Devices, systems and methods for detecting target analytes

Abstract

The present disclosure provides devices for detecting target analytes. In accordance with one aspect of the disclosure, a device for detecting the presence or absence of a target analyte is provided. In accordance with another aspect of the disclosure, a device for detecting presence or absence of a first target analyte and a second target analyte is provided. Devices of the present disclosure include a force sensor and a force transducer. The present disclosure further provides systems for detecting target analytes and methods for detecting target analytes.

Description

[0001] DEVICES, SYSTEMS AND METHODS FOR DETECTING TARGET ANALYTES

[0002] CROSS REFERENCE TO APPLIC TIONS

[0003] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Application No. 63 / 683,877, filed August 16, 2024, the disclosure of which is incorporated herein by reference.

[0004] INTRODUCTION

[0005] Single molecule detection of biological target analytes has been performed using a variety of methods. Single target analytes can be detected directly using methods such as molecular direct electronic detection, wherein nanoscale electronic structures are patterned with aptamers that bind to target molecules with high specificity and produce a measurable electronic signal upon attachment. Single target analytes can also be directly detected via nanoparticle binding in methods such as surface plasmon resonance. However, the signal arising from direct detection is very minimal and as such, 1) may be difficult to detect, and 2) current approaches employed to increase signal often are at the expense of specificity. Further, the specialized nanofabrication methods used for the above direct detection methods are not yet in high volume production manufacturing.

[0006] Biological targets may also be detected using genetic amplification. In genetic amplification, a large number of copies of the target molecule are created using nucleic acid amplification methods such as quantitative polymerase chain reaction (qPCR), real-time reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), nicking enzyme amplification reaction (NEAR) and reverse transcription-nicking enzyme amplification reaction (RT-NEAR). A high copy number of the target molecule allows for the target to be detected more readily. However, genetic amplification methods require time to synthesize the large amount of nucleic acid amplification product needed for detection. Further, genetic amplification is limited to targets that are nucleic acids.

[0007] SUMMARY

[0008] The present disclosure provides devices for detecting target analytes. In accordance with one aspect of the disclosure, a device for detecting the presence or absence of a target analyte is provided. In accordance with another aspect of the disclosure, a device for detecting presence or absence of a first target analyte and a second target analyte is provided. Devices of the present disclosure include a force sensor and a force transducer.

[0009] The present disclosure further provides systems for detecting target analytes and methods for detecting target analytes.

[0010] BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 depicts a schematic of a planar substrate, a first component, a second component and a target analyte, wherein the target analyte is an oligonucleotide.

[0012] FIG. 2 depicts a schematic of steps for detecting a target analyte using the components depicted in FIG. 1.

[0013] FIG. 3A depicts a schematic of a planar substrate, a first component, a second component and a target analyte, wherein the target analyte is a cell, virus, sub-cellular structure or protein.

[0014] FIG. 3B depicts a schematic of a planar substrate, a first component that binds multivalently to the target analyte, a second component and a target analyte, wherein the target analyte is a cell, virus, sub-cellular structure or protein.

[0015] FIG. 3C depicts a schematic of a planar substrate, multiple first components, a second component and a target analyte, wherein the target analyte is a cell, virus, sub-cellular structure or protein.

[0016] FIG. 4 depicts a schematic of steps for detecting a target analyte using the components depicted in FIG. 3A.

[0017] FIG. 5 depicts a schematic of a planar substrate, a first component, a second component and a target analyte, wherein the first component comprises a force sensor that is a micelle.

[0018] FIG. 6 depicts a schematic of steps for detecting a target analyte using the components of FIG. 5.

[0019] FIG. 7 depicts a schematic of a substrate, a first component, a second component, a third component, a first target analyte and a second target analyte.

[0020] FIG. 8 depicts a schematic of detecting a first target analyte and a second target analyte using the components of FIG. 7.

[0021] FIG. 9 illustrates binding of a target analyte to a first component and a second component and then applying a magnetic field to detect the target analyte.

[0022] FIG. 10 illustrates force sensors connected in parallel. FIG. 11 illustrates binding of a target analyte to a first component and a second component and then applying a magnetic field to detect the target analyte, wherein the first component comprises a force sensor that is a micelle.

[0023] FIG. 12 illustrates binding of a first target analyte to a first component and a second component, binding of a second target analyte to the second component and a third component and then applying a magnetic field to detect the target analyte. The first component comprises a force sensor that is a micelle.

[0024] DETAILED DESCRIPTION

[0025] The present disclosure provides devices for detecting target analytes. In accordance with one aspect of the disclosure, a device for detecting the presence or absence of a target analyte is provided. In accordance with another aspect of the disclosure, a device for detecting presence or absence of a first target analyte and a second target analyte is provided. Devices of the present disclosure include a force sensor and a force transducer. The present disclosure further provides systems for detecting target analytes and methods for detecting target analytes.

