Systems and methods for visualizing target molecules - Patents.com

Abstract

Methods and systems are provided for detecting the presence, absence or amount of a target molecule in a sample or on a surface.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 301,784, filed January 21, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Embodiments of the present disclosure relate to systems and methods for detecting the presence, absence or amount of a target molecule in a sample or on a surface. Summary of the Invention

[0003] Aspects of the present disclosure relate to methods for visualizing the presence of one or more pathogens on a surface.

[0004] Provided herein is a method for visualizing the presence of one or more pathogens on a surface. In some embodiments, the method includes the steps of: (a) applying a substantially uniform layer of a liquid composition to a surface, the liquid composition comprising one or more aptamers covalently or non-covalently associated with one or more fluorophores, one or more aptamer conjugates having specific binding affinity for one or more target molecules of one or more pathogens, and a quencher for quenching non-specific fluorescence; (b) exposing the surface to a predetermined temperature for a period of time to allow the population of conjugated aptamers to bind to one or more target molecules; (c) illuminating at least one region of the surface by emitting light in one or more predetermined wavelength ranges around or at an excitation maximum wavelength of one or more phosphors, where the one or more phosphors absorb the emitted light in the one or more predetermined wavelength ranges and emit light in a longer wavelength range; and (e) blocking the emitted light by either (i) blocking the one or more excitation wavelength ranges and transmitting a second wavelength range that includes some or all of the wavelengths emitted by the one or more phosphors, or (ii) blocking wavelengths shorter than the emitted phosphor wavelength range.

[0005] In some embodiments, the applying step comprises spraying the liquid composition onto the surface. In some embodiments, the spraying step comprises a spray distribution with a coefficient of variation (CV) of less than 15%. In some embodiments, the spraying comprises minimizing disturbance of one or more pathogens on the surface. In some embodiments, the applying step comprises atomizing the liquid composition onto the surface. In some embodiments, the spraying or atomizing comprises dispersing droplets onto the surface, the droplets having an average size ranging from about 10 to about 100 μm.

[0006] In some embodiments, the exposing step comprises incubating the surface with one or more aptamer conjugates at room temperature for about 1 minute to about 10 minutes, hi some embodiments, the incubation is for less than 5 minutes.

[0007] In some embodiments, the liquid composition comprises two or more conjugated aptamers, each of the two or more aptamers being conjugated to a different fluorophore. In some embodiments, the illuminating step comprises emitting light in two or more predetermined wavelength ranges at or around two or more excitation maxima of the different fluorophores. In some embodiments, the illuminating step comprises emitting light in one or more predetermined wavelength ranges at or around one or more excitation maxima of the fluorophores at an illuminance that allows visualization of the fluorophores. In some embodiments, the blocking of light comprises using one or more filters, and the one or more filters comprise a long-pass filter.

[0008] In some embodiments, the quencher comprises or consists of graphene oxide.

[0009] In some embodiments, the one or more target molecules comprises a protein.

[0010] In some embodiments, the liquid composition comprises two or more different aptamers. In some embodiments, the two or more different aptamers bind to different target proteins. In some embodiments, the two or more different aptamers bind to the same target protein.

[0011] In some embodiments, the method further comprises imaging the surface.

[0012] In some embodiments, the method further comprises identifying the one or more pathogens.

[0013] In some embodiments, the method further comprises decontaminating the surface.

[0014] In some embodiments, the method further comprises (e) determining the presence or absence of fluorescence from the population of conjugated aptamers, thereby determining the presence or absence of one or more pathogens on the surface.

[0015] In some embodiments, the method further comprises repeating steps (a) through (e).

[0016] In some embodiments, during step (b), a first population of conjugated aptamers binds to one or more targets, and a second population of conjugated aptamers does not bind to the one or more target molecules and remains associated with the quencher.

[0017] In some embodiments, step (c) comprises illuminating at least one region of the surface with an illumination device having a single emitter configured to emit light in one or more predetermined wavelength ranges at or around an excitation maximum wavelength of one or more fluorophores, hi some embodiments, the emitter is configured to emit light in two or more predetermined wavelength ranges at or around an excitation maximum wavelength of two or more different fluorophores.

[0018] In some embodiments, the method includes the steps of: (a) applying a substantially uniform layer of a liquid composition to a surface, the liquid composition comprising (i) one or more aptamers covalently or non-covalently associated with one or more fluorophores, the one or more aptamer conjugates having specific binding affinity for one or more target molecules of one or more pathogens, and (ii) a quencher for quenching non-specific fluorescence; (b) drying the surface at a predetermined temperature for a period of time, wherein (i) a first population of conjugated aptamers bind to the one or more target molecules, and (ii) a second population of conjugated aptamers are not bound to the target molecules and remain associated with the quencher; and (c) drying the surface at one or more predetermined wavelength ranges at or around the excitation maximum wavelength of the one or more fluorophores. wherein the one or more fluorophores absorb light from the illumination device in one or more predetermined wavelength ranges and emit light in a longer wavelength range; (d) blocking or removing light from the illumination device using one or more filters configured to substantially (i) block one or more predetermined excitation wavelength ranges from the illumination device and transmit a second wavelength range including one or more wavelengths emitted by the one or more fluorophores, or (ii) block wavelengths shorter than the emitted wavelength range of the fluorophores; and (e) determining the presence or absence of fluorescence from the first population of conjugated aptamers, thereby determining the presence or absence of one or more pathogens on the surface.

[0019] In some embodiments, the method does not include cleaning the surface prior to illuminating the surface.

[0020] In some embodiments, the method includes imaging the surface using an imaging device. In some embodiments, the method includes identifying one or more pathogens. In some embodiments, the method further includes decontaminating the surface. In some embodiments, the method further includes repeating steps (a) through (e).

[0021] In some embodiments, the liquid composition is an aqueous composition. In some embodiments, the liquid composition comprises a cocktail of aptamers. In some embodiments, the liquid composition comprises two or more different aptamers. In some embodiments, one or more aptamers have binding affinity to one or more target molecules. In some embodiments, one or more aptamers have binding affinity to the same target molecule. In some embodiments, the target molecule is a biomolecule. In some embodiments, the biomolecule is a protein. In some embodiments, the target molecule is a protein. In some embodiments, the target molecule is a surface protein. In some embodiments, the target molecule is an antigen. In some embodiments, the target molecule is a surface antigen.

[0022] In some embodiments, each aptamer is associated / conjugated with one or more fluorophores.In some embodiments, each aptamer is associated / conjugated with two or more different fluorophores.For example, fluorophores can include, but are not limited to, FAM, Cy3, Oregon Green, or any combination of the above.

[0023] In some embodiments, the quencher comprises or consists of graphene oxide.

[0024] In some embodiments, the applying step comprises spraying the surface. In some embodiments, the spraying step comprises a spray distribution with a coefficient of variation (CV) of less than 15%. In some embodiments, the spraying comprises minimizing disturbance of pathogens on the surface. In some embodiments, the spraying is substantially perpendicular to the surface. In some embodiments, the contacting step comprises atomizing the liquid composition onto the surface. In some embodiments, the contacting comprises dispersing droplets onto the surface, the droplets having a size ranging from about 10 to about 100 μm.

[0025] In some embodiments, the methods include incubating the surface with one or more aptamer conjugates at room temperature for about 1 minute to about 10 minutes, 1 minute to 5 minutes, or less than 5 minutes.

[0026] In some embodiments, the illumination device includes a single emitter configured to emit light in two or more predetermined wavelength ranges at or around the excitation maximum wavelength of one or more fluorophores. In some embodiments, the illumination device includes a single emitter configured to emit light in two or more predetermined wavelength ranges at or around the excitation maximum wavelength of two or more fluorophores. In some embodiments, the illumination device is configured to switch the first wavelength to a second, different wavelength. In some embodiments, the emitter is configured to emit light in one or more predetermined wavelength ranges at or around the excitation maximum wavelength of one or more fluorophores at an illuminance that allows visualization of the fluorophores.

[0027] In some embodiments, the excitation wavelength can be optimized for a particular fluorophore or fluorescent dye.

[0028] In some embodiments, blocking or removing light from the illumination device using one or more filters, the one or more filters configured to substantially block excitation wavelengths while passing substantially all emission wavelengths to allow detection and / or visualization and / or imaging. In some embodiments, the filters are placed between the surface and a viewer, detection device, or imager to aid in visualization of the emitted fluorescence and / or improve the signal-to-noise ratio. Depending on the fluorophores used, different filters can be used for optimal visualization. For example, if different fluorophores have sufficiently different emission wavelengths, different filters may be required for optimal visualization. In some embodiments, visualization can be performed in ambient light or in a dark environment.

[0029] In some embodiments, the one or more filters include a long-pass filter.

[0030] In some embodiments, one or more filters can be worn as eyewear, such as goggles and glasses, hi other embodiments, the filter can be a feature of the imaging device (e.g., a camera) used to image the surface.

[0031]

[0023] The embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are likely to be preferred. It is understood, however, that the present disclosure is not limited to the precise arrangements and instrumentalities shown. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 is a table showing excitation and emission wavelengths and visual colors for non-limiting examples of phosphors. [Diagram 2] FIG. 2 is a non-limiting diagram of the absorption-emission spectrum of a typical phosphor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] It will be understood that the present disclosure is not limited to the specific compositions, methods, and experimental conditions described, and that the compositions, methods, and conditions may vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the scope of the appended claims.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein that will be apparent to those skilled in the art upon reading this disclosure.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0036] As used herein, "aptamer", "nucleic acid molecule" or "oligonucleotide" are used interchangeably and refer to a non-naturally occurring nucleic acid molecule having a desired action on a target as defined herein. Aptamers as described herein are small artificial ligands, including DNA, RNA or modifications thereof, that can specifically bind to a target as defined herein with high affinity and specificity. In some embodiments, the aptamer may be a DNA aptamer. For example, the aptamer may be formed from single-stranded DNA (ssDNA). In some embodiments, the aptamer may be an RNA aptamer. For example, the aptamer may be formed from single-stranded RNA (ssRNA). The aptamers of the embodiments of the present disclosure may include modified nucleic acids as described herein.

