reagent 218 are less than 1 microliter, and each spot of colorimetric reagent 218 is separated from the adjacent spot(s) of colorimetric reagent 218 by a distance substantially equal to a width of a test strip device being produced. In addition, the spots of the third colorimetric reagent 218 are sufficiently spatially disposed compared to the spots of the first colorimetric reagent 214 so that a diagnostic instrument can individually and separately detect the spots of the colorimetric reagents 214 and 218 on a test strip device.

[0059] The colorimetric reagents may be deposited on the ribbons of filter paper by any methods known in the art or otherwise contemplated herein that are capable of controlling the diameter of the spot/dot of colorimetric reagent and that are capable of preventing diffusion/bleed through of colorimetric reagent through the filter paper to a site of another colorimetric reagent spot/dot. Non-limiting examples of methods that can be utilized include stamping or dispensing with tight volume control.

[0060] Following disposal of any of the colorimetric reagents on a ribbon of filter paper, the assembly may be exposed to a heating or room temperature incubation step to secure one or more colorimetric reagents to the ribbon(s) of filter paper (i.e., by drying the reagent) and to eliminate the possibility that the colorimetric reagents could wick out of the filter paper upon contact with the fluidic biological sample. For example (but not by way of limitation), after depositing the first colorimetric reagent 214 on the first ribbon 208, after depositing the second colorimetric reagent 216 on the second ribbon 210, and/or after depositing the third colorimetric reagent 218 on the first ribbon 208, the assembly may be exposed to a heating device 220 at a temperature in a range of from about 20°C to about 60°C for a period of time sufficient to secure the colorimetric reagent(s) to their respective ribbon(s). Alternatively, one or more of the colorimetric reagents may simply be allowed to incubate at room temperature for a period of time sufficient to secure the colorimetric reagent(s) to their respective ribbon(s). It will be understood that the method may include one or more heating or room temperature incubation steps or combinations thereof, so as to ensure that all colorimetric reagents are firmly secured to their respective ribbons. In one nonlimiting embodiment, only a single drying step (either a heating or room temperature incubation step) is utilized after all colorimetric reagents have been deposited on their respective ribbons.

[0061] In addition, the unrolled section 206 of the substrate material may be rolled back onto the roll (i.e., opposite of direction 204) at any point during the production process, including (but not limited to) after attachment of one or more ribbons and/or after deposition of one or more colorimetric reagents. Also, the assembly 212 containing one or more colorimetric reagents may be stored in rolled form for any period of time prior to addition of other colorimetric reagents and/or prior to cutting to form test strip devices.

[0062] Lastly, the assembly 212 (containing the unrolled section 206 of substrate material having the ribbons 208 and 210 secured thereto and the colorimetric reagents 214, 216, and 218 deposited on the ribbons 208 and 210) is cut in a direction that is parallel to the direction of roll to form a plurality of test strip devices 224.

[0063] FIG. 5 illustrates another system 200a for preparing the test strip devices (with the assembly steps depicted as proceeding from right to left). The system 200a is similar to the system 200 of FIG. 4 except that the first ribbon 208a is dispensed from a roll 209 of a first reagent pad and the second ribbon 210a is dispensed from a roll 211 of a second reagent pad, and then the first and second ribbons 208a and 210a are cut to width from their respective roll 209 and 211 , respectively, to form strips on the assembly 212a to which the colorimetric reagents 214a, 216a, and 218a can then be secured.

[0064] Certain non-limiting embodiments of the present disclosure are directed to kits that include a plurality of any of the test strip devices disclosed or otherwise contemplated herein. In addition, the kits may further contain one or more other component(s) for performing any of the particular methods described or otherwise contemplated herein. For example, but not by way of limitation, the kits may further contain a reservoir in which the fluidic biological sample may be disposed and into which the test strip devices can be inserted or dipped for adequate contact with the fluidic biological sample. Alternatively (and/or in addition thereto), the kits may further contain a dispensing device (such as, but not limited to, a pipette or dropper) for dispensing the fluidic biological sample onto the reagent pads of the test strip devices. The kits can further include a set of written instructions explaining how to use the kit.