[0026] The devices described herein employ force sensors to carry out highly specific and highly sensitive detection of target analytes. Increased specificity is obtained by requiring binding to two different regions on a target analyte (i.e. a first location and a second location on a target analyte) in order for the target analyte to be detected. Increased sensitivity is obtained by applying an amplification factor to each target that is detected (e.g., multiple fluorophores emit a fluorescent signal for each target that is detected). Utilization of force sensors allows for measurement of mechanical forces at the molecular scale. Devices, systems and methods described herein are capable of single molecule detection.

[0027] Before the present devices, systems and methods are described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present devices, systems and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the devices, systems and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the devices, systems and methods.

[0029] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0030] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present devices, systems and methods, representative illustrative devices, systems and methods are now described.

[0031] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0032] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of’ and “consisting of.” The “consisting essentially of’ allows the presence of the named component(s), along with other components which do not change the function / structure of the named component(s). The “consisting of’ allows the presence of the named component(s).

[0033] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0034] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 ml to 10 ml” is inclusive of the endpoints, 2 ml and 10 ml, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0035] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1.

[0036] It should be noted that many of the terms used herein are relative terms. For example, the terms “top” and “bottom” are relative to each other in location and refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows out of the structure through the outlet. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0037] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0038] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, mRNA or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0039] The terms “oligonucleotide” and “oligo,” used interchangeably herein, refer to multimers up to 200 nucleotides in length. In some embodiments, oligonucleotides may be 5-200 nucleotides in length, including, e.g., be 8-50 nucleotides in length, including 13-25 nucleotides long. Oligonucleotides may be at least 5 nucleotides in length, e.g., at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides or at least 25 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, or a combination thereof. Thus, this term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, mRNA or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) has a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence- specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine / adenosine (A) pairing with thymine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine (G) pairing with cytosine / cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a sensor RNA, etc.): G can also base pair with U.

[0040] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids, the complementary nucleotides, the nucleotide sequences, and the degree of complementarity, variables well known in the art. In general, depending on the nucleic acid sequence, the greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0041] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, a nucleic acid in which 18 of 20 nucleotides are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0043] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0044] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present devices, systems and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0045] DEVICES FOR DETECTING TARGET ANALYTES

[0046] As summarized above, aspects of the present disclosure include devices for detecting target analytes.

[0047] In some embodiments, the device is a device for detecting the presence or absence of a target analyte comprising (a) a substrate, (b) a first component that comprises (i) a first region attached to the substrate, (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected, and (iii) a second region which binds to a first location on the target analyte, and (c) a second component that comprises (i) a first region which binds to a second location on the target analyte and (ii) a second region attached to a force transducer, wherein the force transducer is positionable in contact with the substrate and positionable relative to the first component to facilitate binding of the first and second components to the target analyte, and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

[0048] FIG. 1 shows a first component 2, a second component 30, a substrate 6 and a target analyte 20. First component 2 comprises a force sensor 4, wherein the force sensor 4 comprises a fluorophore 10 and a quencher 8. Fluorophore 10 and quencher 8 are associated such that no fluorescence is detected. First component 2 may comprise one or more force sensors 4. For example, the first component 2 shown in FIG. 1 comprises three force sensors. As shown in FIG. 1, force sensor 4 may comprise an oligonucleotide such that fluorophore 10 and quencher 8 are connected via the oligonucleotide, wherein a portion of the oligonucleotide hybridizes with itself. In some embodiments, the self-hybridizing portion of the oligonucleotide may form a “stem” portion and the non-self-hybridizing portion of the oligonucleotide may form a “loop” portion. In some embodiments, the non-self-hybridizing portion of the oligonucleotide may not form a “loop portion.” First component 2 further comprises a first region 12 attached to the substrate 6 and a second region 14 that binds to first location 22 on the target analyte 20. Second component 30 comprises a first region 32 that binds to a second location 24 on the target analyte 20 and a second region 38 attached to a force transducer 34. As shown in FIG. 1, in some embodiments, force transducer 34 can be positioned to be in contact with the substrate 6. However, in some embodiments, the force transducer is not in contact with the substrate. In some cases, the target analyte 20 may be an oligonucleotide, as depicted in FIG. 1, such that the second region 14 of the first component hybridizes to first location 22 on the target analyte and the first region 32 of the second component hybridizes to second location 24 on the target analyte.

[0049] FIG. 2 shows the detection of a target analyte using the components described in FIG. 1. The top panel of FIG. 2 shows the first component 2 attached to the substrate 6 and the second component 30 positioned to be in contact with the substrate 6. A sample comprising the target analyte 20 is added. The middle panel of FIG. 2 shows the first component and the second component binding to the target analyte via complementary oligonucleotide hybridization. The bottom panel of FIG. 2 shows a magnetic force 100 being applied to the force transducer (e.g., a magnetic nanoparticle). Upon application of the magnetic force, the force transducer moves away from the first component, causing the fluorophore and quencher of the force sensor to dissociate. As shown in FIG. 2, the self-hybridizing regions of the force sensor are separated. Thus, the quencher is no longer within a proximal distance to the fluorophore to quench its fluorescent emission, a fluorescent signal can now be detected. Force transducer 34 may be positioned on the substrate prior to contacting the substrate with the sample or force transducer 34 may be positioned on the substrate after component 30 binds to analyte 20.