[0037] As used herein, the terms "detectable label" and "detectable moiety" are used interchangeably. In some embodiments, the detectable label is a luminescent molecule. In some embodiments, the detectable label is a fluorophore.

[0038] As used herein, a "fluorophore" is a molecule that has the property of fluorescing, which is the property of absorbing light of a given wavelength and emitting light of a longer wavelength.

[0039] Aspects of the present disclosure relate to a method for visualizing the presence of one or more pathogens on a surface. Aspects of the present disclosure relate to a method for visualizing the presence of one or more pathogens on a surface. In some embodiments, the method includes the steps of: (a) contacting the surface with a liquid composition, where the contacting results in deposition of a substantially uniform layer of the liquid composition, the liquid composition comprising (i) one or more aptamers covalently or non-covalently associated with one or more fluorophores, one or more aptamer conjugates having specific binding affinity for one or more target molecules of the one or more pathogens, and (ii) a quencher, where non-specific fluorescence is quenched by association with the quencher; (b) drying the surface at a predetermined temperature for a period of time, where (i) a first population of conjugated aptamers bind to the one or more target molecules and (ii) a second population of conjugated aptamers are not bound to the target molecules and remain associated with the quencher; (c) detecting an excitation maximum wavelength or wavelengths of the one or more fluorophores. The method includes the steps of: illuminating at least one region of the surface with an illumination device configured to emit light in one or more predetermined wavelength ranges at or around the one or more predetermined wavelength ranges, where the one or more fluorophores absorb light from the illumination device in the one or more predetermined wavelength ranges and emit light in a longer wavelength range; (d) blocking or removing light from the illumination device using one or more filters configured to substantially (i) block the one or more predetermined excitation wavelength ranges from the illumination device and transmit a second wavelength range that includes one or more wavelengths emitted by the one or more fluorophores, or (ii) block wavelengths shorter than the emitted wavelength range of the fluorophores; and (e) determining the presence or absence of fluorescence from the first population of conjugated aptamers, thereby determining the presence or absence of one or more pathogens on the surface.

[0040] In some embodiments, the method does not include cleaning the surface prior to illuminating the surface.

[0041] In some embodiments, the method includes imaging the surface using an imaging device. In some embodiments, the method includes identifying one or more pathogens. In some embodiments, the method further includes decontaminating the surface. In some embodiments, the method further includes repeating steps (a) through (e).

[0042] In some embodiments, the liquid composition is an aqueous composition. In some embodiments, the liquid composition comprises a cocktail of aptamers. In some embodiments, the liquid composition comprises two or more different aptamers. In some embodiments, one or more aptamers have binding affinity to one or more target molecules. In some embodiments, one or more aptamers have binding affinity to the same target molecule. In some embodiments, the target molecule is a biomolecule. In some embodiments, the biomolecule is a protein. In some embodiments, the target molecule is a protein. In some embodiments, the target molecule is a surface protein. In some embodiments, the target molecule is an antigen. In some embodiments, the target molecule is a surface antigen.

[0043] In some embodiments, each aptamer is associated / conjugated with one or more fluorophores.In some embodiments, each aptamer is associated / conjugated with two or more different fluorophores.For example, fluorophores can include, but are not limited to, FAM, Cy3, Oregon Green, or any combination of the above.

[0044] In some embodiments, the quencher comprises or consists of graphene oxide.

[0045] In some embodiments, the method includes spraying, atomizing, misting, vaporizing, or coating, dipping, directly adding an aliquot of the liquid composition, either by pouring, swabbing, or using a pipetting device. In some embodiments, the liquid composition has a viscosity, ionic strength, and / or pH that can be selected to optimize fluorescence intensity. In some embodiments, the liquid composition has a pH ranging from about 7 to about 8. In some embodiments, the liquid composition comprises a buffer (e.g., Tris buffer) having a pH of about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, or 8. In some embodiments, the liquid composition has an ionic strength ranging from about 1 mmol / L (mM) to about 2 mol / L (M). As non-limiting examples, the liquid composition can include Tris buffer, magnesium chloride, calcium chloride, sodium chloride, or combinations thereof.

[0046] In some embodiments, the method includes depositing or applying a substantially uniform layer of the liquid composition on the surface. In an embodiment, such a layer may also be conformal. In some embodiments, the method includes spraying the surface. In some embodiments, the spray distribution has a coefficient of variation (CV) of less than about 15%. In some embodiments, the spray distribution has a CV ranging from about 1% to about 15%, about 1% to about 10%, about 5% to about 15%, about 5% to about 10%, about 10% to about 15%, or any value therebetween. In some embodiments, the spraying is substantially perpendicular to the surface. In some embodiments, the contacting step includes atomizing the liquid composition onto the surface. In some embodiments, the contacting step includes depositing the liquid composition as a mist to disperse the droplets and promote better adhesion to the surface, for example, a vertical surface where the droplet size and volume are too large and the liquid flows downward. In some embodiments, the contacting comprises dispersing droplets onto the surface, the droplets having a size ranging from about 1 to about 300 μm.In some embodiments, the droplets may be from about 1 to about 50 μmm, from about 1 to about 100 μmm, from about 1 to about 150 μmm, from about 1 to about 200 μmm, from about 1 to about 300 μmm, from about 10 to about 100 μmm, from about 10 to about 150 μmm, from about 10 to about 200 μmm, from about 10 to about 250 μmm, from about 10 to about 300 μmm, from about 20 to about 100 μmm, from about 20 to about 150 μmm, from about 20 to about 200 μmm, from about 20 to about 250 μmm, from about 20 to about 300 μmm, from about 30 to about 100 μmm, from about 30 to about 150 μmm, from about 30 to about 200 μmm, from about 30 to about 250 μmm , about 30 to about 300μmm, about 40 to about 100μmm, about 40 to about 150μmm, about 40 to about 200μmm, about 40 to about 250μmm, about 40 to about 300μmm, about 50 to about 100μmm, about 50 to about 150μmm, about 50 to about 200μmm, about 50 to about 250μ mm, approximately 50 to approximately 300μmm, approximately 60 to approximately 100μmm, approximately 60 to approximately 150μmm, approximately 60 to approximately 200μmm, approximately 60 to approximately 250μmm, approximately 60 to approximately 300μmm, approximately 70 to approximately 100μmm, approximately 70 to approximately 150μmm, approximately 70 to approximately 200μmm, approximately 70 to approximately 25 0μmm, about 70 to about 300μmm, about 80 to about 100μmm, about 80 to about 150μmm, about 80 to about 200μmm, about 80 to about 250μmm, about 80 to about 300μmm, about 90 to about 100μmm, about 90 to about 150μmm, about 90 to about 200μmm, about 90 to about 250μmm, about 90 to about 300μmm, about 100 to about 150μmm, about 100 to about 200μmm, about 100 to about 250μmm, about 100 to about 300μmm, about 110 to about 150μmm, about 110 to about 200μmm, about 110 to about 250μmm, about 110 to about 30 The average size may be about 0 μmm, about 120 to about 150 μmm, about 120 to about 200 μmm, about 120 to about 250 μmm, about 120 to about 300 μmm, about 130 to about 150 μmm, about 130 to about 200 μmm, about 130 to about 250 μmm, about 130 to about 300 μmm, about 140 to about 150 μmm, about 140 to about 200 μmm, about 140 to about 250 μmm, about 140 to about 300 μmm, about 150 to about 200 μmm, about 150 to about 250 μmm, about 150 to about 300 μmm, about 200 to about 250 μmm, or about 250 to about 300 μm.In some embodiments, the droplets may have a diameter of about 1 to about 10 μm, about 10 to about 20 μm, about 20 to about 30 μm, about 30 to about 40 μm, about 40 to about 50 μm, about 50 to about 60 μm, about 60 to about 70 μm, about 70 to about 80 μm, about 80 to about 90 μm, about 90 to about 100 μm, about 100 to about 110 μm, about 110 to about 120 μm, about 120 to about 130 μm, about 130 to about 140 μm, about 140 to about 150 μm, about 150 to about 160 μm, , about 160 to about 170 μm, about 170 to about 180 μm, about 180 to about 190 μm, about 190 to about 200 μm, about 200 to about 210 μm, about 210 to about 220 μm, about 220 to about 230 μm, about 230 to about 240 μm, about 240 to about 250 μm, about 250 to about 260 μm, about 260 to about 300 μm, about 270 to about 280 μm, about 280 to about 290 μm, or about 290 to about 300 μm. For example, the droplets can have an average size of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 34, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 300 μm.

[0047] In some embodiments, the liquid composition can be uniformly coated / deposited onto a surface using a fine mist spray pressure of less than <150 psi. As noted above, in one embodiment, such a coating can be conformal.

[0048] In some embodiments, a liquid spraying device, also referred to herein as a sprayer, is used to deposit a uniform layer of a liquid composition on a surface. In some embodiments, the sprayer is a handheld device. In some embodiments, the sprayer is a manual sprayer. In some embodiments, the sprayer is a cordless handheld device. In some embodiments, the sprayer is an atomizer. In some embodiments, the sprayer is a nanoatomizer. In some embodiments, the sprayer can provide aerosol functionality. In some embodiments, the sprayer can be configured to pressurize the liquid prior to spraying. See, for example, U.S. Pat. No. 8,905,271, which is incorporated herein by reference in its entirety. In some embodiments, the liquid can be pressurized to 500 mbar to 5 bar. In some embodiments, the sprayer is configured to provide a substantially uniform spray with a spray distribution having a coefficient of variation (CV) of less than about 15%. In some embodiments, the sprayer is configured to deliver a continuous spray. In some embodiments, the nozzle of the sprayer can be configured to provide the required discharge and / or droplet size. In some embodiments, the nozzle is configured to provide a substantially homogenous distribution of the liquid composition resulting in a substantially even coverage of the surface.

