Detection of target analyte in sample

Abstract

A device, method, and kit are disclosed for use in detecting a target analyte in a sample. The device includes a region having a first zone, into which the sample can be delivered, and a second zone in fluid communication with the first zone. The device further makes use of a recognition element that is covalently or non-covalently conjugated to a signal molecule, and a probe that is covalently or non-covalently conjugated to the recognition element, or taken together with the recognition element, forms a single unit. The probe, the recognition element, or the signal molecule has an affinity for the target analyte, such that in the presence of the target analyte, the signal molecule is free from or does not attach to the first zone, allowing the signal molecule to move away from the first zone.

Description

DETECTION OF TARGET ANALYTE IN SAMPLERELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 556,085 filed February 21, 2024. the entire disclosure of which is incorporated herein by this reference.TECHNICAL FIELD

[0002] The presently-disclosed subject matter generally relates to on-site detection of a target analyte in a sample. In particular, certain embodiments of the presently-disclosed subject matter relate on-site sample analysis tools, which are adaptable and user-friendly, and which can be used in any convenient location, including the site of a sample of interest, or in a location where a subject providing a biological sample is located.INTRODUCTION

[0003] Providing on-site sample analysis can address a number of important needs. Onsite detection tools allow for environmental testing of water, soil, or air samples in their native location without the need to collect and transport samples to a laboratory'. In the context of patient care, on-site detection tools such as point-of-care (POC) diagnostic systems provide the opportunity for testing at a convenient location, and shorten the time from analysis to diagnosis, thereby improving care and treatment decisions.

[0004] As will be appreciated, in some circumstances, the ability to provide for POC diagnostic systems can be critical for patient care. For example, deployed military personal or individuals who are operating in remote locations are often unable to physically relocate or to deliver uncompromised samples to a facility having diagnostic laboratory equipment. Additionally, traditionally-marginalized communities, rural communities, and socioeconomically-challenged communities carry a disproportionate burden from both communicable and noncommunicable diseases, which is due in part to the lack of early diagnosis and pathology services, which are critical for early detection, diagnosis, anddisease management. Traditionally-marginalized communities may also be less likely to visit and trust medical professionals. Greater access to effective POC diagnostic systems could help individuals in these and other circumstances, providing access to quick, portable, and simple diagnostic and analytical feedback. Opportunities for telehealth would also greatly benefit from the ability' to take a diagnostic test at home and upload or report the results to a medical professional.

[0005] Increased availability of POC diagnostic devices for use at home or in remote areas, without a need for trained professionals, could encourage individuals to seek medical care while providing information for initial diagnosis and disease management. As exemplified by the COVID- 19 pandemic, rapid analysis can greatly slow the spread of communicable disease by identifying infected individuals.

[0006] Early examples of POC systems include a 1950s-era dipstick formulated for glucose quantification. Since then. POC devices have evolved to become common in everyday life, including glucose monitors for diabetics and at-home pregnancy tests. Current POC systems offer the advantages of portability7and quick readout compared to traditional laboratory-based analysis but still suffer from several issues, including complicated protocols that may be difficult for untrained personnel to complete, limited stability7and shelf life of biological components, the requirement of specialized instrumentation, susceptibility to matrix effects, and limited ability to detect certain analytes.

[0007] Accordingly, there remains a need in the art for improved on-site sample analysis tools, which are adaptable and user-friendly, and which can be used in any convenient location, including the site of a sample of interest (such as the site of testing water, soil, air quality, etc.) or in a location where a patient is located (such as when a biological sample will be tested). For diagnostic and other applications involving a biological sample from a patient, such a device would be particularly valuable to those who are unable or less likely to seek medical care in a traditional office setting, and for use in remote regions where advanced medical instrumentation and lab analysis are not available.SUMMARY

[0008] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0009] The presently-disclosed subject matter includes a device for detecting a target analyte in a sample. The device includes a region having a first zone, into which the sample can be delivered, and a second zone in fluid communication with the first zone.

[0010] In some embodiments, the device makes use of a probe bound within the first zone, a recognition element, and a signal molecule that is attached to the probe or the recognition element. In some embodiments, the probe is either covalently or non-covalently conjugated to a recognition element. In some embodiments, the probe taken together with the recognition element forms a single unit. In some embodiments, the probe, the recognition element, or the signal molecule has an affinity for the target analyte. In some embodiments, the target analyte cleaves or allows the release of the recognition element. In this regard, as will be described herein in greater detail, in the presence of the target analyte, the signal molecule is released from the first zone, allowing the signal molecule to move away from the first zone.

[0011] In some embodiments of the device, the signal molecule is attached to the recognition element, and the signal molecule or the recognition element has an affinity for the probe, and a stronger affinity for the target analyte. In such embodiments of the device, in the absence of the target analyte, the signal molecule is bound within the first zone, due to the attachment of the signal molecule to the recognition element and the affinity of the signal molecule or recognition element for the probe, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the signal molecule or the recognition element and the target analyte. In this regard, in some embodiments, the target analyte is a target nucleotide, the probe is a probe nucleotide, and the recognition element is a recognition nucleotide conjugated to the probe nucleotide through complementary base-pairing, wherein the recognition nucleotide has an affinity for the probe nucleotide, and a stronger affinity for the target nucleotide.

[0012] In some embodiments of the device, the probe and the recognition element, taken together, form a single unit that is a polypeptide; and the recognition element is an amino acid sequence recognized for cleaving by a target analyte that is a protease. In such embodiments, in the absence of the target protease, the signal molecule is bound within the first zone due to polypeptide remaining intact, and in the presence of the target protease, the polypeptide is cleaved, releasing the signal molecule from the first zone.

[0013] In some embodiments of the device, the probe or the recognition element has an affinity for the signal molecule, and a stronger affinity for the target analyte. In such embodiments, in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the signal molecule and the probe or the recognition element, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the probe or the recognition element and the target analyte. In this regard, in some embodiments, the target analyte is a polypeptide or small molecule, and the recognition element has an affinity for the signal molecule, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the recognition element and the signal molecule; and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the recognition element and the target analyte.

[0014] In some embodiments of the device, the signal molecule has an affinity for the recognition element, and a stronger affinity for the target analyte. In such embodiments, in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the signal molecule and the recognition element; and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the signal molecule and the target analyte. In some embodiments, the target analyte is a polypeptide or small molecule.

[0015] In some embodiments of the device, in which the target analyte can be recognized by an aptamer, the probe and the recognition element, taken together, form a single unit that is an aptamer bound within the first zone, wherein the aptamer has a stronger affinity for the target analyte than for the first zone. In such embodiments, in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the aptamer and the first zone, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the aptamer and the target analyte.

[0016] In some embodiments of the device, in which the target analyte can be recognized by an aptamer, the signal molecule is attached to the recognition element, the probe is a probe nucleotide, and the recognition element is a nucleotide aptamer conjugated to the probe nucleotide through complementary base-pairing, wherein the nucleotide aptamer has anaffinity for the probe nucleotide, and a stronger affinity for the target analyte. In such embodiments, in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the nucleotide aptamer and the probe, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the nucleotide aptamer and the target analyte.