[0050] FIG. 3A shows a first component 2, a second component 30, a substrate 6 and a target analyte 40, wherein the target analyte 40 is a cell, sub-cellular structure or protein. First component 2 comprises a force sensor 4, wherein the force sensor 4 comprises a fluorophore 10 and a quencher 8. Fluorophore 10 and quencher 8 are associated such that no fluorescence is detected. First component 2 may comprise one or more force sensors 4. For example, the first component 2 shown in FIG. 3A comprises three force sensors. As shown in FIG. 3A, force sensor 4 may comprise an oligonucleotide such that fluorophore 10 and quencher 8 are connected via the oligonucleotide, wherein a portion of the oligonucleotide hybridizes with itself. The selfhybridizing portion of the oligonucleotide may form a “stem” portion and the non-self-hybridizing portion of the oligonucleotide may form a “loop” portion. First component 2 further comprises a first region 12 attached to substrate 6 and a second region 18 that binds to a first location 42 on a target analyte 40. Second component 30 comprises a first region 36 that binds to a second location 44 on a target analyte 40 and a second region 38 attached to a force transducer 34. Force transducer 34 can be positioned to be in contact with the substrate 6. In some cases, the target analyte 40 may be a cell sub-cellular structure or protein (e.g., a cytokine), as depicted in FIG. 3 A, such that the second region 18 is an antigen-binding protein (e.g., an antibody) that binds to the first location 42 which is a first antigen and the first region 36 is an antigen-binding protein (e.g., an antibody) that binds to the second location 44 which is a second antigen.

[0051] FIG. 3B depicts a similar schematic to FIG. 3A, except that FIG. 3B shows a first component 2 comprising regions 18 and 19 that each bind to different locations 42 and 43, respectively on target analyte 40. First component 2 is attached to substrate 6. As such, the first component 2 can multivalently bind to the target analyte, thereby increasing sensitivity of the system. Second component 30 comprises a first region 36 that binds to a second location 44 on a target analyte 40 and a second region 38 attached to a force transducer 34. In some cases, 18, 19 and 36 may be a first binding partner that binds to respective second binding partner 42, 43 and 44. Binding partner pairs include, but are not limited to, antibody- antigen pairs and biotinstreptavidin pairs.

[0052] FIG. 3C depicts a similar schematic to FIG. 3A, except that FIG. 3C shows multiple first components. FIG. 3C shows first component 2 and another first component 3. First component 2 comprises a first region attached to the substrate 6 and a second region 18 that binds to a first location 42 on a target analyte 40. First component 3 comprises a first region attached to the substrate 6 and a second region 19 that binds to a third location 43 on a target analyte 40. Second component 30 comprises a first region 36 that binds to a second location 44 on a target analyte 40 and a second region 38 attached to a force transducer 34. Force transducer 34 can be positioned to be in solution (e.g., not in contact with the substrate). In some cases, 18, 19 and 36 may be a first binding partner that binds to respective second binding partner 42, 43 and 44. Binding partner pairs include, but are not limited to, antibody-antigen pairs and biotin-streptavidin pairs. Multiple first components, that each comprise one or more force sensors, may be used to enhance sensitivity and / or signal.

[0053] FIG. 4 shows the detection of a target analyte using the components described in FIG. 3 wherein the target analyte 40 is a cell, sub-cellular structure or protein. The top panel of FIG. 4 shows the first component 2 attached to the substrate 6 and the second component 30 positioned to be in contact with the substrate 6. A sample comprising the target analyte 40 is added. The middle panel of FIG. 4 shows the first component and the second component binding to the target analyte. Next, a force is applied to the force transducer, such that the force transducer moves away from the first component. For example, as shown in the bottom panel of FIG. 4, the force is a magnetic force 100 and the force transducer is a magnetic nanoparticle. Upon application of the magnetic force, the force transducer moves away from the first component, causing the fluorophore and quencher of the force sensor to dissociate. As shown in FIG. 4, the selfhybridizing regions of the force sensor are separated. Thus, the quencher is no longer within a proximal distance to the fluorophore to quench its fluorescent emission, a fluorescent signal can now be detected.