[0049] It will be appreciated that the spray directed onto the surface undergoing detection, regardless of the mechanism used, should be dispersed with sufficient force to minimize disturbance of the pathogen population on the surface undergoing detection.

[0050] In some embodiments, the surface is a vertical or horizontal or inclined surface. In some embodiments, the surface is a surface in a healthcare context. In some embodiments, the surface is a surface in a food processing context.

[0051] Embodiments include methods for detecting the presence, absence, or amount of a target molecule in or on a sample, in which the sample may interact (i.e., contact) with an aptamer conjugate as described herein.

[0052] In some embodiments, the method includes incubating a surface or sample with one or more aptamer conjugates under conditions sufficient for at least a portion of the aptamers to bind to target molecules in the sample. In some embodiments, the method includes exposing the surface to a predetermined temperature for a period of time to allow the population of conjugated aptamers to bind to one or more target molecules. In some embodiments, the method includes allowing the aptamers to bind to the target molecules at room temperature for about 1 minute to about 10 minutes, 1 minute to 5 minutes, or less than 5 minutes.

[0053] One of skill in the art will understand the conditions required for binding to occur between the aptamers described herein and the target molecules in or on the surface of a sample. In some embodiments, the conjugated aptamer may be incubated at a temperature between about 4°C and about 40°C. In some embodiments, the sample and aptamer may be incubated at a temperature between about 20°C and about 37°C. In some embodiments, the sample and aptamer may be incubated at 22°C or about 22°C. The incubation temperature may be selected from the ranges of 4°C to less than 20°C, 20°C to less than 22°C, 22°C to less than 24°C, 24°C to less than 26°C, 26°C to less than 28°C, 28°C to less than 30°C, 30°C to less than 32°C, 32°C to less than 34°C, 34°C to less than 36°C, 36°C to 37°C, and 37°C to 40°C. In some embodiments, the sample and aptamer may be diluted to different concentrations (e.g., at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% v / v or more) with water or a buffer (exemplary buffers include, but are not limited to, phosphate buffered saline (PBS), Tris buffer, etc.). The dilution concentration may be selected from the following ranges: 1% to less than 5%, 5% to less than 10%, 10% to less than 20%, 20% to less than 30%, 30% to less than 40%, 40% to less than 50%, 50% to less than 60%, 60% to less than 70%, 70% to less than 80%, or 80% to less than 90%. In some embodiments, the aptamer concentration before dilution may be 100 nM to 50 μM. In some embodiments, the aptamer concentration before dilution can be selected from the ranges of 100nM to 500nM, 500nM to 1μM, 1μM to 2μM, 2μM to 5μM, 5μM to 10μM, 10μM to 15μM, 15μM to 20μM, 20μM to 30μM, 30μM to 40μM, 40μM to 50μM, 50μM to 60μM, 60μM to 70μM, 70μM to 80μM, 80μM to 90μM, 90μM to 100μM. In some embodiments, the aptamer concentration before dilution can be a concentration selected from the ranges described herein. The selected value can be selected from 0.1μM increment concentrations within the ranges herein. In some embodiments, the aptamer concentration before dilution can be 2μM.In some embodiments, the sample and aptamer may be incubated during shaking and / or mixing. In some embodiments, the sample and aptamer are incubated for at least 1 minute, at least 5 minutes, at least 15 minutes, at least 1 hour or more. The sample and aptamer may be incubated for 1 to less than 5 minutes, 5 to less than 15 minutes, 15 minutes to less than 1 hour, 1 hour to less than 24 hours, or 24 hours to less than 48 hours.

[0054] Conjugated Aptamers In some embodiments, the aptamer of the present disclosure is used to detect and / or quantify the amount of target molecule in a sample or on a surface.Typically, the aptamer comprises or is conjugated with a detectable label to form a conjugated aptamer.Any label that can facilitate the detection and / or quantification of the aptamer can be used herein.Non-limiting examples of detectable labels are described below.

[0055] In some embodiments, the detectable label is a luminescent molecule.

[0056] In some embodiments, the detectable label is a fluorescent moiety, such as a fluorescent compound (also referred to herein as a fluorophore) or a fluorescent dye. Non-limiting fluorescent dyes are shown in FIG. 1. In some embodiments, the aptamer comprises a fluorescent and a quencher compound. Fluorescent and quencher compounds are known in the art. See, for example, Mary Katherine Johansson, Methods in Molecular Biol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, 2006, Didenko, ed., Humana Press, Totowa, NJ, and Marras et al., 2002, Nucl. Acids Res. 30, el22 (incorporated herein by reference).

[0057] In some embodiments, the detectable label is fluorescein amidite (FAM). In some embodiments, the FAM-label is conjugated to the 5'-end or 3'-end of the aptamer. Those skilled in the art will understand that the label can be located at any suitable position in the aptamer.

[0058] In some embodiments, the aptamer comprises a FAM fluorophore at its 5' end. In some embodiments, the aptamer is synthesized by incorporating phosphoramidites into the nucleic acid strand one after the other, and the FAM-labeled phosphoramidites are incorporated throughout the synthesis process. In some embodiments, the FAM fluorophore is attached to the 5' end of the aptamer via a linker. In some embodiments, the detectable label is attached to the aptamer described herein via a moiety selected from a thiol group, an amine group, an azide, a 6-carbon linker, and an aminoallyl group, and combinations thereof. In some embodiments, the FAM label can be incorporated into the aptamer using a forward primer with FAM at its 5' end. In some embodiments, the aptamer can be prepared by solid phase synthesis with the FAM label already in place, attached to the 5' end in the primer.

[0059] Moieties that produce a detectable increase in signal when in close proximity to one another, for example as a result of fluorescence resonance energy transfer ("FRET"), can be used herein; suitable pairs include, but are not limited to, fluorescein and tetramethylrhodamine, rhodamine 6G and malachite green, and FITC and thiosemicarbazole, to name just a few.

[0060] In some embodiments, the detectable label is and / or includes a moiety selected from at least one of the following non-limiting examples: a fluorophore, a nanoparticle, a quantum dot, an enzyme, a radioisotope, a predefined sequence moiety, biotin, desthiobiotin, a thiol group, an amine group, an azide, an aminoallyl group, digoxigenin, an antibody, a catalyst, a colloidal metal particle, a colloidal non-metallic particle, an organic polymer, a latex particle, a nanofiber, a nanotube, a dendrimer, a protein, and a liposome.

[0061] In some embodiments, the detectable label is a fluorescent protein, such as green fluorescent protein (GFP), or any other fluorescent protein known to one of skill in the art.

[0062] In some embodiments, the detectable label is an enzyme. For example, the enzyme can be selected from horseradish peroxidase, alkaline phosphatase, urease, β-galactosidase, or any other enzyme known to one of skill in the art.

[0063] In some embodiments, the nature of detection depends on the detectable label used. For example, the label may be detectable based on its color, such as gold nanoparticles. The color can be quantitatively detected by an optical reader or camera, such as a camera with imaging software.

[0064] In some embodiments, the detectable label is a fluorescent label, e.g., a quantum dot. In such embodiments, the detection means may include a fluorescent plate reader, stripe reader, or the like configured to record fluorescence intensity.

[0065] In some embodiments where the detectable label is an enzyme label, non-limiting detection means can be, for example, colorimetric, chemiluminescent, and / or electrochemical (including but not limited to using an electrochemical detector). Electrochemical sensing can be by conjugation of a redox reporter (including but not limited to methylene blue or ferrocene) to one end of the aptamer and a sensor surface to the other end. The conformational change of the aptamer upon target binding can change the distance between the reporter and the sensor, generating a readout.

[0066] In some embodiments, the detectable label may further comprise an enzyme, including but not limited to, horseradish peroxidase (HRP), alkaline phosphatase (APP), or the like, which catalytically turns over a substrate to generate an amplified signal.

[0067] Embodiments include complexes (conjugates) comprising an aptamer of the present disclosure and a detectable molecule. Typically, the aptamers of the present disclosure are covalently or physically conjugated to the detectable molecule.

[0068] In some embodiments, the detectable molecule is a visually, optically, photonically, electronically, acoustically, photoacoustically, mass, electrochemically, electrooptically, spectroscopically, enzymatically, or other physically, chemically, or biochemically detectable label.

[0069] In some embodiments, the detectable molecule is detected by luminescence, UV / VIS spectroscopy, enzymatically, electrochemically or radioactively. Luminescence refers to the emission of light. For example, photoluminescence, chemiluminescence and bioluminescence are used to detect the label. In photoluminescence or fluorescence, excitation occurs by the absorption of photons. Representative fluorophores include, but are not limited to, bisbenzimidazole, fluorescein, acridine orange, Cy5, Cy3 or propidium iodide, which can be covalently bound to aptamers, tetramethyl-6-carboxyrhodamine (TAMRA), Texas Red (TR), rhodamine, Alexa Fluor dyes. Fluorescent dyes that are excited and emit light of different wavelengths have been developed by various companies.

[0070] In some embodiments, the detectable molecule is a colloidal metal particle, including but not limited to gold nanoparticles, a colloidal non-metallic particle, a quantum dot, an organic polymer, a latex particle, a nanofiber (non-limiting example is carbon nanofiber), a nanotube (non-limiting example is carbon nanotube), a dendrimer, a protein, or a liposome with a signal generating agent. Colloidal particles can be detected colorimetrically.