[0017] The presently-disclosed subject matter further includes a kit for detecting a target analyte in a sample. In some embodiments, the kit includes a device and preparation reagents. In some embodiments, the kit includes a device and preparation reagents provided in a sample preparation container including, for example, lysis buffers, binding buffers, extraction buffers, and sample collection swabs. The device comprises a first zone, into which the sample can be delivered, and a second zone in fluid communication with the first zone. The preparation reagents are designed for preparing the sample before it is delivered to the first zone of the device. The preparation reagents can be provided, for example, in a container that is packaged together with the device.

[0018] In some embodiments of the kit, the preparation reagents include a probe that is covalently or non-covalently conjugated to a recognition element, or a probe that, taken together with the recognition element, forms a single unit. In some embodiments of the kit, the preparation reagents further include a signal molecule attached to the probe or the recognition element, wherein the probe or the recognition element selectively binds the target analyte.

[0019] In some embodiments of the kit, the probe is covalently or non-covalently conjugated to a recognition element. In some embodiments, the device further includes a binding molecule that recognizes the probe. In some embodiments, the probe, recognition element, and binding molecule could be nucleotides that recognize one another through complementary’ base-pairing. In some embodiments, the probe, recognition element, and binding molecule could be a combination of polypeptides and aptamers, antibodies, or fragments thereof. In some embodiments, the probe, recognition element, and binding molecule could be a combination of nucleotides and aptamers.

[0020] In some embodiments of the kit, wherein the probe and the recognition element, taken together, form a single unit attached to the signal molecule. The single unit can be, forexample, an aptamer, an antibody, or an antibody fragment that selectively binds the target analyte.

[0021] In some embodiments of the kit, confirmed target analytes are obtained from a source distinct from any sample, and are covalently or non-covalently bound within the first zone of the device. In some embodiments, the probe and the recognition element, taken together, form a single unit attached to the signal molecule, and the confirmed target analytes bound within the first zone can be recognized by that single unit. In such embodiments, in the absence of the target analyte in the sample, when the preparation is delivered to the first zone, the signal molecule attached to the single unit remains within the first zone due to the affinity between the single unit and the confirmed target analytes bound within the first zone, and in the presence of the target analyte in the sample, the signal molecule attached to the single unit in the preparation becomes bound to the target analyte in the sample, such that when the preparation is delivered to the first zone, the signal molecule is free to migrate away from the first zone because the single unit is unable to bind the confirmed target analytes bound within the first zone.

[0022] In some embodiments of the kit, the device further includes a binding molecule that recognizes the target analyte covalently or non-covalently bound within the first zone. In some embodiments, the probe and the recognition element, taken together, form a single unit attached to the signal molecule. When using such an embodiments, the sample is delivered to the first zone of the device. Subsequently, the preparation reagents are added to the first zone of the device. In such embodiments, in the absence of the target analyte in the sample, upon delivery of the sample to the first zone, no target analyte is available to attach to the binding molecule, and when the preparation reagents are delivered to the first zone the signal molecule attached to the single unit is free to migrate away from the first zone. However, in such embodiments, in the presence of the target analyte in the sample, upon delivery of the sample to the first zone, the target analyte attaches to the binding molecule in the first zone and when the signal molecule attached to the single unit is delivered to the first zone, it remains within the first zone due to the affinity between the single unit and the target analyte.

[0023] The presently-disclosed subject matter further includes a method for detecting a target analyte in a sample, which involves delivering the sample to a device as disclosed herein such that the sample enters the first zone, and detecting a location of the signal molecule. In some embodiments, the method further involves determining the amount of thesignal molecule present in the first zone and / or determining the amount of the signal molecule outside of the first zone. In some embodiments, the method further involves comparing the amount of the signal molecule present in the first zone to the amount of the signal molecule outside of the first zone.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0025] FIG. 1A and IB are schematics illustrating the operation of an exemplary embodiment of the presently-disclosed subject matter in the absence of a target analyte (FIG. 1 A) and in the presence of a target analyte (FIG. IB).

[0026] FIG. 2 is a schematic illustrating an embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a nucleotide.

[0027] FIG. 3 is a schematic illustrating an embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a polypeptide having proteolytic enzymatic activity.

[0028] FIG. 4 is a schematic illustrating an embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a polypeptide.

[0029] FIG. 5 is a schematic illustrating another embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a polypeptide.

[0030] FIG. 6 is a schematic illustrating an embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer.

[0031] FIG. 7 is a schematic illustrating another embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer.

[0032] FIG. 8 is a schematic illustrating an embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer, antibody, or antibody fragment.

[0033] FIG. 9A-9C are schematics illustrating another embodiment of a device provided in accordance with the presently-disclosed subject matter for use in detecting a target analyte that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer, antibody, or antibody fragment.DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0034] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary' limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0035] The presently-disclosed subject matter includes devices, kits, and methods, for use in detecting a small molecule, a nucleotide, or a polypeptide target analyte in a sample. Relevant samples include any that may contain a target analyte of interest. Examples of relevant samples include biological samples, such as urine, serum, blood, plasma, saliva, sputum, feces, tear, hair, nails, whole cells, soil, water, air, manufacturing materials, and food / beverage industry samples. In some embodiments, the sample could be a non-fluid sample, in which case it could be prepared prior to analysis, for example, by using appropriate reagents, such as solubilization reagents and lysis buffers. In some embodiments, sample preparation could also include extraction (e.g., whole cell extraction; extraction from aqueous or organic solutions), filtration (e g., to remove red blood cells), and / or amplification steps (e.g., nucleotide amplification by PCR).

[0036] As will be appreciated by one of ordinary skill in the art, where biological samples are being employed, the device, kit. and method disclosed herein have diagnostic and prognostic applications. For example, embodiments of the presently-disclosed subject matter could be used in connection with alpha-1 antitrypsin, a deficiency of which can lead to severe lung and liver disease, could be detected. For another example, embodiments of the presently-disclosed subject matter could be used in connection with high-risk genotypes of HPV 16 and HPV 18 / 45. which are associated with 74% of cervical cancer cases. For another example, embodiments of the presently-disclosed subject matter could be used in connection with detection and risk assessment of prostate cancer. For another example, embodiments of the presently-disclosed subject matter could be used in connection with pre-diabetes screen and kidney function. For another example, embodiments of the presently -disclosed subject matter could be used in connection with identification of bacterial or yeast populations in the ethanol production industry. For another example, embodiments of the presently-disclosed subject matter could be used in connection with the detection of cortisol levels in saliva of human or other mammal subjects. Of course, there are many applications for other sample types, for example, samples obtained from water, soil, or air sources. Additional examples include, for example, samples obtained in connection with the food and beverage industry, including the di sti 11 ery industry, and samples obtained in the manufacturing industry or chemical production industry.