[0054] FIG. 5 shows a first component 202, a second component 230, a substrate 206 and a target analyte 220. First component 202 comprises a force sensor 204, wherein the force sensor 204 is a micelle. For example, as shown in FIG. 5, the micelle comprises multiple fluorophores (e.g., fluorophore 210) and one quencher (e.g., quencher 208). In some cases, as shown in FIG. 5, the fluorophores and quencher are located in the hydrophobic center of the micelle, such that no fluorescent signal is detected. First component 202 further comprises a first region 212 attached to the substrate 206 and a second region 214 that binds to a first location 222 on the target analyte 220. Second component 230 comprises a first region 232 that binds to a second location 224 on the target analyte 220 and a second region 238 attached to a force transducer 234. Force transducer 234 can be positioned to be in contact with the substrate 206. In some cases, the target analyte 220 may be an oligonucleotide, as depicted in FIG. 5, such that the second region 214 of the first component hybridizes to the first location 222 on the target analyte and the first region 232 of the second component hybridizes to the second location 224 on the target analyte. FIG. 6 shows the detection of a target analyte using the components described in FIG. 5. The top panel of FIG. 6 shows the first component 202 attached to the substrate 206 and the second component 230 positioned to be in contact with the substrate 206. A sample comprising the target analyte 220 is added. The middle panel of FIG. 6 shows the first component and the second component binding to the target analyte. Next, a force is applied to the force transducer, such that the force transducer moves away from the first component. For example, as shown in the bottom panel of FIG. 6, the force is a magnetic force 300 and the force transducer is a magnetic nanoparticle. When the force transducer moves away from the first component, the quencher is removed from the hydrophobic center of the micelle. As such, the quencher is no longer within a proximal distance to the fluorophores to quench their fluorescent emission, and a fluorescent signal can now be detected.

[0055] A “force sensor” is a sensor that produces a detectable fluorescent signal when a force is applied to the sensor. Force sensors of the present disclosure comprise a fluorophore and a quencher. Prior to application of a force, the fluorophore and quencher of the force sensor are associated such that no fluorescent signal is detected. When the fluorophore and quencher are located within a certain distance from each other, the energy from an excited fluorophore is transferred to the quencher, such that no radiative emission is emitted from the fluorophore. In some examples, a relatively low background fluorescent signal may be present when the fluorophore and quencher are associated, this relatively low background fluorescent signal is also encompassed by the phrases “baseline fluorescence signal’ or “no fluorescent signal is detected.” However, when the fluorophore and quencher are moved apart such that they are located further than said distance from each other, the excited fluorophore is no longer quenched by the quencher, and a fluorescent emission can be detected. In cases where a low background fluorescent signal is present when the fluorophore and quencher are associated, dissociation of the fluorophore and quencher results in an increase in the fluorescent signal. Such an increase in fluorescent signal relative to a low or no fluorescent signal detected when the fluorophore and quencher are associated is encompassed by the phrase “a fluorescent signal is detected” and grammatical equivalents thereof.

[0056] In some embodiments, the fluorophore is a small molecule, fluorescent protein or quantum dot. In some embodiments, the fluorophore is a small molecule. Small molecule fluorophores include, but are not limited to, cyanines, oxazines, boron-dipyrromethenes (BODIPYs), perylenes, diketopyrrolopyrroles (DPPs), and xanthenes, with the latter including fluorescein and rhodamine derivatives, such as the ATTO dyes (see, e.g., Leake et al. (2003) Chem Phys Rev 4:01 1302). In some embodiments, the fluorophore is a fluorescent protein. Many types of fluorescent proteins are known in the art (see, e.g., Day et al. (2009) Chem Soc Rev 38:2887-2921). In some embodiments, the fluorophore is a quantum dot. Many types of quantum dots are known in the art (see, e.g., Bera et al. (2010) Materials 3:2260-2345).

[0057] In some embodiments, the quencher is a metal nanoparticle including, but not limited to, a gold nanoparticle. Gold nanoparticles are ultra-efficient fluorescence quenchers. In nanometal surface energy transfer (NSET), energy from an excited fluorophore is transferred to the metal nanoparticle (e.g., a gold nanoparticle), and thus no emission is radiated. The efficiency of NSET is inversely proportional to the fourth power of the distance between the fluorophore and the quencher. Metal nanoparticle (e.g., gold nanoparticle) size and shape can be tailored to affect the quenching distance (see, e.g., Swierczewska et al. (2011) Phys Chem Chem Phys 12:9929-9941).

[0058] In some embodiments, the quencher is a second fluorophore. In Forster resonance energy transfer (FRET), energy from an excited fluorophore (i.e., a donor fluorophore) is transferred to a second acceptor fluorophore that emits a longer wavelength emission than the donor fluorophore emits. When the donor fluorophore and acceptor fluorophore are no longer within the required distance for FRET to occur (i.e., the donor fluorophore and acceptor fluorophore are at a distance apart that is larger than the Forster radius), emission from the donor fluorophore with be detected. The efficiency of FRET is inversely proportional to the sixth power of the distance between the fluorophore and the quencher. Many FRET donor and acceptor fluorophore pairs are known in the art (see, e.g., Bajar et al. (2016) Sensors 16: 1488, Roy et al. (2008) Nat Methods 5:507-516, We et al., (2020) Chem Soc Rev 49:51110-5139, etc.).