[0071] In some embodiments, the detectable molecule is an enzyme. In some embodiments, the enzyme can convert a substrate into a colored product. Examples of enzymes include, but are not limited to, peroxidase, luciferase, β-galactosidase, or alkaline phosphatase. For example, the colorless substrate X-gal is converted by the activity of β-galactosidase into a blue product whose color can be visually detected.

[0072] In some embodiments, the detection molecule is a radioisotope. Detection can be performed by detecting a labeled aptamer. 3 H, 14 C. 32 P, 33 P, 35 S or 125It can also be performed by radioisotopes, including but not limited to I. In some embodiments, scintillation counting can be performed, which indirectly measures the radioactivity emitted by radioactively labeled aptamer-target complex. The scintillator material is excited by the radiation emission of the isotope. During the transition of the scintillation material back to the ground state, the excitation energy is again emitted as a flash of light, amplified, and counted by a photomultiplier tube.

[0073] In some embodiments, the detectable molecule is selected from digoxigenin and biotin.Thus, the aptamer can also be labeled with digoxigenin or biotin, which can be bound by, for example, antibody or streptavidin, and then can have a label, for example, an enzyme conjugate.The covalent binding (conjugation) of the above aptamer with an enzyme can be achieved by several known methods.

[0074] In some embodiments, detection of binding of the aptamer is accomplished by detecting the aptamer with a radioisotope in an RIA (radioactive immunoassay), preferably 125 This can also be achieved by labeling with I, or by fluorescence in FIA (fluorescence immunoassay) using a fluorophore. In some embodiments, the fluorophore is fluorescein or fluorescein isothiocyanate (FITC).

[0075] In some embodiments, the detectable moiety comprises a fluorescent moiety, and visualizing comprises visualizing the fluorescence and / or measuring the level of fluorescence. In some embodiments, the detectable moiety comprises biotin having binding affinity for a streptavidin protein conjugate, such as streptavidin / horseradish peroxidase, and visualizing comprises visualizing using a colorimetric reaction. In some embodiments, the detectable moiety, a gold nanoparticle, is conjugated to an aptamer, and visualizing comprises visualizing using a colorimetric assay. In some embodiments, the detectable moiety comprises a fluorescent quantum dot.

[0076] In some embodiments, binding of an aptamer to a target as defined results in the formation of an aptamer-target complex. The binding or binding event can be detected, for example, visually, optically, photonically, electronically, acoustically, photoacoustically, by mass, electrochemically, electro-optically, spectroscopically, enzymatically, or otherwise chemically, biochemically, or physically, as described herein.

[0077] The binding of aptamer and target can be detected using any suitable technique.As discussed above, for example, the binding of aptamer and target can be detected using biosensor.In some embodiments, the binding of aptamer and target is detected using non-limiting examples of SPR, RlfS, BLI, LFD or ELONA as described herein.

[0078] In some embodiments, the aptamer can be attached to the surface of the biosensor using a biotin group. In some embodiments, the biotin group is attached to the 5' or 3' end of the aptamer. In some embodiments, the surface of the biosensor has avidin / streptavidin attached thereto, and the immobilization of the aptamer to the surface of the biosensor is via biotin-avidin interaction. In some embodiments, the surface of the biosensor is coated with avidin / streptavidin.

[0079] In some embodiments, the aptamer is linked or conjugated to a fluorescent moiety. In some embodiments, the aptamer is an aptamer conjugate comprising an aptamer conjugated to a fluorescent moiety. In some embodiments, the fluorophore is at the 5'-end or 3'-end of the aptamer. In some embodiments, the aptamer is associated with an antisense oligonucleotide having a fluorophore. In some embodiments, the fluorophore is at the 5'-end or 3'-end of the aptamer. In some embodiments, the antisense oligonucleotide is complementary to the 5'-end or 3'-end of the aptamer. In some embodiments, the fluorophore is at the 5'-end or 3'-end of the antisense oligonucleotide.

[0080] In some embodiments, the aptamers of the present disclosure are prepared using in vitro selection principles known in the art, including repeated cycles of target binding, separation and preferential amplification of target binding sequences. Selection can be performed using immobilized target proteins. Immobilization can include, but is not limited to, immobilization to a solid surface. In a non-limiting example, the solid surface can be a bead. In a non-limiting example, the solid surface can be a magnetic bead.

[0081] Non-limiting examples of amplification methods include polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR), strand displacement amplification, nucleic acid sequence-based amplification, and amplification methods based on the use of Q-beta replicase. In non-limiting embodiments, at least one of the aptamers can be immobilized on a solid surface during amplification. Each of these exemplary methods is well known in the art.

[0082] In some embodiments, the aptamer is selected from a nucleic acid molecule library, such as a single-stranded DNA or RNA nucleic acid molecule library. The aptamer can be selected from a "universal aptamer selection library" designed to ensure that any selected aptamer requires little or no adaptation to any of the listed assay formats.

[0083] Once selected, the aptamers can be further modified before use to remove either or both of the primer sequence and / or portions of the randomized sequence that are not required for target binding.

[0084] Typically, the aptamers of the disclosed embodiments include a first primer region (e.g., at the 5' end), a second primer region (e.g., at the 3' end), or both. The primer regions can serve as primer binding sites for PCR amplification of the library and selected aptamers.

[0085] The first primer region and / or the second region may include a detectable label as described herein. As used herein, the terms "detectable label" and "detectable moiety" are used interchangeably. In some embodiments, the first and / or second primer region may be labeled with a luminescent molecule. In some embodiments, the first and / or second primer region may be fluorescently labeled. Non-limiting examples of fluorescent labels include, but are not limited to, fluorescein, green fluorescent protein (GFP), yellow fluorescent protein, cyan fluorescent protein, and the like. In some embodiments, fluorescein labels are used. In some embodiments, other forms of detecting primers may be used, including, but are not limited to, phosphate (PO4) labels, isotopic labels, electrochemical sensors, colorimetric biosensors, and the like.

[0086] Aptamers may include natural or non-natural nucleotides and / or base derivatives (or combinations thereof). In some embodiments, aptamers include one or more modifications to include chemical structures other than deoxyribose, ribose, phosphate, adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U). Aptamers may be modified at the nucleobase, sugar or phosphate backbone.

[0087] In some embodiments, aptamer comprises one or more modified nucleotides.Representative modifications include, for example, alkylation, arylation or acetylation, alkoxylation, halogenation, nucleotides that contain amino groups or other functional groups.Examples of modified nucleotides include, but are not limited to, 2'-fluororibonucleotides, 2'-NH2-, 2'-OCH3- and 2'-O-methoxyethylribonucleotides, which are used for RNA aptamers.

[0088] Aptamers can be entirely or partially phosphorothioate or DNA, phosphorodithioate or DNA, phosphoroselenoate or DNA, phosphorodiselenoate or DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), N3'-P5' phosphoramidate RNA / DNA, cyclohexene nucleic acid (CeNA), tricycloDNA (tcDNA) or spiegelmer, or phosphoramidate morpholine (PMO) moieties or any other modification known to those of skill in the art (see also Chan et al., Clinical and Experimental Pharmacology and Physiology (2006) 33, 533-540).

[0089] Some of the modifications may stabilize the aptamer against nucleic acid cleaving enzymes. In aptamer stabilization, a distinction can generally be made between subsequent modification of the aptamer and selection with already modified RNA / DNA. Stabilization will not affect the affinity of the modified RNA / DNA aptamer, but may prevent rapid degradation of the aptamer in organisms, biological fluids or solutions by RNases / DNases. An aptamer is said to be stabilized if the half-life of the aptamer in a sample (e.g. biological medium, organism, solution) is greater than 1 minute, greater than 1 hour, or greater than 1 day. Aptamers may be modified with reporter molecules that may allow detection of the labeled aptamer. Reporter molecules may also contribute to increased stability of the aptamer.

[0090] Aptamers form three-dimensional structures according to their nucleic acid sequences. The three-dimensional structures of aptamers can arise due to Watson and Crick intramolecular base pairs, Hoogsteen base pairs (quadruplex structures), wobble-pair formation or other non-canonical base interactions. In some embodiments, the three-dimensional structures allow aptamers to bind precisely to target structures, similar to antigen-antibody binding. The nucleic acid sequences of aptamers can have three-dimensional structures specific to defined target structures under defined conditions.

[0091] The embodiments include competitive aptamers that compete with the aptamers described herein for binding to the target protein as defined herein. The embodiments include competitive one or more aptamers that compete with one or more of the aptamers described above for binding to the target protein as defined herein. In some embodiments, a competitive assay can be used to identify competitive aptamers that compete for binding to the target protein as defined herein. In an exemplary, non-limiting competitive assay, the immobilized target protein as defined herein is incubated in a solution that includes a first labeled aptamer that binds to the target protein as defined herein and a second unlabeled aptamer that is tested for its ability to compete with the first aptamer for binding to the target protein as defined herein. As a control, the immobilized target protein as defined herein can be incubated in a solution that includes the first labeled aptamer but does not include the second unlabeled aptamer. After incubation under conditions that allow for the binding of the first aptamer to the target protein as defined herein, excess unbound aptamer can be removed, and the amount of label associated with the immobilized target protein as defined herein can be measured.If the amount of label associated with the immobilized target as defined herein is substantially reduced in the test sample compared to the control sample, this indicates that the second aptamer competes with the first aptamer for binding to the target protein as defined herein.