[0037] The device of the presently-disclosed subject matter includes a region having a first zone, into which the sample can be delivered, and a second zone in fluid communication with the first zone.

[0038] In some embodiments, the device makes use of a probe bound within the first zone, a recognition element, and a signal molecule that is attached to the probe or the recognition element. In some embodiments, the probe is either covalently or non-covalently conjugated to a recognition element. In some embodiments, the probe taken together with the recognition element forms a single unit. In some embodiments, the probe, the recognition element, or the signal molecule has an affinity for the target analyte. In some embodiments, the target analyte cleaves or allow s the release of the recognition element. In this regard, as will be described herein in greater detail, in the presence of the target analyte, the signal molecule is released from the first zone, allowing the signal molecule to move away from the first zone.

[0039] The structure and material of the device employed in connection with the presently-disclosed subject matter can vary, so long as it includes the features as disclosed herein. Examples of types of devices that could be employed include, but are not limited to, lateral flow immunoassay (LFIA) devices, lateral flow devices including, for example, nitrocellulose membranes, capillary tube, paper-based microfluidic devices (pPAD), other microfluidic devices such as, for example, lab-on-a-chip or lab-on-a-disc devices. As will be appreciated by the skilled artisan upon studying this document, in certain embodiments of the device of the presently-disclosed subject matter, the first zone and second zone that are in fluid communication could be part of a multi-channel or multi-zone region or chamber having any of a variety of patterns for multiplexed analysis. Such analysis, and preferential direction with the multi-channel or multi-zone design could be performed with volume based, pressure based, centripetal force based and / or time based strategies. Also contemplated are multiplexed analyses involving manipulation of the signal molecule (e.g., changing the fluorophore attached, wherein each fluorophore would correspond to a particular target analyte).

[0040] Operation of an embodiment of a device of the presently-disclosed subject matter is described with reference to FIG. 1A and IB. The exemplary device 10 includes a region having a first zone, identified in FIG. 1A and IB as “Zone 1,'’ into which a sample is delivered for detecting a target analyte 20 in that sample. Also provided in this first zone is a probe 14, which covalently or non-covalently bound within the first zone. The probe 14 may be attached to the surface of the first zone by any number of covalent or non-covalent interactions. Solid surfaces including but not limited to polymers, glass, cellulose (and modified varieties), silicon, carbon, metals (gold, silver, etc), can be modified through mechanisms including but not limited to physical adsorption, surface morphology, electrostatic interactions, chemical modifiers of hydroxyl, carboxyl, amino, aldehyde, epoxide, and hydrazide functional groups for covalent conjugation (EDC / NHS conjugation), click chemistry, photochemical methods, modifications of materials with biotin / streptavidin for non-covalent but permanent immobilization, metal-thiol covalent bonds, hydrophobic interactions, and pi stacking to immobilized (bio)molecules to the surface.30'34

[0041] Depending on the embodiment of the device 10, the probe 14 is covalently or non- covalently conjugated to a recognition element 18, or the probe 14, taken together with the recognition element 18, forms a single unit 14, 18. As will be appreciated by one of ordinaryskill in the art upon study of this document, the probe 14, recognition element 18, and signal molecule can take different forms depending on the target analyte and the desired operation of the device. The exemplary device pictured in FIG. 1A and IB can also be found in FIG. 4, which is described in more detail below.

[0042] Notwithstanding distinct embodiments, as disclosed herein, the probe, the recognition element, or the signal molecule of the device has an affinity for the target analyte, such that in the presence of the target analyte, the signal molecule will accumulate in a particular location and / or in particular amounts, allowing for detection of and / or intensity' determination of the signal molecule to correlate with presence and / or amount of target analyte in the sample.

[0043] With continued reference to FIG. 1A and IB, the exemplar}' device 10 also includes a second zone, identified in as “analysis zone 2,’’ which is in fluid communication with the first zone and towards which the signal molecule 16 can migrate when it is free from the first zone.

[0044] As will be appreciated by one of ordinary' skill in the art, any number of signal molecules can be employed, so long as they are capable of being attached to a nucleotide or to a polypeptide and capable of producing a detectable signal. Examples of signal molecules that can be used in accordance with the presently-disclosed subject matter include, but are not limited to, fluorescent molecules, colorimetric, dyes, detection systems, magnetic molecules, electro chemical molecules, redox-active molecules, mass-based tags, and combinations thereof. With regard to fluorescent molecules, there are many examples that will be known to one of ordinary' skill in the art, including polypeptide and small molecule examples. Cy3 and Cy5 fluorophores are two examples. There are also many examples of dyes that could be employed, which include, but are not limited to, acridine, anthraquinone, azo, thiazole, and phenol based dyes. Examples of detection systems that can be used will also be known to one of ordinary skill in the art and include, but are not limited to gold (Au), quantum dots, and cobalt (Co) detection systems. Magnetic molecules can also be used with examples including, but not limited to, manganese (Mn), gadolinium (Gd), iron oxide, and platinum (Pt) compounds. As will be appreciated by the skilled artisan, various devices could be employed to detect the signal molecule, depending on the type of signal molecule being used. Examples include fluorimeters, such as a charge coupled device (CCD) or a photomultiplier tube (PMT) detector with a light-emitting diode (LED) or other light source. Additional examples includenuclear magnetic resonance (NMR) spectrometer, x-ray fluorescence spectrometer, infrared (IR) spectrometer, mass spectrometer, color or light detecting cameras or sensors, and resistance / current / potential electrochemical detection.

[0045] Referring again to FIG. 1A, when the sample is added to the device 10, the probe 14 will be held in the first zone (Zone 1) by an appropriate conjugation method. However, upon the introduction of a sample including the target analyte 20. the interaction of the probe 14, the recognition element 18, or the signal molecule 16 with the remainder of the device 10 is altered, resulting in the signal molecule 16 being free from (either alone or together with other components of the device) the first zone. With reference to FIG. IB, when the signal molecule 16 is free from the first zone, the conjugated portion of the probe 14 will continue to be held in the first zone, while the signal molecule 16 can migrate away from the first zone, e.g., to a second zone (Zone 2). Therefore, in such embodiments, detection of the signal molecule 16 outside of the first zone is indicative of a presence and / or amount of the target analyte 20 in the sample.

[0046] As noted above, depending on the nature of the target analyte, the probe, recognition element, and signal molecule can take different forms. As will be appreciated by one of ordinary skill in the art upon studying this document, there are a number of different molecule that can have utility for use as the probe and / or the recognition element of the presently disclosed subject matter. Examples include, but are not limited to nucleotides, such as DNA, for use in detecting nucleotide; polypeptides, for detecting enzy matic proteins; antibodies, for detecting proteins; binding proteins (binding polypeptides) for detecting proteins, small molecules, and nucleotide analytes; antigens, for detection of antibody- or antibody-like proteins, aptamers for detecting, small molecules, peptides, or proteins, molecularly imprinted polymers (MIP), for detecting proteins or small chemical molecule. Examples will be discussed in more detail with reference to FIG. 2-8.