[0065] The devices, kits, and methods of the present disclosure allow, by way of example and not by way of limitation, for: (1 ) the improved detection of the presence (or non-presence) of a plurality of analytes of interest that may be present in a low- volume of a patient’s liquid test sample; (2) the improved detection of the presence (or non-presence) of the plurality of analytes of interest present in samples of patient populations that produce low-volumes of liquid test sample output (including, but not limited to, newborns, infants, toddlers, young adults, adults, and elderly populations, as well as persons suffering from conditions that restrict urine output, such as dehydration, kidney disease, urethral strictures, and obstructive uropathies); (3) the ability to incorporate smaller and more numerous analyte testing sites on the test strip to thereby increase the number of analytes that can be detected in a low-volume of a patient’s liquid test sample; and (4) a reduction in the manufacturing costs associated with the production of such test strips due to a decrease in the amount reagent(s) and materials needed to conduct such diagnostic tests. It is to such devices and methods, as well as kits related thereto, that the presently disclosed and claimed inventive concept(s) is directed.

EXAMPLE

[0066] An Example is provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Example is simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0067] In this Example, urinalysis test strip devices are produced that have any of the exemplary configurations of FIGS. 1-3 for paper compatible micro-deposition strips produced with 12 different reagent types. The strips are prepared based on the reagent/paper/adhesive/desiccant compatibilities shown in Table 1.

TABLE 1

Abbreviations: W, Whatman; A, Ahlstrom; Y Y9494 (3M); K, 3028EK (3M); (*), gram minimum recommended for single-pad strips; SG, silica gel; MS, molecular sieve. [0068] The SG (specific gravity)/pH ‘stamped’ reagents were dried in the oven and imaged after dipping in water to check for wicking. Four dispense/dry cycles at 50°C for 30 minutes were utilized to provide adequate color with the concentration used. The resultant strip is shown in FIG. 6.

[0069] Process of Preparing the Test Strips (FIG. 4)

[0070] Filter Papers for forming the ribbons of reagent pads are received in 12” to 24” rolls. Adhesive backer is placed onto each roll. These rolls are slit into 60/120 1/5” and optionally 120/240 1/10” half-length reels. The slit reels have their adhesive’s protective film removed and are then processed with rolls of plastic substrate material to make new single rolls of un-singulated strips. Batch preparation of reagents can be laborious, so a few reagents may need to be deposited at a time with the roll being stored desiccated between depositing batches. Depending on reagent stability, a drying step may be utilized between sets or once upon completion. For example (but not by way of limitation), after application of one or more reagents, the strips may be dried at 50°C for at least about 30 minutes to effectively eliminate wicking of reagent after being dipped in a sample, as demonstrated with the specific gravity (SG) and pH reagents of FIG. 6.

[0071] Reagent spot diameter can be controlled by stamping or dispensing the reagents onto the reagent pad with tight volume control. When stamping, a submicroliter volume is used, and the diameter is maintained by the low volume utilized, thereby reducing or eliminating wicking away from the surface deposit. Dispensing a drop may require slightly more volume to provide consistency between replicates, and the dispensed volume may need to be varied to control diameter if a uniform spot diameter between different reagents is desired.

NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0072] The following is a list of non-limiting illustrative embodiments disclosed herein:

[0073] Illustrative embodiment 1 . A test strip device for detecting the presence of a plurality of target analytes in a fluidic biological sample, the test strip device comprising: (i) a substrate; (ii) a plurality of reagent pads secured to the substrate, wherein at least two of the plurality of reagent pads are formed of a separate material; (iii) a plurality of colorimetric reagents, wherein each colorimetric reagent detects one of the target analytes, wherein each of the plurality of colorimetric reagents is deposited at a location on a reagent pad that is spatially separate from other colorimetric reagents, and wherein each colorimetric reagent is deposited on the reagent pad at a volume of less than 1 microliter; and wherein at least one of the reagent pads has at least two colorimetric reagents deposited thereon at spatially separate locations that allow for detection of separate target analytes at each of the at least two spatially separate locations; and wherein the test strip device is structured and configured for positioning within a diagnostic instrument for optical interrogation of the plurality of colorimetric reagents.

[0074] Illustrative embodiment 2. The test strip device of illustrative embodiment 1 , wherein each colorimetric reagent is deposited on the reagent pad at a volume in a range of from about 1 nl to about 900 nl.