[0059] In some embodiments, the force sensor comprises a fluorophore and quencher that are connected via a linker. The linker may comprise any type of linker including, but not limited to, polymer linkers, peptide linkers or oligonucleotide linkers. In some embodiments, the force sensor comprises a fluorophore and quencher that are connected via an oligonucleotide linker. In some embodiments, the oligonucleotide linker comprises DNA, RNA or a combination thereof. In some embodiments, at least a portion of the oligonucleotide linker hybridizes with itself. In other words, the oligonucleotide linker is “self-hybridizing”. The self-hybridizing portion of the oligonucleotide may form a “stem” portion and the non-self-hybridizing portion of the oligonucleotide may form a “loop” portion, such that the oligonucleotide linker has a stem-loop structure. In some embodiments, the oligonucleotide linker may be single stranded or at least partially single stranded. When the oligonucleotide linker is self-hybridized, the fluorophore and quencher of the force sensor are located within close proximity to each other, such that no fluorescent signal is detected (see, e.g., the top panel of FIG. 2). However, upon applying a force to the force sensor comprising a self-hybridizing oligonucleotide linker, the self-hybridizing regions separate such that the fluorophore and quencher dissociate, causing a fluorescent signal to be detected (see, e.g., the bottom panel of FIG. 2).

[0060] In some embodiments, the force sensor comprises a fluorophore and quencher that are connected via polymer linker, such as a mechanopolymer linker. By mechanopolymer, it is meant a polymer that can undergo structural changes when a mechanical force is applied. In some cases, the mechanopolymer is a ladderane that can be mechanically unzipped (see, e.g., Chen et al. (2017) Science 357:475-479). When a force is applied to a force sensor comprising a fluorophore and quencher linked by a mechanopolymer linker, the mechanopolymer linker can “unzip” such that the fluorophore and quencher dissociate.

[0061] In some embodiments, the force sensor is a micelle. By micelle, it is meant an aggregate of amphipathic lipid molecules. By amphipathic, it is meant that the lipid molecules have both hydrophilic and hydrophobic regions. In some embodiments, the micelle has a hydrophobic center. In some embodiments, the micelle has a hydrophobic center comprising multiple fluorophores and one quencher. For example, as shown in FIG. 6 and FIG. 8, the micelle is tethered to the substrate (i.e., attached to the substrate via a first region of the first component) and the quencher is attached to a second region of the first component that binds to the target analyte. The quencher may be attached to a second region of the first component that binds to the target analyte using any type of attachment including, but not limited to, covalent and non-covalent bonding. Attachment methods include, but are not limited to, oligonucleotide hybridization, click chemistry, phosphite chemistry, NHS-ester chemistry, thiol-chemistry and silane-chemistry, streptavidin-biotin interactions, antibody-antigen interactions and the like.

[0062] In some embodiments, the fluorophores and quencher are hydrophobic and thus associate in the hydrophobic center of the micelle, while the outer surface of the micelle is hydrophilic. As a result, in some embodiments, when a force is applied to the micelle, the quencher is pulled out of the hydrophobic center of the micelle, and the micelle reforms with only fluorophores in the hydrophobic center, thus greatly amplifying the fluorescent signal. In some embodiments, when a force is applied to the micelle, the micelle breaks apart, leading to the release of fluorophores that can be detected.

[0063] In some embodiments, the micelle has a hydrophobic center comprising one or more fluorophores (e.g., one fluorophore, two fluorophores, three fluorophores, four fluorophores, five fluorophores, six fluorophores, seven fluorophores, eight fluorophores, nine fluorophores, ten fluorophores, 11 fluorophores, 12 fluorophores, 13 fluorophores, 14 fluorophores, 15 fluorophores, 20 fluorophores, 25 fluorophores, 30 fluorophores, 35 fluorophores, 40 fluorophores, 45 fluorophores, 50 fluorophores, 60 fluorophores, 70 fluorophores, 80 fluorophores, 90 fluorophores, 100 fluorophores, 200 fluorophores, 300 fluorophores, 400 fluorophores, 500 fluorophores, 1000 fluorophores) including, e.g., two or more fluorophores, three or more fluorophores, four or more fluorophores, five or more fluorophores, six or more fluorophores, seven or more fluorophores, eight or more fluorophores, nine or more fluorophores, ten or more fluorophores, 15 or more fluorophores, 20 or more fluorophores, 25 or more fluorophores, 30 or more fluorophores, 35 or more fluorophores, 40 or more fluorophores, 45 or more fluorophores, 50 or more fluorophores, 60 or more fluorophores, 70 or more fluorophores, 80 or more fluorophores, 90 or more fluorophores, 100 or more fluorophores, 200 or more fluorophores, 300 or more fluorophores, 400 or more fluorophores, 500 or more fluorophores, and 1000 or more fluorophores. In such embodiments, the hydrophobic center may further comprise one or more quenchers (e.g., one quencher, two quenchers, three quenchers, four quenchers, five quenchers, six quenchers, seven quenchers, eight quenchers, nine quenchers, ten quenchers, 11 quenchers, 12 quenchers, 13 quenchers, 14 quenchers, 15 quenchers, 20 quenchers, 25 quenchers, 30 quenchers, 35 quenchers, 40 quenchers, 45 quenchers, 50 quenchers, 60 quenchers, 70 quenchers, 80 quenchers, 90 quenchers, 100 quenchers, 200 quenchers, 300 quenchers, 400 quenchers, 500 quenchers, 1000 quenchers) including, e.g., two or more quenchers, three or more quenchers, four or more quenchers, five or more quenchers, six or more quenchers, seven or more quenchers, eight or more quenchers, nine or more quenchers, ten or more quenchers, 15 or more quenchers, 20 or more quenchers, 25 or more quenchers, 30 or more quenchers, 35 or more quenchers, 40 or more quenchers, 45 or more quenchers, 50 or more quenchers, 60 or more quenchers, 70 or more quenchers, 80 or more quenchers, 90 or more quenchers, 100 or more quenchers, 200 or more quenchers, 300 or more quenchers, 400 or more quenchers, 500 or more quenchers, and 1000 or more quenchers.