[0092] Full-length aptamer sequences have the ability to form multiple three-dimensional conformations (also referred to herein as "shapes") at room temperature. These three-dimensional conformations are fluid, based on the energy landscape of possible conformations. The structural elements of aptamers are involved in binding to targets. In some embodiments, truncation and sequence changes of aptamer sequences to stabilize structural elements involved in binding can improve aptamer performance by reducing the energy landscape of aptamers optimized to exist only in conformations that bind to targets, or at least by increasing the probability (and therefore the existence over time) of such conformations.

[0093] In some embodiments, aptamers are modified to form secondary and / or tertiary structures to improve the binding affinity of the aptamer to a target molecule.

[0094] It should be understood that a given aptamer may exist in dynamic equilibrium between many possible shapes or conformations. These structures may be fluid with respect to each other. The binding affinity of an aptamer to a target protein depends on the structure of the aptamer. In some embodiments, the aptamer structure comprises one or more stems and loops.

[0095] To optimize the binding effectiveness of a given structure to a target protein, it is desirable that the structure of the selected aptamer is not fluid with other structures (e.g., in different environments) and is the structure in which it exists primarily. Therefore, even though the aptamer is selected using an affinity-based selection assay as described herein, further optimization may be required to achieve the desired binding affinity to the target protein. The predicted conformation / structure of each aptamer can be obtained in silico from the primary sequence. In some embodiments, the primary structure of the aptamer can be manipulated (e.g., substituted, deleted) to stabilize the secondary or tertiary structure. In some embodiments, the aptamer can be truncated to stabilize the secondary structure.

[0096] In some embodiments, aptamers are selected using affinity-based selection assays, predicted conformations are generated in silico, and primary sequences are optimized (e.g., truncations / deletions, substitutions, etc.) such that the optimized aptamers exhibit optimized conformations and are stabilized. The resulting optimized aptamers have less flexible conformations or exhibit less flexible conformational range or difference than non-optimized aptamers. These optimized aptamers can be retested for binding efficacy to determine whether the stabilized conformation is the desired conformation that binds to the target protein.

[0097] Aptamers can have a secondary structure (referred to herein as a "stem") that has at least two complementary regions of the same nucleic acid strand that base-pair to form a double helix. Stems as described herein can be referenced by the location of the 5' side of the stem in the 5' to 3' direction of the aptamer compared to the 5' side of additional stems present in the aptamer (i.e., the stem sequence closer to the 5' end of the aptamer).

[0098] For example, stem 1 may refer to the stem sequence closest to the 5' end of the aptamer, its complementary stem sequence, or both stem sequences collectively. Similarly, stem 2 may refer to the next stem sequence located 3' relative to stem 1, its complementary stem sequence, or both stem sequences collectively. In some cases, the aptamers of the present disclosure have one or more stems. For example, the aptamers of the present disclosure can have one, two, three, or more stems. Each additional stem may be referenced by its location on the aptamer as described above in the 5' to 3' direction. For example, stem 2 may be located 3' on the aptamer relative to stem 1, stem 3 may be located 3' on the aptamer relative to stem 2, etc. The stems may be adjacent to unpaired regions. The unpaired regions may be at the ends of the aptamer or in internal regions of the aptamer.

[0099] A stem as described herein can be referred to by its position in the 5' to 3' direction on the aptamer. A stem as described herein can be referred to by its length (1, 2, 3, 4, 5, 6 or more base pairs). For example, stem (4f) refers to the 4 base pair stem structure on the 5' side. Stem (4r) refers to the 4 base pair stem structure on the 3' side.

[0100] As used herein, the term "loop" generally refers to the internal unpaired region of an aptamer.The term "loop" generally refers to any unpaired region of an aptamer that is adjacent to the stem region at both the 5' and 3' ends.In some cases, the loop sequence may be adjacent to a single base-paired stem, so that the loop and stem structures together resemble a hairpin.In such cases, the primary sequence of an aptamer generally contains a first stem sequence adjacent to the 5' end of the loop sequence and a second stem sequence adjacent to the 3' end of the loop sequence, and the first and second stem sequences are complementary to each other.

[0101] A loop as described herein can be referred to by its position in the 5' to 3' direction on the aptamer.A loop as described herein can be referred to by its length (1, 2, 3, 4, 5, 6 or more nucleotides).For example, loop(4) refers to a loop structure having 4 nucleotides.

[0102] As used herein, the term "stem-loop" generally refers to the secondary structure of the aptamer of the present disclosure having at least one stem and at least one loop. In some cases, the stem-loop secondary structure includes a structure having two stems, which may include a terminal stem, an internal loop, an internal stem and a terminal loop. As used herein, "terminal stem" generally refers to a stem that encompasses both the 5' and / or 3' ends of the aptamer. In some cases, the "terminal stem" is flanked at one or both ends by a "tail" that includes one or more unpaired nucleotides. For example, the terminal stem present in an aptamer may be flanked at its 5' end by a tail of one or more unpaired nucleotides (or other structures). Similarly, the terminal stem present in an aptamer may be flanked at its 3' end by a tail of one or more unpaired nucleotides (or other structures). In some cases, the stem-loop secondary structure includes a structure having two or more stems. Unless otherwise indicated, when an aptamer comprises one or more stems and / or one or more loops, the stems and loops are numbered consecutively in ascending order from the 5' to the 3' end of the primary nucleotide sequence.

[0103] In some embodiments, the aptamer is an aptamer beacon that undergoes a conformational change when the aptamer binds to a target protein, and detection of binding of the aptamer to the target protein relies on the conformational change of the aptamer.

[0104] In some embodiments, the aptamer conjugate is an aptamer that comprises a fluorescent moiety at the first end of the aptamer and a quencher moiety at the second end of the aptamer.In some embodiments, the aptamer comprises a loop, a first nucleic acid fragment that is complementary to a second nucleic acid fragment, and the first fragment and the second fragment form a stem portion when the first fragment and the second fragment are hybridized, and the first aptamer fragment comprises a fluorophore and the second aptamer fragment comprises a quencher.

[0105] In some embodiments, antisense oligonucleotide can be designed to hybridize with the first fragment, the second fragment or a combination thereof and to disrupt the stem and loop structure of aptamer.For example, antisense oligonucleotide can be complementary to the 5' end, the 3' end, the 5' end and the 3' end of aptamer or any related sequence.In some embodiments, two antisense oligonucleotides are provided, the first antisense oligonucleotide comprises a fluorophore and hybridizes with the first fragment of aptamer, and the second antisense comprises a quencher and hybridizes with the second fragment of aptamer.

[0106] In some embodiments, the quencher comprises a "dark" quencher. In some embodiments, the quencher comprises a Black Hole Quencher® (BHQ). For example, the 3' end of the antisense oligonucleotide can be linked to a Black Hole Quencher®.

[0107] In some embodiments, the antisense oligonucleotide acts to compete with the binding of the aptamer to the target protein.

[0108] In some embodiments, upon binding of the aptamer to a target protein, the aptamer undergoes a conformational change that alters the distance between the fluorophore and the quencher, resulting in the emission of a fluorescent signal.

[0109] In some embodiments, two or more different aptamers are provided configured to bind to two or more different target proteins in a sample, each aptamer comprising a different fluorophore.

[0110] Graphene oxide In some embodiments, a composition is provided that includes one or more aptamer conjugates and graphene oxide (GO). Graphene oxide self-assembles into two-dimensional sheets in an aqueous environment (see He et al., Chemical Physical Letters, Volume 287, Issues 1-2, 24 April 1998, Pages 53-56). In some embodiments, the composition is a suspension. In some embodiments, the composition includes a buffer. In some embodiments, the composition further includes a blocking agent to minimize or eliminate non-specific binding. In some embodiments, the blocking agent includes, but is not limited to, polyethylene glycol (PEG) (including polymer chains of various lengths), Tween (e.g., Tween 20, Tween 40, Tween 80), nucleic acid (e.g., oligonucleotides, fragmented salmon sperm DNA), polyvinylpyrrolidine, or any blocking agent known in the art.

[0111] In some embodiments, fluorescently labeled aptamers are provided that allow for surface detection of target molecules in combination with graphene oxide. Without being bound to any particular theory, the labeled aptamers bind to graphene oxide (GO) with electrostatic repulsion by balancing binding by π-π stacking and hydrogen bonding. The proximity of the fluorophore label on the aptamer to the GO surface results in quenching of fluorescence. The introduction of a mixture of aptamer and graphene oxide to a target molecule to which the aptamer binds or a sample containing the target molecule changes the equilibrium formed between the aptamer and GO such that less aptamer binds to GO and more aptamer binds to the target. The fluorescence of the aptamer bound to the target is not quenched, or at least not to the same extent as when the aptamer binds to GO.

[0112] In some embodiments, this aptamer / GO mixture can be used for the detection of visual changes on a surface. In some embodiments, when the aptamer / GO equilibration mixture is sprayed onto a surface in the absence of target molecules, no fluorescence is visible / detectable. In some embodiments, fluorescence is observed / detected in the presence of target molecules (or pathogens).

[0113] The equilibrated mixture of aptamer / GO refers to a mixture of aptamer / GO that has a relative fluorescence that does not change over time. In some embodiments, this equilibration can be achieved in less than 24 hours after aptamer and GO are mixed. For example, equilibration can be achieved in about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours or less.

[0114] In some embodiments, the composition comprises one or more different aptamers that have binding affinity to the same target molecule.In some embodiments, the composition comprises one or more different aptamers that have binding affinity to one or more different target molecules.In some embodiments, each aptamer can comprise a different detectable moiety.

[0115] Graphene oxide can be prepared by the synthesis of graphene from oxygen donor sources, such as NaNO3, H2SO 4、 The graphene oxide is prepared by exposure to H3PO4 and KMnO4. In some embodiments, the graphene oxide comprises an oxygen content of about 36%. In some embodiments, the graphene oxide comprises an oxygen content greater than 36%. In some embodiments, the graphene oxide comprises an oxygen content of about 44-45%.