[0075] Illustrative embodiment 3. The test strip device of illustrative embodiment 1 or 2, wherein at least two of the reagent pads has at least two colorimetric reagents deposited thereon.

[0076] Illustrative embodiment 4. The test strip device of illustrative embodiment 3, wherein at least one of the reagent pads has at least three colorimetric reagents deposited thereon.

[0077] Illustrative embodiment 5. The test strip device of illustrative embodiment 4, wherein at least one of the reagent pads has at least four colorimetric reagents deposited thereon.

[0078] Illustrative embodiment 6. The test strip device of any of illustrative embodiments 1-5, wherein the substrate is formed of a material selected from the group consisting of acrylic, polystyrene, styrene-acrylonitrile, polycarbonate, polyethylene terephthalate, and combinations thereof.

[0079] Illustrative embodiment 7. The test strip device of any of illustrative embodiments 1-6, wherein each of the reagent pads is formed of a quantitative or qualitative natural or synthetic filter paper.

[0080] Illustrative embodiment 8. The test strip device of any of illustrative embodiments 1-7, wherein each of the reagent pads is formed of a material independently selected from the group consisting of cellulose, nitrocellulose, cotton, spun glass, silica glass, soda lime glass, glass microfiber, cellulose acetate, cellulose nitrate, nylon, polyester film, polyethylene terephthalate, polycarbonate, and polystyrene, and combinations thereof. [0081] Illustrative embodiment 9. The device of any of illustrative embodiments 1- 8, wherein the device is configured for use with a fluidic biological sample selected from the group consisting of urine, whole blood or any portion thereof, synovial fluid, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0082] Illustrative embodiment 10. The device of illustrative embodiment 9, further defined as a urinalysis test strip device.

[0083] Illustrative embodiment 1 1. The device of any of illustrative embodiments 1- 10, wherein the plurality of target analytes comprises at least two target analytes, and wherein each of the at least two analytes is selected from the group consisting of glucose, bilirubin, ketones, blood, proteins, urobilinogen, nitrites, leukocytes, albumin, creatinine, ascorbic acid, specific gravity, and pH.

[0084] Illustrative embodiment 12. The device of illustrative embodiment 11 , wherein (ii) is further defined as at least two reagent pads secured to the substrate, wherein each of the at least two reagent pads are selected from the group consisting of: (a) a reagent pad comprising colorimetric reagents for detection of leukocyte, occult blood, creatinine, and glucose; (b) a reagent pad comprising colorimetric reagents for detection of ketone, specific gravity, pH, and protein; (c) a reagent pad comprising colorimetric reagents for detection of bilirubin and whole blood cells; (d) a reagent pad comprising a colorimetric reagent for detection of urobilinogen; and (e) a reagent pad comprising a colorimetric reagent for detection of nitrite.

[0085] Illustrative embodiment 13. The device of illustrative embodiment 12, wherein (ii) is further defined as at least five reagent pads secured to the substrate, and wherein the at least five reagent pads comprise (a)-(e).

[0086] Illustrative embodiment 14. The device of illustrative embodiment 11 , wherein (ii) is further defined as at least two reagent pads secured to the substrate, wherein each of the at least two reagent pads are selected from the group consisting of: (a) a reagent pad comprising colorimetric reagents for detection of leukocyte and occult blood; (b) a reagent pad comprising colorimetric reagents for detection of ketone, specific gravity, and pH; (c) a reagent pad comprising a colorimetric reagent for detection of protein/albumin; (d) a reagent pad comprising colorimetric reagents for detection of bilirubin and whole blood cells; (e) a reagent pad comprising a colorimetric reagent for detection of urobilinogen; (f) a reagent pad comprising a colorimetric reagent for detection of nitrite; (g) a reagent pad comprising a colorimetric reagent for detection of creatinine; and (h) a reagent pad comprising a colorimetric reagent for detection of glucose.

[0087] Illustrative embodiment 15. The device of illustrative embodiment 14, wherein (ii) is further defined as at least eight reagent pads secured to the substrate, and wherein the at least eight reagent pads comprise (a)-(h).

[0088] Illustrative embodiment 16. The device of any of illustrative embodiments 1-

15, wherein the plurality of colorimetric reagents comprises at least 12 colorimetric reagents for the detection of at least twelve target analytes.