[0064] In some embodiments, the first component comprises one or more force sensors (e.g., one force sensor, two force sensors, three force sensors, four force sensors, five force sensors, six force sensors, seven force sensors, eight force sensors, nine force sensors, ten force sensors, 11 force sensors, 12 force sensors, 13 force sensors, 14 force sensors, 15 force sensors, 20 force sensors, 25 force sensors, 30 force sensors, 35 force sensors, 40 force sensors, 45 force sensors, 50 force sensors) including, e.g., two or more force sensors, three or more force sensors, four or more force sensors, five or more force sensors, six or more force sensors, seven or more force sensors, eight or more force sensors, nine or more force sensors, ten or more force sensors, 15 or more force sensors, 20 or more force sensors, 25 or more force sensors, 30 or more force sensors, 35 or more force sensors, 40 or more force sensors, 45 or more force sensors, and 50 or more force sensors. In some embodiments, multiple force sensors are connected in series. By connected in series, it is meant that that the force sensors are connected along a single path. In other words, the force sensors are connected end-to-end. For example, FIG. 1 and FIG. 3 illustrate force sensors connected in series. In some embodiments, two or more force sensors are connected in series, including, e.g., two or more force sensors, three or more force sensors, four or more force sensors, five or more force sensors, six or more force sensors, seven or more force sensors, eight or more force sensors, nine or more force sensors, ten or more force sensors, 15 or more force sensors, 20 or more force sensors, 25 or more force sensors, 30 or more force sensors, 35 or more force sensors, 40 or more force sensors, 45 or more force sensors, and 50 or more force sensors connected in series. In some cases, one force sensor, two force sensors, three force sensors, four force sensors, five force sensors, six force sensors, seven force sensors, eight force sensors, nine force sensors, ten force sensors, 11 force sensors, 12 force sensors, 13 force sensors, 14 force sensors, 15 force sensors, 20 force sensors, 25 force sensors, 30 force sensors, 35 force sensors, 40 force sensors, 45 force sensors, or 50 force sensors are connected in series.

[0065] In some embodiments, multiple force sensors are connected in parallel. By connected in parallel, it is meant that that the force sensors are connected along multiple paths. For example, FIG. 10 illustrates force sensors comprising self-hybridized oligonucleotides connected in parallel. FIG. 10 shows force sensors arranged in a cube shape. By applying a force, the force sensors can unhybridize simultaneously, allowing for an amplified fluorescent signal to be detected. In some embodiments, two or more force sensors are connected in parallel, including, e.g., two or more force sensors, three or more force sensors, four or more force sensors, five or more force sensors, six or more force sensors, seven or more force sensors, eight or more force sensors, nine or more force sensors, ten or more force sensors, 15 or more force sensors, 20 or more force sensors, 25 or more force sensors, 30 or more force sensors, 35 or more force sensors, 40 or more force sensors, 45 or more force sensors, and 50 or more force sensors connected in parallel. In some cases, one force sensor, two force sensors, three force sensors, four force sensors, five force sensors, six force sensors, seven force sensors, eight force sensors, nine force sensors, ten force sensors, 11 force sensors, 12 force sensors, 13 force sensors, 14 force sensors, 15 force sensors, 20 force sensors, 25 force sensors, 30 force sensors, 35 force sensors, 40 force sensors, 45 force sensors, or 50 force sensors are connected in parallel.