[0047] Embodiments of the presently-disclosed subject matter can be used for detecting target analytes that are nucleotides or polypeptides. FIG. 2 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a nucleotide. The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 2, and includes a probe 14. a signal molecule 16, and a recognition element 18.

[0048] The probe 14 is a nucleotide conjugated to the first zone. Such conjugation can be achieved by methods know n to those of ordinary skill in the art, for example, by those previously described.30-32

[0049] The recognition element 18 is a nucleotide and is conjugated to the signal molecule 16. Such conjugation can be achieved by methods know n to those of ordinary’ skill in the art. for example, by those previously described in methods accessible at the following link: www.thermofisher.com / us / en / home / references / molecular-probes-the-handbook / nucleic- acid-detection-and-genomics-technology / labeling-oligonucleotides-and-nucleic-acids.html.

[0050] The recognition element 18 is conjugated to the probe 14 through complementary base-pairing. Notably, in the embodiment depicted in FIG. 2, the probe 14 has an affinity for the recognition element 18, but the probe 14 and / or recognition element 18 nucleotides have been selected and / or engineered such that the probe 14 has a stronger affinity for the target nucleotide 20 than for the recognition element 18. In other embodiments, the probe and / or recognition element nucleotides can be engineered such that the recognition element has a stronger affinity for the target nucleotide than for the probe. As will be appreciated by one of ordinary’ skill in the art, designing nucleotides having the desired differential affinities, in view of the sequence of the target analyte, can be accomplished by modifying the length and thus number of complementary bases, nucleotide base content, and / or overall % complementary' bases (to include mismatched base(s)).33’34

[0051] With reference to the bottom portion of FIG. 2, when a sample containing the target nucleotide 20 is introduced, because the probe 14 has a stronger affinity for the target nucleotide 20 than for the recognition element 18, the target nucleotide 20 binds the probe 14 and displaces the recognition element 18. Through this process, the signal molecule 16, which is conjugated to the recognition element 18, is free from the first zone. Thus, as depicted, there is a resulting combination of the probe 14 and the target nucleotide 20 that will remain held in the first zone; and there is another resulting combination including the signal molecule 16 and the recognition element 18 that is free to migrate away from the first zone. Therefore, detection of the signal molecule 16 outside of the first zone will occur w hen the sample contains the target nucleotide 20.

[0052] As noted above, in other embodiments, the probe and / or recognition element nucleotides can be engineered such that the recognition element has a stronger affinity’ for thetarget nucleotide than for the probe. In such an embodiment, when a sample containing the target nucleotide is introduced, because the recognition element has a stronger affinity for the target nucleotide than for the probe, the target nucleotide binds the recognition element, displacing it from the probe. Through this process, the signal molecule, which is conjugated to the recognition element that is bound to the target nucleotide, is free from the first zone. Thus, the probe will remain held in the first zone; and the resulting combination including the signal molecule, the recognition element, and the target nucleotide is free to migrate away from the first zone. Therefore, detection of the signal molecule outside of the first zone will occur when the sample contains the target nucleotide.

[0053] FIG. 3 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a polypeptide that is a protease. The device 10, prior to receiving a sample is depicted in the top portion of FIG. 3, and includes a probe 14, a recognition element 18, and a signal molecule 16.

[0054] In the embodiment depicted in FIG. 3, the probe 14 and the recognition element 18 are provided as a single polypeptide 14, 18. In this regard, the recognition element 18 portion of the polypeptide 14, 18 is an amino acid sequence recognized by the target protease 20 for cleaving, such that, in the presence of the target protease 20, the polypeptide 14. 18 is cleaved. Polypeptide sequences can be determined for specific protease recognition from the literature describing the protease discovery and / or characterization, as exemplified but not limited examples previously published.21'23

[0055] One end of the polypeptide 14. 18 is conjugated to the first zone, while the other end of the polypeptide 14, 18 is conjugated to the signal molecule 16. Accordingly, and with reference to the right portion of FIG. 3, when the amino acid sequence that is contained within the polypeptide 14, 18 is recognized by the target protease 20 and cleaved there are two resulting particles. One resulting particle is bound to the first zone, to which a portion of the cleaved polypeptide 14, 18 is conjugated, which that will remain held in the first zone. The other resulting particle includes the signal molecule 16, to which another portion of the cleaved polypeptide 14, 18 is conjugated. Therefore, detection of the signal molecule 16 outside of the first zone will occur when the sample contains the target protease 20.

[0056] As noted, in the example depicted in FIG. 3, one end of the polypeptide 14, 18 is conjugated to the surface of the first zone, while the other end of the polypeptide 14, 18 isconjugated to the signal molecule 16. Such conjugation can be achieved by methods known to those of ordinary’ skill in the art. for example, by those previously described to attach proteins to the surface of materials30'32and to fluorescently label the polypeptide signal molecule.13

[0057] FIG. 4 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a polypeptide, which can be a protein that is not a protease. The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 4, including a probe 14, a recognition element 18, and a signal molecule 16. In the embodiment depicted in FIG. 4, the signal molecule 16 can be a labelled ligand binding domain of the target protein 20, which is initially attached to the probe 14 via the recognition element 18.

[0058] The conjugation between the signal molecule 16 and the recognition element 18 can occur through non-covalent interactions with the target polypeptide’s 20 ligand, but could also be conjugated by making use of molecularly imprinted polymers, or by other mechanism known to those of ordinary skill in the art, including those referenced herein. Whatever the mechanism, notably, in the embodiment depicted in FIG. 4, the recognition element 18 has an affinity for the signal molecule 16, but the recognition element 18 and the signal molecule 16 have been selected and / or engineered such that the recognition element 18 has a stronger affinity for the target polypeptide 20 than for the signal molecule 16. Designing a recognition element and signal molecule having the desired differential affinities, in view of the target polypeptide, can be accomplished by computational modelling of the protein-ligand interactions as described by Du, et al.,24or by experimental methods, including but not limited to those discussed by Zer, et al., Parker, et al., and Nguyen, et al.25' 27

[0059] With reference to the bottom portion of FIG. 4, when a sample containing the target polypeptide 20 is introduced, because the recognition element 18 has a stronger affinity for the target polypeptide 20 than for the signal molecule 16, the target polypeptide 20 binds the recognition element 18 and displaces the signal molecule 16. Through this process, the signal molecule 16 is released from the first zone.

[0060] Thus, as depicted, there is a resulting combination including the probe 14, the recognition element 18, and the target polypeptide 20 that will remain held in the first zone. Meanwhile, the signal molecule 16 is free to migrate away from the first zone. Therefore,detection of the signal molecule 16 outside of the first zone will occur when the sample contains the target polypeptide 20.