[0089] Illustrative embodiment 17. The device of any of illustrative embodiments 1-

16, wherein at least one of the plurality of colorimetric reagents is selected from the group consisting of glucose oxidase, peroxidase, potassium iodide, 2,4-dichloroaniline diazonium salt, sodium nitroprusside, bromothymol blue, methyl vinyl ether, maleic anhydride, sodium hydroxide, diisopropylbenzene dihydroperoxide, 3, 3’, 5, 5’- tetramethylbenzidine, methyl red, tetrabromophenol blue, p-diethylamino- benzaldehyde, p-arsanilic acid, 1 , 2, 3, 4-tetrahydrobenzo(h) quinolin-3-ol, derivatized pyrrole amino acid ester, bis (3’,3”-diiodo-4’,4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6- tetrabromosulfonepthalein, copper sulfate, and diazonium salt, or combinations thereof.

[0090] Illustrative embodiment 18. A method of determining the presence of at least one target analyte in a fluidic biological sample, comprising the steps of: contacting the test strip device of any of illustrative embodiments 1-17 with a fluidic biological sample whereby each of the reagent pads of the test strip device is directly wetted with the fluidic biological sample such that the fluidic biological sample interacts with each of the colorimetric reagents of the test strip device; positioning the test strip device into a diagnostic instrument; and actuating the diagnostic instrument to optically interrogate each of the colorimetric reagents to determine if the at least one target analyte is present in the fluidic biological sample.

[0091] Illustrative embodiment 19. The method of illustrative embodiment 18, wherein the fluidic biological sample is selected from the group consisting of urine, whole blood or any portion thereof, synovial fluid, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof. [0092] Illustrative embodiment 20. A method of preparing a batch of test strip devices, wherein each of the test strip devices is structured and configured for positioning within a diagnostic instrument for detection of the presence of a plurality of target analytes in a fluidic biological sample, wherein the method comprises the steps of: (i) unrolling a portion of a roll of substrate material; (ii) securing at least a first ribbon to the unrolled portion of substrate material at a perpendicular angle to a direction of roll; (iii) securing at least a second ribbon to the unrolled portion of substrate material at a perpendicular angle to a direction of roll, wherein the first and second ribbons are spatially separated from one another and are formed of a separate material, thereby forming an assembly of the unrolled portion of substrate material and the first and second ribbons; (iv) depositing at least a first colorimetric reagent in a row of spots on the at least first ribbon, wherein the colorimetric reagent is deposited at each spot in a volume of less than 1 microliter, and wherein the spots are separated by a distance substantially equal to a width of a test strip device; (v) depositing at least a second colorimetric reagent in a row of spots on the at least second ribbon, wherein the colorimetric reagent is deposited at each spot in a volume of less than 1 microliter, and wherein the spots are separated by a distance substantially equal to a width of a test strip device; and (vi) cutting the assembly in a direction parallel to the direction of roll to form a plurality of test strip devices.

[0093] Illustrative embodiment 21. The method of illustrative embodiment 20, further comprising the step of, after at least one of steps (iv) and (v), exposing the assembly to a temperature in a range of from about 20°C to about 60°C for a period of time sufficient to secure the first colorimetric reagent to the first ribbon.

[0094] Illustrative embodiment 22. The method of illustrative embodiment 21 , wherein the exposure step is performed before step (v).

[0095] Illustrative embodiment 23. The method of illustrative embodiment 21 or 22, wherein the exposure step is performed after step (v).

[0096] Illustrative embodiment 24. The method of any of illustrative embodiments 21-23, wherein the exposure step is performed before step (v) and repeated after step (v).

[0097] Illustrative embodiment 25. The method of any of illustrative embodiments 20-24, wherein the at least first and second ribbons have an adhesive applied to a lower surface thereof for securing the ribbon to the roll of substrate material. [0098] Illustrative embodiment 26. The method of any of illustrative embodiments 20-25, further comprising the step of rolling the assembly prior to at least one of steps (iii), (iv), (v), and/or (vi).

[0099] Illustrative embodiment 27. The method of any of illustrative embodiments 20-26, wherein in step (iv) and/or (v), the reagents are deposited by stamping or dispensing.

[0100] Illustrative embodiment 28. The method of any of illustrative embodiments 20-27, wherein one or more of steps (i)-(v) are repeated prior to step (vi).