[0116] Once formed, the graphene oxide self-assembles into two-dimensional sheets of various sizes. In some embodiments, the total surface area of ​​the graphene oxide is about 736.6 m. 2 / g. In some embodiments, the total surface area of ​​graphene oxide is a function of the amount of graphene oxide used. In some embodiments, the amount of graphene oxide used is optimized based on the level of fluorescence quenching. Without being bound by theory, determining the appropriate amount of GO to use for a desired level of aptamer quenching can be a consideration of the total surface area of ​​GO in solution. In some embodiments, the feasible range is defined as a function of the desired quenching range, taking into account the need to visualize fluorescence in the presence of virus and not in its absence.

[0117] In some embodiments, the graphene oxide is in the form of nanoparticles, hi some embodiments, the average size of the nanoparticles is between 10 and 500 nm.

[0118] Aptamers (APTs) are attached to graphene oxide (GO) sheets based on van der Waals forces and hydrogen bonds. In some embodiments, a detectable moiety, e.g., a fluorescent moiety, is conjugated to the aptamer, and the fluorescence of the fluorescent moiety is quenched upon association with the graphene oxide surface.

[0119] In some embodiments, in the presence of a target surface protein to which the aptamer binds with a binding affinity greater than the binding affinity to graphene oxide, the aptamer is "displaced" from the graphene oxide surface and binds to the target protein. In some embodiments, the aptamer binds with a binding affinity that is greater than 2x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 500x, 1000x, or more than the binding affinity to graphene oxide. In some embodiments, the aptamer binds with a binding affinity between 2-1000 times, between 10-1000 times, between 50-1000 times, between 100-1000 times, between 2-100 times, between 10-100 times, between 50-100 times, between 100-200 times, between 100-500 times, between 100-1,000 times the binding affinity to graphene oxide. When the aptamer is "displaced" from the graphene by its specific binding to the target protein, fluorescence is emitted. A minimum of 2 times, the most preferred usability would be 100 times, and an acceptable range would be between 10-1000 times.

[0120] Without being bound by theory, in physical terms, the aptamer is not transported (it rapidly binds and dissociates dynamically) and the relationship between the aptamer and the graphene oxide surface can be described by the following associated differential equation: Dx1=-a * x1 * x2+b * x3 Dx2=-a * x1 * x2+b * x3 Dx3=a * x1 * x2-b * x3 (where x1 corresponds to the concentration of aptamer, x2 corresponds to the concentration of graphene oxide, and x3 corresponds to the concentration of aptamer-graphene oxide complex, and "D" represents the derivative as a function of time.) D is the derivative of these concentrations over time, and "a" and "b" are the binding and dissociation rates for complex formation.

[0121] These equations describe the movement of the system towards equilibrium, where the system is not static, the molecules still associate and dissociate at the same rate, and the overall measurement of the system simply does not result in a change in the expression of fluorescence.

[0122] Therefore, the introduction of an additional factor [T], the concentration of the target protein to which the aptamer binds, shifts this equilibrium such that the concentration of aptamer / GO decreases relative to the amount of aptamer / T formed.

[0123] So we can construct a new system of equations as follows: Dx1=-a * x1 * x2+b * x3-c * x1 * x4+d * x5 Dx2=-a * x1 * x2+b * x3 Dx3=a * x1 * x2-b * x3 Dx4=-c * x1 * x4+d * x5 Dx5=c * x1 * x4-d * x5 (wherein x1 corresponds to the concentration of aptamer, x2 corresponds to the concentration of graphene oxide, x3 corresponds to the concentration of aptamer-graphene oxide complex, x4 corresponds to the concentration of target, x5 corresponds to the concentration of the complex between free target and aptamer, and "c" and "d" are the binding and dissociation rates for the complex formation between the aptamer and target).

[0124] When d / c is a smaller value than b / a, the equilibrium shifts towards a greater amount of x5 and a lesser amount of x3, resulting in the appearance of fluorescence.

[0125] In some embodiments, the methods provided herein allow for a detection limit of 25 nM or greater.

[0126] In some embodiments, the ratio of aptamer to target protein may play a role in enhancing signal at lower concentrations.Without being bound by theory, this is because lower concentrations of aptamer may require less target protein to significantly change recovery (or change proportionality).In some embodiments, using less aptamer may help to detect concentrations of less than about 25nM, for example about 10pM or about 100fM.

[0127] In some embodiments, there is provided a method for detecting the presence or absence of a target molecule, comprising: providing one or more aptamer conjugates comprising one or more aptamers conjugated to one or more detectable moieties; combining the aptamer conjugate with a predetermined concentration of graphene oxide; contacting the aptamer conjugate-graphene oxide combination with the sample to be tested; and Visualizing the detectable portion of the aptamer conjugate bound to the target molecule A method is provided, comprising:

[0128] In some embodiments, the method further comprises incubating the aptamer conjugate with the sample for a predetermined period of time to allow the aptamer conjugate to bind to the target molecule, if present.

[0129] In some embodiments, the sample is first contacted with a blocking agent, and then contacted with the aptamer conjugate-graphene oxide combination.

[0130] In some embodiments, the visualizing step comprises imaging the sample. In some embodiments, the visualizing step comprises measuring a level of fluorescence. In some embodiments, the visualizing step comprises comparing the level of fluorescence to a negative control sample. In some embodiments, the method does not include a washing step prior to the visualizing step.

[0131] visualization In some embodiments, the method includes illuminating a site (e.g., a region of a surface or a sample) with an illumination device. In some embodiments, the illumination device used is configured to emit light at a wavelength or range of wavelengths that allows visualization of the detectable molecule conjugated to the aptamer. In some embodiments, the detection molecule is a fluorophore that absorbs and is excited by the wavelength output of the illumination device.

[0132] In some embodiments, the illuminator is configured to emit light in a predetermined wavelength range, the predetermined wavelength corresponding to the wavelength of light that is absorbed and excites a detectable moiety of the aptamer conjugate. For example, the aptamer may be conjugated to a fluorophore that absorbs at a wavelength of about 505 nm, and the illuminator produces light having a wavelength of about 505 nm.

[0133] A typical fluorophore absorption-emission spectrum diagram is illustrated in FIG. 2. Fluorophores are photoreactive chemical compounds that absorb light energy at a specific wavelength and emit light at a longer wavelength. Due to their novel electronic conformation, fluorophores have their own characteristic absorption (excitation) and emission spectra. These absorption and emission spectra indicate the relative intensity of fluorescence, which is classically plotted on the vertical axis against wavelength on the horizontal axis. For a given fluorophore, the manufacturer indicates the wavelength of the peak of excitation irradiance (maximum excitation) and the wavelength of the peak of fluorescence emission intensity (maximum emission). In some embodiments, when the fluorophore is conjugated to an aptamer, the excitation and emission spectra are shifted to lower or higher excitation or emission wavelengths.

[0134] In some embodiments, the lighting device may have a single emitter. In some embodiments, the lighting device may have multiple identical light emitters, e.g., to increase brightness. In some embodiments, multiple lighting devices with identical light emitters may be used, e.g., to increase brightness. In some embodiments, the emitter is a light emitting diode (LED). In some embodiments, the emitter is a laser, etc.

[0135] In some embodiments, the single emitter is configured to emit light at a single predetermined wavelength. In some embodiments, the single emitter is configured to emit light at a single predetermined wavelength range. In some embodiments, the single predetermined wavelength or the single predetermined wavelength range is at or around an excitation maximum wavelength of one or more fluorophores. For example, the wavelength range has a peak wavelength at or around an excitation maximum wavelength of one or more fluorophores. In some embodiments, the single light emitter is configured to emit light at two or more predetermined wavelengths or two or more predetermined wavelength ranges. In some embodiments, the two or more predetermined wavelengths or the two or more predetermined wavelength ranges are at or around an excitation maximum wavelength of one or more fluorophores. In some embodiments, the two or more predetermined wavelengths or the two or more predetermined wavelength ranges are at or around an excitation maximum wavelength of two or more fluorophores.

[0136] For example, the emitter may emit light within a particular wavelength range or may be designed to generate light anywhere between about 350 nm and 740 nm. For example, in some embodiments, the emitter may emit light in a wavelength range between about 360 nm and 385 nm (UV light). In some embodiments, the emitter may generate light with a wavelength between about 405 nm and 420 nm. In some embodiments, the emitter may generate light with a wavelength between about 435 nm and 465 nm. In some embodiments, the emitter may generate light with a wavelength between about 485 nm and 515 nm. In some embodiments, the light source may generate light with a wavelength between about 490 nm and 505 nm. In some embodiments, the emitter may generate light with a wavelength between about 510 nm and 545 nm. In some embodiments, the emitter may generate light with a wavelength between about 530 nm and 560 nm. In some embodiments, the emitter may generate light with a wavelength between about 585 nm and 605 nm. In some embodiments, the emitter may generate light with a wavelength between about 615 nm and 635 nm. In some embodiments, the emitter may generate light with a wavelength between about 400 nm and 700 nm. In some embodiments, the emitter may generate light with a wavelength between about 835 nm and 865 nm. In some embodiments, the emitter may generate light with a wavelength between about 935 nm and 965 nm. In some embodiments, the emitter is configured to generate narrow bands of light having center wavelengths of 365 nm, 415 nm, 450 nm, 485 nm, 505 nm, 530 nm, 545 nm, 620 nm, and 850 nm. In some embodiments, the emitter is configured to generate a precise band of light of 485 nm with a variation of + / - 2%. In some embodiments, the emitter is configured to generate a narrow band of light having a center wavelength of 505 nm.

[0137] In some embodiments, a single emitter is configured to modulate / switch from a first predetermined wavelength to a second predetermined wavelength.