[0066] In some embodiments, multiple force sensors are connected in combinations of parallel and series connections. Any combination of parallel connections and series connections may be used. In some embodiments, three or more force sensors are connected in a combination of parallel and series connections, including, e.g., four or more force sensors, five or more force sensors, six or more force sensors, seven or more force sensors, eight or more force sensors, nine or more force sensors, ten or more force sensors, 15 or more force sensors, 20 or more force sensors, 25 or more force sensors, 30 or more force sensors, 35 or more force sensors, 40 or more force sensors, 45 or more force sensors, and 50 or more force sensors connected in a combination of parallel and series connections. In some cases, three force sensors, four force sensors, five force sensors, six force sensors, seven force sensors, eight force sensors, nine force sensors, ten force sensors, 11 force sensors, 12 force sensors, 13 force sensors, 14 force sensors, 15 force sensors, 20 force sensors, 25 force sensors, 30 force sensors, 35 force sensors, 40 force sensors, 45 force sensors, or 50 force sensors are connected in a combination of parallel and series connections. For example, in the case where there are three sensors, a first sensor may be connected in series to a second sensor, and the first and second sensors may be connected in parallel to a third sensor. In another example, in the case where there are four sensors, a first sensor may be connected in series to a second sensor, a third sensor may be connected in series to a fourth sensor, and the first and second sensors may be connected in parallel to the third and fourth sensors. In another example, in the case where there are four sensors, a first sensor may be connected in series to a second sensor and a third sensor, and a the first, second and third sensors may be connected in parallel to a fourth sensor.

[0067] By “force transducer” it is meant a component that converts one type of force to another (e.g., a component that converts a magnetic force to a mechanical force, or a component that converts a flow force to a mechanical force, etc.).

[0068] In some embodiments, the force transducer is a magnetic nanoparticle. Magnetic nanoparticles are nanoparticles that can be manipulated using magnetic fields. In other words, a magnetic force may be applied to a magnetic nanoparticle such that the magnetic nanoparticle moves. When the magnetic nanoparticle is connected to one or more force sensors, as described herein, and a magnetic force is applied, the movement of the magnetic nanoparticle applies a mechanical force to the force sensor it is connected to, thereby dissociating the fluorophore from the quencher. In certain embodiments, the magnetic nanoparticle is an iron oxide nanoparticle. Nanoparticles (e.g., iron oxide nanoparticles) may be modified in a variety of different ways including, but not limited to, the addition of a shell and / or functionalization with ligands and / or surfactants (see, e.g., Zhu et al. (2018) Nanomaterials 8:810). Nanoparticles may be modified so they are hydrophobic or hydrophilic. In some embodiments, the force transducer is a magnetic nanoparticle (e.g., an iron oxide nanoparticle) that is hydrophobic and / or modified to be hydrophobic.

[0069] In some embodiments, the force transducer is a kite component. By “kite component” it is meant a component that is activated by the flow or a liquid, gas or other media. In other words, by flowing a liquid, gas or other media over the kite component, the kite component moves, such that the movement of the kite component applies a mechanical force to the force sensor it is connected to, thereby dissociating the fluorophore from the quencher.

[0070] In some embodiments, the force transducer is positionable in contact with the substrate. By positionable in contact with the substrate it is meant that the force transducer is movable such that it can be positioned to be in contact with the substrate.

[0071] In some embodiments, the force transducer is in contact with the substrate. In some embodiments, the force transducer is in contact with the substrate due to a gravitational force. In some embodiments, the force transducer is weakly attached to the substrate. By weakly attached, it is meant that the attachment force between the force transducer and the substrate is weaker than the force applied to the force transducer. The force transducer may be weakly attached to the substrate using any suitable method including, but not limited to physical adsorption, non-covalent bonding and covalent bonding. In some embodiments, the force transducer is in contact with the substrate due to a magnetic force. In some embodiments, the magnetic force that holds the force transducer in contact with the substrate is a weaker magnetic force than the force applied to the force transducer.

[0072] In some embodiments, the force transducer is not attached to the substrate. In some embodiments, the force transducer is not in contact with the substrate. In some embodiments, the force transducer is not in contact with nor attached to the substrate. In some embodiments, the force transducer is in solution.

[0073] In some embodiments, the force transducer is positioned relative to the first component to facilitate binding of the first and second components to the target analyte and the force transducer is configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected. As such, the force transducer is able to be moved to a different position after binding to the target analyte.

[0074] In some embodiments, a region of one of the components of the device is attached to a force transducer. In embodiments wherein a device detects presence or absence of a target analyte, the device comprises a second component comprising a second region attached to a force transducer. In embodiments wherein a device detects presence or absence of two target analytes, the device comprises a third component comprising a second region attached to a force transducer. This region of a component that attaches to a force transducer may comprise a polymer, peptide, oligonucleotide or combinations thereof. The force transducer may be attached using any method including, but not limited to, covalent and non-covalent bonding. Covalent bonding attachment methods include, but are not limited to, click chemistry, phosphite chemistry, NHS-ester chemistry, thiol-chemistry and silane-chemistry. Non-covalent bonding includes, but is not limited to, streptavidin-biotin interactions, antibody- antigen interactions and the like.