[0061] FIG. 5 depicts another exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a polypeptide, which can be a protein that is not a protease. The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 5, and includes a probe 14. a recognition element 18, and a signal molecule 16. As with the embodiment depicted in FIG. 4, in the embodiment depicted in FIG. 5, the signal molecule 16 can be a labelled ligand of the target protein 20, which is initially attached to the probe 14 via the recognition element 18.

[0062] Notably, in the embodiment depicted in FIG. 5, the signal molecule 16 has an affinity for the recognition element 18, but the recognition element 18 and the signal molecule 16 have been selected and / or engineered such that the signal molecule 16 has a stronger affinity for the target polypeptide 20 than for the recognition element 18 through compatibility of the binding site with regard to size, electrostatic interactions, hydrogen bonding, hydrophobic interactions, or shape. Designing a recognition element and signal molecule having the desired differential affinities, in view of the target polypeptide, can be accomplished as describe with reference to the embodiment depicted in FIG. 4.

[0063] With reference to the bottom portion of FIG. 5, when a sample containing the target polypeptide 20 is introduced, because the signal molecule 16 has a stronger affinity for the target polypeptide 20 than for the recognition element 18, the target polypeptide 20 binds and displaces the signal molecule 16. Through this process, the signal molecule 16 is free from the first zone.

[0064] Thus, as depicted, there is a resulting combination including the probe 14 and the recognition element 18 that will remain held in the first zone. Meanwhile, the signal molecule 16, to which the target polypeptide 20 is bound, is free to migrate away from the first zone. Therefore, detection of the signal molecule 16 outside of the first zone will occur when the sample contains the target polypeptide 20.

[0065] FIG. 6 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer.

[0066] As is known to those of ordinary skill in the art, an aptamer is often a singlestranded nucleic acid (DNA or RNA) or sometimes a polypeptide that is designed to bind to a specific target analyte with high affinity and specificity. Aptamers are sometime referred to as chemical antibodies or synthetic receptors due to their ability7to mimic the targeting capabilities of antibodies in a w ide range of applications.

[0067] Aptamers can be created, for example, using a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). During SELEX, a large library of random nucleic acid sequences is exposed to the target analyte of interest. The sequences that bind most strongly to the target are isolated, amplified, and subjected to several rounds of selection and amplification. This iterative process eventually leads to obtaining aptamers with strong binding affinity to the target analyte.

[0068] Aptamers can be tailored to bind to a diverse array of targets, including, for example, proteins / peptides / polypeptides, nucleotides, small molecules, virus particles, whole cells, metal ions, and biological compounds and macromolecules, such as carbohydrates, lipids, lipoproteins, and other complex biomolecular assemblies. Accordingly, they can be used to detect and quantify specific analytes in complex mixtures.

[0069] In the embodiment depicted in FIG. 6, the probe 14 and the recognition element 18 are provided as a single unit 14, 18 that is an aptamer. The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 6, in w hich the aptamer-containing single unit (aptamer unit) 14, 18 is attached to the first zone, such as through electrostatic or other non- covalent interactions. In the first zone, the aptamer-containing single unit 14.18 is also attached to the signal molecule 16, such as by covalent conjugation.

[0070] In the embodiment depicted in FIG. 6, the aptamer unit 14,18 and signal molecule 16 are initially attached to the first zone, but the aptamer unit 14,18 has been selected and / or engineered such that the aptamer unit 14,18 has a stronger affinity for the target analyte 20 than for the first zone. Designing an aptamer having the desired differential affinities, in view of the target analyte, can be accomplished as previously described.28, 29

[0071] With reference to the bottom portion of FIG. 6, when a sample containing the target analyte 20 is introduced, because the aptamer unit 14,18 has a stronger affinity7for the target analyte 20 than for the first zone, the target analyte 20 binds and displaces the aptamerunit 14,18 to which the signal molecule 16 is conjugated. Through this process, the signal molecule 16 and aptamer unit 14,18 are free from the first zone.

[0072] Thus, as depicted, the signal molecule 16, to which the aptamer unit 14,18 and target analyte 20 are bound, is free to migrate away from the first zone. Therefore, detection of the signal molecule 16 outside of the first zone will occur when the sample contains the target analyte 20.

[0073] FIG. 7 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer. The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 7, and includes a probe 14, and a recognition element 18 conjugated to a signal molecule 16. In the embodiment depicted in FIG. 7, the recognition element 18 is an aptamer.

[0074] With continued reference to the embodiment in FIG. 7, the aptamer recognition element 18 is conjugated to the probe 14 through complementary base-pairing. Notably, the recognition element 18 has an affinity for the probe 14, but the probe and / or recognition element nucleotides have been selected and / or engineered such that the recognition element 18 has a stronger affinity for the target analyte 20 than for the probe 14.

[0075] With reference to the bottom portion of FIG. 7, when a sample containing the target analyte 20 is introduced, because the recognition element 18 has a stronger affinity' for the target analyte 20 than for the probe 14, the target analyte 20 binds and displaces the recognition element 18 to which the signal molecule 16 is conjugated. Through this process, the signal molecule 16 and the recognition element 18 are free from the first zone.

[0076] Thus, as depicted, the probe 14 will remain held in the first zone. Meanwhile, the signal molecule 16, to which the recognition element 18 and target analyte 20 are bound, is free to migrate away from the first zone. Therefore, detection of the signal molecule 16 outside of the first zone will occur when the sample contains the target analyte 20.

[0077] FIG. 8 depicts an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer or antibody, which term is inclusive of an antibody fragment (e.g., Fab portion of an antibody). While the embodiment depicted in FIG. 8 makes use of anantibody as the probe 14 and the recognition element 18, taken together, form a single unit 14, 18, as will be appreciated by the skilled artisan, in other embodiments, any other probe / recognition element could be used.

[0078] The device 10, prior to receiving a sample, is depicted in the top portion of FIG. 8. The device 10 includes confirmed target analytes 21, obtained from a source distinct from any sample (e.g.. in vitro source), which are covalently or non-covalently bound within the first zone.

[0079] Prior to being introduced to the embodiment of the device 10 depicted in FIG. 8, the sample is prepared as follows. With reference to the middle portion of FIG. 8, sample preparation reagents include the single unit 14, 18, which is an antibody that selectively binds the target analyte 20. The single unit (antibody unit) 14,18 is also attached to the signal molecule 16, such as by covalent conjugation. To prepare the sample, it is contacted with the antibody unit 14.18 attached to the signal molecule 16. If there is target analyte 20 in the sample, the target analyte 20 will bind with the antibody unit 14,18. If there is not target analyte 20 in the sample, the antibody unit 14,18 will remain unbound.

[0080] The prepared sample is then introduced to the device 10 containing the first zone presenting the confirmed target analyte 21 (see middle and bottom of FIG. 8). If there is not target analyte 20 in the sample, the antibody unit 14,18 is free and will bind the confirmed target analyte 21 on the first zone, such that the attached signal molecule 16 is held in the first zone. However, if there is target analyte 20 in the sample, it was bound to the antibodyunit 14,18 during sample preparation, in which case the antibody unit 14.18 cannot become bound to the confirmed target analyte 21 in the first zone.