[0138] In some embodiments, the excitation wavelength can be optimized for a particular fluorophore or fluorescent dye. Depending on the fluorophore, the excitation wavelength can range from about 350 nm to about 650 nm (including but not limited to about 440-460 nm, about 490-515 nm, about 505 nm, about 480 nm, etc.). As illustrated in Figures 1 and 2, depending on the fluorophore, the excitation wavelength can range from about 450 nm to about 670 nm, with the emission wavelength being longer than the excitation wavelength. In some embodiments, the excitation wavelength range for FAM fluorophores can include but is not limited to 475-515 nm, and the excitation wavelength range for Cy3 fluorophores can include but is not limited to 490-560 nm. In some embodiments, the illuminator is configured to emit light at a wavelength or wavelength range close to the maximum excitation wavelength of the fluorophore to enable visualization of the output fluorescence. For example, the maximum excitation wavelength for FAM is 495 nm excitation wavelength, and an illumination device configured to visualize FAM-aptamer conjugates may have a wavelength ranging from about 485 nm to about 515 nm. In some embodiments, the emitter can generate light of sufficient illuminance to allow visualization of fluorophores at a predetermined wavelength or range of wavelengths. In some embodiments, the emitter has sufficient power (or wattage) to allow visualization of fluorophores at a predetermined wavelength or range of wavelengths.

[0139] In some embodiments, the illuminator is configured to emit light at a predetermined lumen value or range of values ​​that allows visualization of the aptamer conjugate. In some embodiments, the illuminator is configured to provide between about 4500 and about 6500 lumens. For example, an illuminator for visualizing a FAM-aptamer conjugate can be configured to provide about 5500 lumens.

[0140] In some embodiments, the illuminator is configured to emit light at a predetermined lux value or range of values ​​that allows visualization of the aptamer conjugate. In some embodiments, the illuminator is configured to provide an intensity of about 200,000 to 280,000 lux at 505 nm. For example, an illuminator for visualizing FAM-aptamer conjugates can be configured to provide an intensity of about 234,000 lux at 505 nm.

[0141] In some embodiments, blocking or removing light includes using one or more filters configured to substantially block excitation wavelengths while passing substantially all emission wavelengths for detection and / or visualization and / or imaging. In some embodiments, the filter is between the surface and the viewer, detection device or imaging device to aid in visualization of the fluorescence emitted from the surface and / or to improve the signal-to-noise ratio. Depending on the fluorophores used, different filters can be used for optimal visualization. For example, if different fluorophores have sufficiently different emission wavelengths, different filters may be required for optimal visualization. In some embodiments, visualization can be performed in ambient light or in some embodiments, visualization can be performed in ambient light or in a dark environment.

[0142] In some embodiments, the filter is a bandpass filter, a longpass filter, or a combination thereof. A bandpass filter passes a band of light from the filter to the detector (e.g., a 580 nm bandpass passes light from about 555 nm to 605 nm). A longpass filter passes longer wavelengths of light to the detector (e.g., a 580 nm longpass allows light from about 580 nm to the end of the visible spectrum).

[0143] The method according to some embodiments includes a set of conditions for illuminating the site using an illumination device as described herein. In some embodiments, the illumination device includes a single emitter, and the illumination device is configured to allow switching between two or more different wavelengths, each wavelength suited to a particular detectable molecule (e.g., a fluorophore) and / or compatible filter.

[0144] In some embodiments, the lighting device is a portable lighting device. In some embodiments, the lighting device is a handheld lighting device. In some embodiments, the lighting device is a cordless device. In some embodiments, the lighting device is battery powered and rechargeable. In some embodiments, the battery is built into the device. In some embodiments, the battery is removable. Non-limiting lighting devices include flashlights, flares, and the like.

[0145] Illumination devices are well known and available to those skilled in the art. In some embodiments, the illumination device can be in the form of a battery-powered, handheld LED light source (e.g., available from Rofin Forensic, etc.). For example, the illumination device is a Polilight-Flare® Plus2 with an output of 485 nm to 515 nm and a peak wavelength of 505 nm, for example, to allow visualization of FAM-aptamer conjugates.

[0146] In some embodiments, the illuminator is a NightSea Xite Royal Blue (XRB) emitting 440-460 nm, a NightSea Prototype Blue Light emitting 470-490 nm, or a NightSea Cyan (CY) emitting 490-515 nm, e.g., to allow visualization of FAM-aptamer conjugates.

[0147] In some embodiments, the illuminator is custom made to emit a precise wavelength of 485 nm with a variation of + / - 2%.

[0148] In some embodiments, the method further includes imaging (eg, photographing) the site and detecting the presence or absence of a target molecule (or pathogen).

[0149] In some embodiments, the method can include the use of a bandpass filter in combination with the illumination device. The bandpass filter can be configured to transmit light in a particular wavelength band and block stray light outside of the predetermined wavelength band. In some embodiments, the bandpass filter is a 590 nm bandpass filter that passes light between 570 and 610 nm to the detector.

[0150] In some embodiments, the method can include the use of a long pass filter in combination with the illumination device. The long pass filter transmits wavelengths of light longer than or greater than a specified wavelength and blocks or blocks shorter wavelengths of light. In some embodiments, the long pass filter is 550 nm and blocks light shorter than 550 nm but allows longer wavelengths of light to pass to the detector.

[0151] In some embodiments, one or more filters can be worn as eyewear, such as goggles and glasses, hi other embodiments, the filter can be a feature of the imaging device (e.g., a camera) used to image the surface.

[0152] In some embodiments, visualizing the site may occur under ambient light or dark conditions.

[0153] In some embodiments, the method may further include visualizing the site using viewing goggles, glasses, etc. In some embodiments, the goggles are orange in color and thus suitable for use in combination with an illumination device that produces light having a wavelength between about 485 nm and 515 nm, e.g., 505 nm, and an aptamer containing detection molecule that emits at a wavelength of approximately 505 nm.

[0154] In some embodiments, the fluorescence can be observed visually. In some embodiments, the fluorescence can be observed using an instrument. In some embodiments, the instrument is a fluorometer. In some embodiments, the fluorometer can be calibrated to allow for observation of a signal of a predetermined wavelength and / or a predetermined intensity.

[0155] kit Embodiments also provide kits for detecting and / or quantifying a target molecule, the kits comprising one or more aptamers associated with a detectable molecule as described herein.

[0156] Embodiments provide kits further comprising an illumination device as described herein. In some embodiments, the kits may further comprise a bandpass or longpass filter as described herein. In some embodiments, the kits may further comprise viewing goggles or glasses, etc. as described herein. In some embodiments, the kits may further comprise a spray device, etc. as described herein.

[0157] In some embodiments, the kit comprises: a) a solution comprising an aptamer or a cocktail of aptamers comprising one or more fluorophores capable of emitting at one or more emission wavelengths; b) an illumination device configured to emit light at one or more predetermined excitation wavelengths; c) One or more filters (bandpass or longpass filters) d) Spray device Includes.

[0158] In some embodiments, the kit further comprises viewing eyewear comprising a bandpass or longpass filter.

[0159] In some embodiments, the kit further comprises a polarizing filter. The polarizing filter can be used to reduce glare or reflection of light coming from a surface (e.g., glass or metal). In some embodiments, the polarizing filter is a circular polarizing filter. In some embodiments, the polarizing filter can be part of the imaging device. For example, the circular polarizing filter can be attached to the lens of a camera.

[0160] For example, the kit may include: a) A solution containing a detection molecule conjugated to an aptamer, for example an aptamer having a fluorophore capable of emitting at a wavelength between about 485-515 nm. In some embodiments, the fluorophore is capable of emitting at a wavelength having a peak at 517 and is capable of being excited at a peak between about 490-505 nm. In some embodiments, the fluorophore is capable of being excited at a peak at 495 nm. b) Illumination Device. In some embodiments, the illumination device is configured to emit light having a wavelength between about 485-515 nm. In some embodiments, the illumination device is configured to emit light having a wavelength between about 490-505 nm. c) Filter: In some embodiments, the filter comprises or consists of a 590 nm bandpass filter, and in some embodiments, the filter is a 550 nm longpass filter. d) Vision goggles. In some embodiments, the visibility goggles are orange visibility goggles. In some embodiments, the visibility goggles are band pass filters. In some embodiments, the visibility goggles are long pass filters. e) Polarizing Filter: In some embodiments, the polarizing filter is a circular polarizing filter used to image the surface. f) Spraying device. In some embodiments, the spraying device is a manual sprayer. In some embodiments, the spraying device has aerosol functionality. In some embodiments, the spraying device is an atomizer sprayer. In some embodiments, the spraying device is a fogger sprayer.

[0161] In some embodiments, the kit further comprises instructions for use according to any of the methods described herein.

[0162] The kit may include additional components for the reaction intended to be carried out by the kit or method, such as components for the intended detection of concentration, separation and / or isolation procedures.Non-limiting examples include buffers, substrates for color reactions, dyes or enzyme substrates.In the kit, the aptamer may be provided in various forms, including pre-immobilized on a support (e.g., solid support), lyophilized, or in a liquid medium.

[0163] The kits herein can be used to carry out any of the methods described herein. The parts of the kits can be packaged individually in vials or combined in a container or multi-container unit. Typically, the preparation of the kits follows standard procedures known to those skilled in the art.

[0164] use In some embodiments, a method for detecting target molecules is provided that uses conjugated aptamers.The method can include allowing a sample to interact with the conjugated aptamers described herein and detecting the presence, absence and / or amount of one or more target molecules (of one or more pathogens).The method can be for detecting the presence, absence and / or amount of target molecules in a sample using detection methods including but not limited to photonic detection, electronic detection, acoustic detection, electrochemical detection, electro-optical detection, enzymatic detection, chemical detection, biochemical detection or physical detection.