[0101] Illustrative embodiment 29. The method of any of illustrative embodiments 20-28, further defined as producing the test strip device of any of illustrative embodiments 1-17.

[0102] Thus, in accordance with the present disclosure, there have been provided devices and kits, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. A test strip device for detecting the presence of a plurality of target analytes in a fluidic biological sample, the test strip device comprising:

(i) a substrate;

(ii) a plurality of reagent pads secured to the substrate, wherein at least two of the plurality of reagent pads are formed of a separate material;

(iii) a plurality of colorimetric reagents, wherein each colorimetric reagent detects one of the target analytes, wherein each of the plurality of colorimetric reagents is deposited at a location on a reagent pad that is spatially separate from other colorimetric reagents, and wherein each colorimetric reagent is deposited on the reagent pad at a volume of less than 1 microliter; and wherein at least one of the reagent pads has at least two colorimetric reagents deposited thereon at spatially separate locations that allow for detection of separate target analytes at each of the at least two spatially separate locations; and wherein the test strip device is structured and configured for positioning within a diagnostic instrument for optical interrogation of the plurality of colorimetric reagents.

2. The test strip device of claim 1 , wherein each colorimetric reagent is deposited on the reagent pad at a volume in a range of from about 1 nl to about 900 nl.

3. The test strip device of claim 1 , wherein at least two of the reagent pads has at least two colorimetric reagents deposited thereon.

4. The test strip device of claim 3, wherein at least one of the reagent pads has at least three colorimetric reagents deposited thereon.

5. The test strip device of claim 4, wherein at least one of the reagent pads has at least four colorimetric reagents deposited thereon.

6. The test strip device of claim 1 , wherein the substrate is formed of a material selected from the group consisting of acrylic, polystyrene, styrene-acrylonitrile, polycarbonate, polyethylene terephthalate, and combinations thereof.

7. The test strip device of claim 1 , wherein each of the reagent pads is formed of a quantitative or qualitative natural or synthetic filter paper.

8. The test strip device of claim 1 , wherein each of the reagent pads is formed of a material independently selected from the group consisting of cellulose, nitrocellulose, cotton, spun glass, silica glass, soda lime glass, glass microfiber, cellulose acetate, cellulose nitrate, nylon, polyester film, polyethylene terephthalate, polycarbonate, and polystyrene, and combinations thereof.

9. The device of claim 1 , wherein the device is configured for use with a fluidic biological sample selected from the group consisting of urine, whole blood or any portion thereof, synovial fluid, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cysticfluid, sweat, interstitial fluid, extracellularfluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

10. The device of claim 9, further defined as a urinalysis test strip device.

11 . The device of claim 1 , wherein the plurality of target analytes comprises at least two target analytes, and wherein each of the at least two analytes is selected from the group consisting of glucose, bilirubin, ketones, blood, proteins, urobilinogen, nitrites, leukocytes, albumin, creatinine, ascorbic acid, specific gravity, and pH.

12. The device of claim 11 , wherein (ii) is further defined as at least two reagent pads secured to the substrate, wherein each of the at least two reagent pads are selected from the group consisting of:

(a) a reagent pad comprising colorimetric reagents for detection of leukocyte, occult blood, creatinine, and glucose;

(b) a reagent pad comprising colorimetric reagents for detection of ketone, specific gravity, pH, and protein; (c) a reagent pad comprising colorimetric reagents for detection of bilirubin and whole blood cells;

(d) a reagent pad comprising a colorimetric reagent for detection of urobilinogen; and

(e) a reagent pad comprising a colorimetric reagent for detection of nitrite.

13. The device of claim 12, wherein (ii) is further defined as at least five reagent pads secured to the substrate, and wherein the at least five reagent pads comprise

(a)-(e).

14. The device of claim 11 , wherein (ii) is further defined as at least two reagent pads secured to the substrate, wherein each of the at least two reagent pads are selected from the group consisting of:

(a) a reagent pad comprising colorimetric reagents for detection of leukocyte and occult blood;

(b) a reagent pad comprising colorimetric reagents for detection of ketone, specific gravity, and pH;

(c) a reagent pad comprising a colorimetric reagent for detection of protein/albumin;

(d) a reagent pad comprising colorimetric reagents for detection of bilirubin and whole blood cells;

(e) a reagent pad comprising a colorimetric reagent for detection of urobilinogen;

(f) a reagent pad comprising a colorimetric reagent for detection of nitrite;

(g) a reagent pad comprising a colorimetric reagent for detection of creatinine; and

(h) a reagent pad comprising a colorimetric reagent for detection of glucose.