[0081] Thus, when the target analyte 20 is present in the sample, there is a lower resulting portion of the antibody unit 14,18 that will remain held in the first zone. Meanwhile, the signal molecule 16, to which the antibody unit 14,18 and target analyte 20 are bound, is free to migrate away from the first zone. Therefore, detection and / or greater relative intenstity of the signal molecule 16 outside of the first zone will occur when the sample contains the target analyte 20.

[0082] FIG. 9A-9C depict an exemplary embodiment of a device 10 for use in detecting a target analyte 20 that is a small molecule, polypeptide, or other analyte that can be recognized by an aptamer or antibody, which term is inclusive of an antibody fragment (e.g..Fab portion of an antibody). While the embodiment depicted in FIG. 9A-9C makes use of an antibody as the probe 14 and the recognition element 18, taken together, form a single unit 14, 18, as will be appreciated by the skilled artisan, in other embodiments, any other probe / recognition element could be used.

[0083] The device 10, as it exists prior to introduction of the sample, is shown in FIG. 9A. The device 10 includes binding molecules 23 covalently or non-covalently bound within the first zone. The binding molecules 23 are selected for their ability to recognize the target analyte 20. As will be appreciated by the skilled artisan, various types of binding molecules 23 can be employed, depending on the nature of the target analyte 20. For example, sandwich-type assays or biotin / avidin interactions could be employed.

[0084] Moving from FIG. 9A to FIG. 9B, when the sample is introduced to the device 10, if the target analyte 20 is present in the sample, the target analyte 20 will bind to the binding molecules 23 and will be held in the first zone, as depicted in FIG. 9B.

[0085] After the sample has been introduced to the device 10, preparation reagents will be introduced to the device 10. Such preparation reagents include the single unit 14, 18, which is an antibody that selectively binds the target analyte 20. The single unit (antibody unit) 14,18 is also attached to the signal molecule 16, such as by covalent conjugation. Turning to FIG. 9C, when the antibody unit 14,18 with the attached signal molecule 16 are introduced to the device 10, the target analyte 20 will bind with the antibody unit 14,18, such that it remains in the first zone. If there is not target analyte 20 in the sample, the antibody unit 14, 18 will remain unbound.

[0086] Thurs, if there is not target analyte 20 in the sample, there is nothing to bind to the binding molecule 23, such that when the antibody unit 14,18 is introduced, it is free to migrate out of the first zone, such that the attached signal molecule 16 is outside the first zone. However, if there is target analyte 20 in the sample, it binds to the binding molecule 23 in the first zone, where it will be recognized by the antibody unit 14,18, in which case the antibody unit 14,18 with the signal molecule 16 will be bound in the first zone. Any unbound, leftover antibody unit 14,18 will be free to migrate out of the first zone. Accordingly, signal molecule 16 within the first zone is indicative of a presence of target analyte 20 in the sample; and, w ith a defined quantity of preparation reagents, the relativeintensities of signal within the first zone and outside the first zone will be indicative of amount of target analyte 20 in the sample.

[0087] In some embodiments, in addition to use of aptamers and antibodies, the presently-disclosed subject matter can make use of a variety of molecular recognition elements, such as, for example, the following. Short chains of amino acids (relatively shorter polypeptides, which are often referred to in the art as peptides) can be designed or selected to bind to specific targets. Peptides can be engineered to mimic the binding properties of antibodies or aptamers. Custom-designed synthetic polymers, also known as molecularly imprinted polymers (MIPs), can be created to specifically bind to certain target molecules. MIPs are polymer matrices with binding sites that are molecularly shaped to match the target molecule. Nanobodies, also known as single-domain antibodies or VHHs (variable heavy domains of heavy-chain antibodies), nanobodies are derived from the unique heavy-chain- only antibodies found in camelids. They are small, stable, and can be engineered to bind with high specificity to a wide range of target molecules. Ribozymes and deoxyribozymes, which are RNA and DNA molecules, respectively, not only bind to target molecules but also possess enzymatic activity. They can catalyze specific reactions in the presence of their target molecules, making them useful in certain applications. Chemical ligands are a ty pe of small molecule that can be chemically designed to specifically interact with certain target molecules, making them useful for molecular recognition applications. Instead of using whole antibodies, specific binding domains of antibodies (such as Fab fragments or singlechain variable fragments, scFv) can be engineered for target binding. These antibody fragments retain the binding specificity' but are smaller and can be easier to w ork w ith.

[0088] The presently-disclosed subject matter further includes a method for detecting a target analyte in a sample, which involves delivering the sample to a device as disclosed herein such that the sample enters the first zone, and detecting a location of the signal molecule. In some embodiments, the method further involves determining the amount of the signal molecule present in the first zone and / or determining the amount of the signal molecule outside of the first zone. In some embodiments, the method further involves comparing the amount of the signal molecule present in the first zone to the amount of the signal molecule outside of the first zone.

[0089] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0091] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0092] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0093] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).

[0094] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0095] In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the filing date of this Application.

[0096] The present application can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or theirequivalent in structure or function, plus any other element or elements which are not recited. The terms "‘having'’ and “including” are also to be construed as open ended unless the context suggests otherwise.

[0097] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0098] Unless otherw ise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary' depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0099] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%. in some embodiments ±0. 1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0100] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10"’ is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0101] The terms “nucleotide”, “polynucleotide”, “nucleic acid” and “nucleic acid sequence” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0102] The term “complementary ” refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences. As is know i in the art, the nucleic acid sequences of two complementary strands are the reverse complement of each other when each is view ed in the 5 ' to 3’ direction. As is also known in the art, two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% fully complementary’. Indeed, it can be useful to design certain nucleotide sequences to be less than fully complementary, such that they have a reduced affinity7for a particular sequence.

[0103] As used herein, the term “polypeptide” means any polymer comprising any of the 20 protein amino acids, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted.

[0104] The term “small molecule” as used herein, refers to organic or inorganic molecules either synthesized or found in nature, generally having a molecular weight less than 10,000 grams per mole, optionally less than 5,000 grams per mole, and optionally less than 2,000 grams per mole.