[0165] In some embodiments, the method is for detecting the presence, absence or amount of one or more target molecules on a surface.In some embodiments, the provided aptamer and method can be useful for detecting pathogens on surfaces in hospitals and health facilities.Non-limiting examples of surfaces can include bed linen, medical equipment, clothing, floors, walls, etc.

[0166] In some embodiments, the methods may be for use in detecting target molecules from a surface as described herein in a previously obtained sample.

[0167] In some embodiments, the methods of the present disclosure may be used to detect surface proteins, antigens, etc. In some embodiments, the methods of the present disclosure may be used to detect pathogens, including but not limited to, C. difficile.

[0168] In some embodiments, the method may be used to detect one or more target surface proteins or one or more pathogens in real time. Following detection and / or quantification of one or more pathogens, actions may be taken to kill and / or remove the pathogens. Non-limiting examples of such actions may include washing or discarding bed linen, and / or cleaning surfaces, including but not limited to medical equipment, beds, walls, floors, etc. Measures such as isolating patients and implementing strict hygiene protocols may also be taken.

[0169] In one aspect of the present disclosure, a composition is provided that comprises at least one aptamer, where at least one of the aptamers is as described herein, and the composition optionally comprises at least one of water, salt, one or more of the buffers herein, detergent, and BSA.In one aspect of the present disclosure, a composition is provided that comprises at least one aptamer and graphene oxide, where at least one of the aptamers is as described herein, and the composition optionally comprises at least one of water, salt, one or more of the buffers herein, detergent, and BSA.In some embodiments, the composition is a solution.In one aspect of the present disclosure, a composition is provided that comprises at least one aptamer, and optionally comprises at least one of water, salt, one or more of the buffers herein, detergent, and BSA.

[0170] In one aspect of the disclosure, there is provided an apparatus for detecting the presence, absence or level of a surface antigen in a sample, comprising: i. a support; and ii. Aptamers as described herein An apparatus is provided that includes:

[0171] In an embodiment, the device is for detecting the presence, absence or level of a surface antigen in a sample.

[0172] In embodiments, the sample may be a sample previously obtained from a subject suspected of or diagnosed with a bacterial or viral infection, hi embodiments, the sample may be an object in a hospital environment, such as bedding, furniture, building structures.

[0173] In embodiments, the support is a bead, a microtiter plate or other assay plate, a strip, a membrane, a film, a gel, a chip, a microparticle, a nanoparticle, a nanofiber, a nanotube, a micelle, a micropore, a nanopore, or a biosensor surface.

[0174] In embodiments, the instrument is suitable for surface plasmon resonance (SPR), biolayer interferometry (BLI), lateral flow assays and / or enzyme-linked oligonucleotide assays (ELONA).

[0175] In one aspect of the disclosure, there is provided the use of an aptamer, a conjugate, a biosensor or test strip, a composition or a device as described herein for detecting, concentrating, separating and / or isolating a surface antigen.

[0176] In one aspect of the disclosure, a method is provided for detecting the presence, absence or amount of a surface antigen in a sample, the method comprising interacting the sample with an aptamer, complex, or composition as described herein, and detecting the presence, absence or amount of the surface antigen.

[0177] In some embodiments, the method is for detecting the presence, absence or amount of a surface protein in a sample.

[0178] In some embodiments, the presence, absence or amount of a surface antigen is detected by photonic, electronic, acoustic, electrochemical, electro-optical, enzymatic, chemical, biochemical or physical detection.

[0179] In one aspect of the disclosure, a kit for detecting and / or quantifying a surface antigen is provided, the kit comprising an aptamer as described herein.

[0180] The present disclosure is now described in more detail by way of non-limiting examples of specific embodiments. The experimental examples use uncontaminated standard reagents and buffers. EXAMPLES

[0181] [Example 1] Visualizing pathogens on patient room surfaces Surfaces in a patient's room (non-limiting examples of surfaces are doorknobs, bed guards, chair arms, and sink faucet knobs) were analyzed for contamination with pathogens of interest, e.g., Clostridioides difficile. Identified surfaces were sprayed with a solution containing FAM-aptamer conjugates and graphene oxide, which coats the surface with a thin, uniform deposit of the solution. The solution dries within minutes, and visualization was performed by passing a forensic torch (Polilight-Flare® Plus 2 cyan light with an output ranging from 485 nm to 515 nm and a peak wavelength of 505 nm) over the dried surfaces. Areas of pathogen contamination were determined by the user wearing appropriate filter glasses.

[0182] [Example 2] Visualizing pathogens on patient room surfaces Surfaces within a patient's room were analyzed for contamination with a pathogen of interest, e.g., Clostridioides difficile. Identified surfaces were sprayed with a solution containing Cy3-aptamer conjugate and graphene oxide, which coats the surface with a thin, uniform deposit of the solution. The solution dries within minutes and visualization was performed by passing it through a forensic torch (NIGHTSEA cyan light, with output ranging from 490-515 nm). Alternatively, a Poliflare "green" light (with output ranging from 510-545 nm, with a peak wavelength of 530 nm) can be used. Areas of pathogen contamination were determined by the user wearing appropriate filter glasses.

[0183] References cited throughout this application are incorporated herein in their entirety as if each reference were fully set forth, for all purposes apparent in the specification and the references themselves. For presentation purposes, certain of these references are cited at specific points in this specification. The citation of a reference at a specific point indicates the manner in which the teachings of the reference are incorporated. However, the citation of a reference at a specific point does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0184] It is understood, therefore, that the disclosure is not limited to the particular embodiments disclosed, but is intended to encompass all modifications that are within the spirit and scope of the disclosure as defined by the appended claims, the above description, and / or as illustrated in the accompanying drawings.

Claims

1. 1. A method for visualizing the presence of one or more pathogens on a surface, comprising: a. applying a substantially uniform layer of a liquid composition to the surface, the liquid composition comprising: (i) one or more aptamer conjugates comprising one or more aptamers covalently or non-covalently associated with one or more fluorophores, the aptamer conjugates having specific binding affinity for one or more target molecules of the one or more pathogens; and (ii) a quencher to quench non-specific fluorescence A process comprising: b. exposing the surface to a predetermined temperature for a period of time to allow the population of conjugated aptamers to bind to the one or more target molecules; c) illuminating at least one region of the surface by emitting light in one or more predetermined wavelength ranges around or at an excitation maximum wavelength of the one or more phosphors, wherein the one or more phosphors absorb the emitted light in the one or more predetermined wavelength ranges and emit light in a longer wavelength range; d. blocking the emitted light by (i) blocking the one or more excitation wavelength ranges and transmitting a second wavelength range that includes some or all of the wavelengths emitted by the one or more fluorophores, or (ii) blocking wavelengths shorter than the emitted wavelength range of the fluorophores. A method comprising:

2. The method of claim 1 , wherein the applying step comprises spraying the liquid composition onto the surface.

3. 3. The method of claim 2, wherein the step of spraying comprises a spray distribution with a coefficient of variation (CV) of less than 15%.

4. The method of claim 2 , wherein the spraying comprises minimizing disturbance of the one or more pathogens on the surface.

5. The method of claim 1 , wherein the applying step comprises atomizing the liquid composition onto the surface.

6. The method of claim 2 or 5, wherein said spraying or atomizing comprises dispersing droplets onto said surface, said droplets having an average size ranging from about 10 to about 100 μm.

7. 6. The method of any one of claims 1 to 5, wherein step (b) comprises incubating the surface with the one or more aptamer conjugates at room temperature for about 1 minute to about 10 minutes.

8. The method of claim 7, wherein the incubation is for less than 5 minutes.

9. 6. The method of claim 1, wherein the liquid composition comprises two or more conjugated aptamers, each of the two or more aptamers being conjugated to a different fluorophore.

10. 10. The method of claim 9, wherein the illuminating step comprises emitting the light in two or more predetermined wavelength ranges at or around two or more excitation maxima of the different fluorophores.

11. 6. The method of claim 1, wherein the illuminating step comprises emitting the light in the one or more predetermined wavelength ranges at or around an excitation maximum wavelength of the one or more fluorophores at an illuminance that allows visualization of the fluorophores.

12. 6. The method of claim 1, wherein the blocking of light comprises using one or more filters, the one or more filters comprising a long pass filter.

13. 6. The method of claim 1 , wherein the quencher comprises or consists of graphene oxide.

14. The method of claim 1 , wherein the one or more target molecules comprises a protein.

15. The method of claim 1 , wherein the liquid composition comprises two or more different aptamers.

16. The method of claim 15, wherein the two or more different aptamers bind to different target proteins.

17. The method of claim 15 , wherein the two or more different aptamers bind to the same target protein.

18. The method of claim 1 , further comprising imaging the surface.

19. 6. The method of claim 1, further comprising identifying the one or more pathogens.

20. 6. The method of claim 1, further comprising decontaminating the surface.

21. 21. The method of claim 20, further comprising the step of: (e) determining the presence or absence of fluorescence from the population of conjugated aptamers, thereby determining the presence or absence of the one or more pathogens on the surface.

22. 21. The method of claim 20, further comprising repeating steps (a) through (e).

23. 6. The method of any one of claims 1 to 5, wherein during step (b), a first population of conjugated aptamers binds to the one or more targets and a second population of conjugated aptamers does not bind to the one or more target molecules and remains associated with the quencher.

24. 6. The method of claim 1, wherein step (c) comprises illuminating the at least one region of the surface with an illumination device having a single emitter configured to emit light in the one or more predetermined wavelength ranges at or around an excitation maximum wavelength of the one or more phosphors.

25. 25. The method of claim 24, wherein the emitter is configured to emit the light in two or more predetermined wavelength ranges at or around two or more excitation maxima of different fluorophores.

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