15. The device of claim 14, wherein (ii) is further defined as at least eight reagent pads secured to the substrate, and wherein the at least eight reagent pads comprise (a)-(h).

16. The device of claim 1 , wherein the plurality of colorimetric reagents comprises at least 12 colorimetric reagents for the detection of at least twelve target analytes.

17. The device of claim 1 , wherein at least one of the plurality of colorimetric reagents is selected from the group consisting of glucose oxidase, peroxidase, potassium iodide, 2,4-dichloroaniline diazonium salt, sodium nitroprusside, bromothymol blue, methyl vinyl ether, maleic anhydride, sodium hydroxide, diisopropylbenzene dihydroperoxide, 3, 3’, 5, 5’ -tetramethylbenzidine, methyl red, tetrabromophenol blue, p-diethylamino-benzaldehyde, p-arsanilic acid, 1 , 2, 3, 4- tetrahydrobenzo(h) quinolin-3-ol, derivatized pyrrole amino acid ester, bis (3’, 3”- diiodo-4’,4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetrabromosulfonepthalein, copper sulfate, and diazonium salt, or combinations thereof.

18. A method of determining the presence of at least one target analyte in a fluidic biological sample, comprising the steps of: contacting the test strip device of any one of claims 1-17 with a fluidic biological sample whereby each of the reagent pads of the test strip device is directly wetted with the fluidic biological sample such that the fluidic biological sample interacts with each of the colorimetric reagents of the test strip device; positioning the test strip device into a diagnostic instrument; and actuating the diagnostic instrument to optically interrogate each of the colorimetric reagents to determine if the at least one target analyte is present in the fluidic biological sample.

19. The method of claim 18, wherein the fluidic biological sample is selected from the group consisting of urine, whole blood or any portion thereof, synovial fluid, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

20. A method of preparing a batch of test strip devices, wherein each of the test strip devices is structured and configured for positioning within a diagnostic instrument for detection of the presence of a plurality of target analytes in a fluidic biological sample, wherein the method comprises the steps of: (i) unrolling a portion of a roll of substrate material;

(ii) securing at least a first ribbon to the unrolled portion of substrate material at a perpendicular angle to a direction of roll;

(iii) securing at least a second ribbon to the unrolled portion of substrate material at a perpendicular angle to a direction of roll, wherein the first and second ribbons are spatially separated from one another and are formed of a separate material, thereby forming an assembly of the unrolled portion of substrate material and the first and second ribbons;

(iv) depositing at least a first colorimetric reagent in a row of spots on the at least first ribbon, wherein the colorimetric reagent is deposited at each spot in a volume of less than 1 microliter, and wherein the spots are separated by a distance substantially equal to a width of a test strip device;

(v) depositing at least a second colorimetric reagent in a row of spots on the at least second ribbon, wherein the colorimetric reagent is deposited at each spot in a volume of less than 1 microliter, and wherein the spots are separated by a distance substantially equal to a width of a test strip device; and

(vi) cutting the assembly in a direction parallel to the direction of roll to form a plurality of test strip devices.

21. The method of claim 20, further comprising the step of, after at least one of steps (iv) and (v), exposing the assembly to a temperature in a range of from about 20°C to about 60°C for a period of time sufficient to secure the first colorimetric reagent to the first ribbon.

22. The method of claim 21 , wherein the exposure step is performed before step (v).

23. The method of claim 21 , wherein the exposure step is performed after step (v).

24. The method of claim 21 , wherein the exposure step is performed before step (v) and repeated after step (v).

25. The method of claim 20, wherein the at least first and second ribbons have an adhesive applied to a lower surface thereof for securing the ribbon to the roll of substrate material.

26. The method of claim 20, further comprising the step of rolling the assembly prior to at least one of steps (iii), (iv), (v), and/or (vi).

27. The method of claim 20, wherein in step (iv) and/or (v), the reagents are deposited by stamping or dispensing.

28. The method of claim 20, wherein one or more of steps (i)-(v) are repeated prior to step (vi).