[0105] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0106] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:REFERENCES1. Kennedy, S. M.; O’Driscoll, L.; Purcell, R.; Fitz-Simons, N.; McDermott, E. W.; Hill, A. D.; O’Higgins, N. J.; Parkinson, M.; Linehan, R.; Clynes, M., Prognostic importance of survivin in breast cancer. Br J Cancer 2003, 88 (7), 1077-1083.2. Jaiswal, P. K.; Goel, A.; Mittal, R. D., Survivin: A molecular biomarker in cancer. Indian J Med Res 2015, 141 (4), 389-397.3. Smith, S. D.; Wheeler, M. A.; Plescia, J.; Colberg, J. W.; Weiss, R. M.; Altieri. D. C., Urine Detection of Survivin and Diagnosis of Bladder Cancer. JAMA 2001, 285 (3), 324-328.4. Gunaldi, M.; Isiksacan, N.; Kocoglu, H.; Okuturlar, Y .; Gunaldi, O.; Topcu, T.; Karakul ut, M., The value of serum survivin level in early diagnosis of cancer. Journal of Cancer Research and Therapeutics 2018, 14 (3), 570-573.5. Ross, G. M. S.; Filippini, D.; Nielen, M. W. F.; Salentijn, G. I. J., Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects in Sandwich Lateral Flow Immunoassays. Analytical Chemistry 2020, 92 (23), 15587-15595.6. Chen, Y.; Ding, X.; Zhang, Y.; Natalia, A.; Sun, X.; Wang, Z.; Shao, H., Design and synthesis of magnetic nanoparticles for biomedical diagnostics. Quantitative imaging in medicine and surgery 2018, 8 (9), 957-970.7. Bilal, M.: Zhao, Y.; Rasheed, T.: Iqbal, H. M. N., Magnetic nanoparticles as versatile carriers for enzy mes immobilization: A review. International Journal of Biological Macromolecules 2018, 120, 2530-2544.8. Lee, J. W.; Choi, S.-R.; Heo, J. H., Simultaneous Stabilization and Functionalization of Gold Nanoparticles via Biomolecule Conjugation: Progress and Perspectives. ACS Applied Materials & Interfaces 2021, 13 (36), 42311-42328.9. Eivazzadeh-Keihan, R.; Bahreinizad. H.; Amiri, Z ; Aliabadi, H. A. M.; Salimi-Bani, M.; Nakisa, A.; Davoodi, F.; Tahmasebi, B.; Ahmadpour, F.; Radinekiyan, F.; Maleki, A.: Hamblin, M. R.; Mahdavi, M.; Madanchi, H., Functionalized magnetic nanoparticles for the separation and purification of proteins and peptides. TrAC Trends in Analytical Chemistry 2021, 141, 116291.10. Zhao, L.; Li, L.; Zhu, C.; Ghulam, M.; Qu, F., pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins. Analytica Chimica Acta 2020, 1097, 161-168.11. Fresco-Cala, B.; Batista, A. D.: Cardenas, S., Molecularly Imprinted Polymer Micro- and Nano-Particles. A review. Molecules (Basel, Switzerland) 2020, 25 (20).12. Kozlowski, R.; Ragupathi, A.; Dyer, R. B., Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates. Bioconjugate chemistry 2018, 29 (8), 2691-2700.13. Toseland, C. P., Fluorescent labeling and modification of proteins. Journal of chemical biology 2013, 6 (3), 85-95.14. Zhang, W.; Wang, R.; Luo, F.; Wang, P.; Lin, Z., Miniaturized electrochemical sensors and their point-of-care applications. Chinese Chemical Letters 2020, 31 (3), 589-600.15. Bilal. M.: Zhao, Y.; Rasheed, T.: Iqbal, H. M. N., Magnetic nanoparticles as versatile carriers for enzy mes immobilization: A review. International Journal of Biological Macromolecules 2018, 120, 2530-2544.16. Lee, J. W.; Choi, S.-R.; Heo, J. H., Simultaneous Stabilization and Functionalization of Gold Nanoparticles via Biomolecule Conjugation: Progress and Perspectives. ACS Applied Materials & Interfaces 2021, 13 (36), 42311-42328.17. Eivazzadeh-Keihan, R.; Bahreinizad. H.; Amiri, Z.; Aliabadi, H. A. M.; Salimi-Bani, M.; Nakisa, A.; Davoodi, F.; Tahmasebi, B.; Ahmadpour, F.; Radinekiyan, F.; Maleki, A.; Hamblin, M. R.; Mahdavi, M.; Madanchi, H , Functionalized magnetic nanoparticles for the separation and purification of proteins and peptides. TrAC Trends in Analytical Chemistry 2021, 141, 116291.18. Zhao, L.; Li, L.; Zhu, C.; Ghulam, M.; Qu, F., pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins. Analytica Chimica Acta 2020, 1097, 161-168.19. Fresco-Cala, B.; Batista, A. D.i Cardenas, S., Molecularly Imprinted Polymer Micro- and Nano-Particles. A review. Molecules (Basel, Switzerland) 2020, 25 (20).20. Hurst, S. I; Lytton-Jean, A. K.; Mirkin, C. A., Maximizing DNA loading on a range of gold nanoparticle sizes. Anal Chem 2006, 78 (24), 8313-8.21. Olsen, J. V.; Ong, S.-E.; Mann, M., Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues *. Molecular & Cellular Proteomics 2004, 3 (6), 608-614.22. Yu, Z.; Visse, R.; Inouye, M.; Nagase, H.; Brodsky, B., Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system. J Biol Chem 2012, 287 (27), 22988-22997.23. Coombs, G. S.; Bergstrom, R. C.; Pellequer, J.-L.; Baker, S. L; Navre, M.; Smith, M. M.; Tainer, J. A.; Madison, E. L.; Corey, D. R., Substrate specificity of prostatespecific antigen (PSA). Chemistry & Biology 1998, 5 (9). 475-488.24. Du X, Li Y, Xia YL, Ai SM. Liang J, Sang P, Ji XL. Liu SQ. Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods. Int J Mol Sci. 2016 Jan 26; 17(2): 144.25. Zer C, Avery KN, Meyer K, Goodstein L, Bzymek KP, Singh G, Williams JC. Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A. Protein Eng Des Sei. 2017 Jun l;30(6):409-417.26. Parker, B. W.; Goncz, E. J.; Krist, D. T.; Statsyuk, A. V.; Nesvizhskii, A. I.; Weiss, E. L. Mapping low-affinity / high-specificity peptide-protein interactions using ligandfootprinting mass spectrometry. Proceedings of the National Academy of Sciences 2019. 1 16 (42), 21001-21011.27. Nguyen, H. Q.; Roy, J.; Harink, B.; Damle, N. P.; Latorraca, N. R.; Baxter, B. C.; Brower, K.; Longwell, S. A.; Kortemme, T.; Thom, K. S.; et al. Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads. eLife 2019, 8, e40499.28. Kohlberger, M.; Gadermaier, G. SELEX: Critical factors and optimization strategies for successful aptamer selection. Biotechnology and Applied Biochemistry 2022, 69 (5), 1771-1792. DOI: 10. 1002 / bab.2244.29. Yiice, M.; Ullah, N.; Budak, H. Trends in aptamer selection methods and applications. Analyst 2015, 140 (16), 5379-5399, 10.1039 / C5AN00954E. DOI: 10.1039 / C5AN00954E.30. Sonawane. M. D.; Nimse, S. B. Surface Modification Chemistries of Materials Used in Diagnostic Platforms with Biomolecules. Journal of Chemistry 2016, 2016, 9241378. DOI: 10.1155 / 2016 / 9241378.31. Fleischmann, S.; Hinrichs, ; Oertel, U.; Reichelt, S.; Eichhorn, K.-J.; Voit, B. Modification of Polymer Surfaces by Click Chemistry. Macromolecular Rapid Communications 2008, 29 (12-13), 1177-1185. DOI: 10.1002 / marc.200800095.32. Tischer, T.; Claus, T. K.; Bruns, M.; Trouillet, V.; Linkert, K.; Rodriguez- Emmenegger, C.; Goldmann, A. S.; Perrier, S.; Borner, H. G; Bamer-Kowollik, C. Spatially Controlled Photochemical Peptide and Polymer Conjugation on Biosurfaces. Biomacromolecules 2013, 14 (12), 4340-4350. DOI: 10.1021 / bm401274v.33. Kuhn, H.; Sahu, B.; Rapireddy, S.; Ly. D. H.; Frank-Kamenetskii, M. D. Sequence specificity at targeting double-stranded DNA with a y-PNA oligomer modified with guanidinium G-clamp nucleobases. Artif DNA PNA XNA 2010, 1 (1), 45-53. DOI: 10.4161 / adna. 1.1. 12444.34. Ochoa, S.; Milam, V. T. Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides. Molecules (Basel, Switzerland) 2020, 25 (20). DOI: 10.3390 / molecules25204659.

[0107] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is:

1. A device for detecting a target analyte in a sample, comprising: a) a first zone, into which the sample can be delivered; b) a second zone in fluid communication with the first zone; c) a probe bound within the first zone, which (i) is covalently or non-covalently conjugated to a recognition element, or (ii) taken together with the recognition element, forms a single unit; and d) a signal molecule attached to the probe or the recognition element; wherein the probe, the recognition element, or the signal molecule has an affinity for the target analyte, or wherein the target analyte cleaves or allows the release of the recognition element, such that in the presence of the target analyte, the signal molecule is released from the first zone.

2. The device of claim 1, wherein the signal molecule is attached to the recognition element, and wherein the signal molecule or the recognition element has an affinity7for the probe, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone, due to the attachment of the signal molecule to the recognition element and the affinity7of the signal molecule or recognition element for the probe, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the signal molecule or the recognition element and the target analyte.

3. The device of claim 2, wherein the target analyte is a target nucleotide; the probe is a probe nucleotide; and the recognition element is a recognition nucleotide conjugated to the probe nucleotide through complementary7base-pairing; wherein the recognition nucleotide has an affinity7for the probe nucleotide, and a stronger affinity for the target nucleotide.

4. The device of claim 1, wherein the target analyte is a protease; the probe and the recognition element, taken together, form a single unit that is a polypeptide; and the recognition element is an amino acid sequence recognized by the protease for cleaving, such that, in the absence of the target protease, the signal molecule is bound within the first zone due to polypeptide remaining intact, and in the presence of the target protease, the polypeptide is cleaved, releasing the signal molecule from the first zone.

5. The device of claim 1, wherein the probe or the recognition element has an affinity for the signal molecule, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the signal molecule and the probe or the recognition element, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the probe or the recognition element and the target analyte.

6. The device of claim 5, wherein the target analyte is a polypeptide or small molecule; the recognition element has an affinity for the signal molecule, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the recognition element and the signal molecule; and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the recognition element and the target analyte.

7. The device of claim 1, wherein the signal molecule has an affinity for the recognition element, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity between the signal molecule and the recognition element; and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity' between the signal molecule and the target analyte.

8. The device of claim 7, wherein the target analyte is a polypeptide or small molecule.

9. The device of claim 1, wherein the target analyte can be recognized by an aptamer; and the probe and the recognition element, taken together, form a single unit that is an aptamer bound within the first zone; wherein the aptamer has a stronger affinity' for the target analyte than for the first zone, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity' between the aptamer and the first zone, and in the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the aptamer and the target analyte.

10. The device of claim 1, wherein the target analyte can be recognized by an aptamer; the probe is a probe nucleotide; the recognition element is a nucleotide aptamer conjugated to the probe nucleotide through complementary base-pairing; and the signal molecule is attached to the recognition element; wherein the nucleotide aptamer has an affinity for the probe nucleotide, and a stronger affinity for the target analyte, such that in the absence of the target analyte, the signal molecule is bound within the first zone due to the affinity' between the nucleotide aptamer and the probe, andin the presence of the target analyte, the signal molecule is released from the first zone due to the stronger affinity between the nucleotide aptamer and the target analyte11. A kit for use in detecting a target analyte in a sample, comprising: a) a device according to claim 1, and b) preparation reagents provided in a sample preparation container.

12. A kit for detecting a target analyte in a sample, comprising: a) a device comprising(i) a first zone, into which the sample can be delivered;(ii) a second zone in fluid communication with the first zone; and b) preparation reagents to which the sample can be added prior to delivery to the first zone, comprising(i) a probe that (A) is covalently or non-covalently conjugated to a recognition element, or (B) taken together with the recognition element, forms a single unit; and(ii) a signal molecule attached to the probe or the recognition element, wherein the probe or the recognition element selectively binds the target analyte.

13. The kit of claim 12, the probe is covalently or non-covalently conjugated to a recognition element.

14. The kit of claim 13, and further comprising a binding molecule analyte covalently or non-covalently bound within the first zone, which recognizes the probe or the recognition element.

15. The kit of claim 12, wherein the probe and the recognition element, taken together, form a single unit attached to the signal molecule.

16. The kit of claim 15, wherein the single unit is an aptamer, an antibody, or an antibody fragment that selectively binds the target analyte.

17. The kit of claim 12, where target analytes that can be recognized by the single unit are covalently or non-covalently bound within the first zone, such that in the absence of the target analyte in the sample, when the preparation is delivered to the first zone, the signal molecule attached to the single unit remains within the first zone due to the affinity between the single unit and the target analytes bound within the first zone, and in the presence of the target analyte in the sample, the signal molecule attached to the single unit in the preparation becomes bound to the target analyte in the sample, such that when the preparation is delivered to the first zone, the signal molecule is free to migrate away from the first zone because the single unit is unable to bind the target analytes bound within the first zone.

18. The kit of claim 12, and further comprising a binding molecule that recognizes the target analyte covalently or non-covalently bound within the first zone, such that in the absence of the target analyte in the sample, no target analyte is available to attach to the binding molecule, and when the preparation is delivered to the first zone the single signal molecule attached to the single unit is free to migrate away from the first zone, and in the presence of the target analyte in the sample, it attaches to the binding molecule in the first zone and the signal molecule attached to the single unit remains within the first zone due to the affinity between the single unit and the target analyte.

19. A method for detecting a target analyte, comprising: a) delivering a sample to the device of any one of claims 1-10, such that the sample enters the first zone; b) detecting a location of the signal molecule.

20. The method of claim 19, and further comprising determining the amount of the signal molecule present in the first zone and / or determining the amount of the signal molecule outside of the first zone.

21. The method of claim 20, and further comprising comparing the amount of the signal molecule present in the first zone to the amount of the signal molecule outside of the first zone.