[0075] In some embodiments, the device comprises substrate, wherein the substrate is substantially planar. By “planar” it is meant that the substrate is flat, i.e., two-dimensional. The substrate may be a rigid or flexible. The substrate may be made of any suitable material including, but not limited to, glass, metal and plastic. The substrate may be a coverslip, a planar surface of a well plate, a planar surface of a petri dish or another surface. Substrates may be treated and / or coated to prepare the substrates for attachment to other components using any variety of methods. Substrate treatments include, but are not limited to, acid treatment, base treatment, oxidizing treatment, plasma treatment, detergent treatment, heat treatment or combinations thereof. Substrate coatings include, but are not limited to, polylysine coating and glutaraldehyde coating.

[0076] The first component comprises a first region attached to the substrate. The first region may comprise a polymer, peptide, oligonucleotide or combination thereof. In some embodiments, the first region comprises a polymer. Polymers may be any suitable synthetic or naturally-occurring polymer including, but not limited to, polyethylene glycol (PEG). In some embodiments, the first region comprises a peptide. In some embodiments, the first region comprises an oligonucleotide. The oligonucleotide may be DNA, RNA or a combination thereof. DNA may be genomic DNA. RNA may be mRNA. In some embodiments, the oligonucleotide is single stranded or at least partially single stranded.

[0077] The first region may be attached to the substrate using any attachment method including, but not limited to, physical adsorption, covalent bonding and non-covalent bonding. Physical adsorption attachment methods include, but are not limited to, hydrogen bonding, van der Waals forces, electrostatic forces and hydrophobic interactions. Covalent bonding attachment methods include, but are not limited to, click chemistry, phosphate chemistry, NHS-ester chemistry, thiol- chemistry and silane-chemistry. Non-covalent bonding includes, but is not limited to, streptavidinbiotin interactions, antibody- antigen interactions and the like.

[0078] In some embodiments, one or more first components may be attached to the substrate. Increasing the number of first components attached to the substrate allows for target analytes, if present, to more readily bind since there are more first components available for binding. In cases where more first components are available than target analytes are present, not all first components may bind to a target analyte (see, e.g., FIG. 9). In some embodiments, the substrate comprises one or more first components (e.g., one first component, two first components, three first components, four first components, five first components, six first components, seven first components, eight first components, nine first components, ten first components, 11 first components, 12 first components, 13 first components, 14 first components, 15 first components, 20 first components, 25 first components, 30 first components, 35 first components, 40 first components, 45 first components, 50 first components, 60 first components, 70 first components, 80 first components, 90 first components, 100 first components, 200 first components, 300 first components, 400 first components, 500 first components, 1000 first components) including, e.g., two or more first components, three or more first components, four or more first components, five or more first components, six or more first components, seven or more first components, eight or more first components, nine or more first components, ten or more first components, 15 or more first components, 20 or more first components, 25 or more first components, 30 or more first components, 35 or more first components, 40 or more first components, 45 or more first components, 50 or more first components, 60 or more first components, 70 or more first components, 80 or more first components, 90 or more first components, 100 or more first components, 200 or more first components, 300 or more first components, 400 or more first components, 500 or more first components, and 1000 or more first components.

[0079] In some embodiments, the target analyte is an oligonucleotide. The oligonucleotide may be DNA, RNA or a combination thereof. DNA may be genomic DNA. RNA may be mRNA. In some embodiments, the target analyte is an oligonucleotide that is single stranded or at least partially single stranded.

[0080] In some embodiments, the target analyte (e.g., an oligonucleotide) is subjected to amplification methods. In some embodiments, the target analyte is amplified before it is combined with the other components of the device or system.

[0081] In some embodiments, the target analyte (e.g., an oligonucleotide) is not subjected to amplification methods. Target analytes that are not subjected to amplification methods are therefore unaffected by amplification bias.

[0082] In some embodiments, the target analyte is a cell, sub-cellular structure or protein. In some embodiments, the target analyte is a cell. Cells may be prokaryotic or eukaryotic. Cells may be any variety of cell including, but not limited to, bacterial cells, fungal cells, cells comprising viral components, erythrocytes, platelets, bone marrow cells, endothelial cells, lymphocytes, hepatocytes, neurons, glia, epidermal cells, interstitial cells, adipocytes, fibroblasts, muscle cells, etc. In some embodiments, the target analyte is a sub-cellular structure. Sub-cellular structures may be any variety of sub-cellular structure including, but not limited to, vesicles, organelles or extracellular matrix structures. In some embodiments, the target analyte is a protein. Proteins may be any variety of protein including, but not limited to antibodies, cytokines or other plasma proteins. In some embodiments, the target analyte an antibody. In some embodiments, the target analyte is a cytokine. Cytokines include, but are not limited to, IL-la, IL-ip, IL-IRa, IL-18, 11-2, 11-4, IL-7, 11-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-a, IFN-0, IFN-y, CD154, LT- , TNF-a, TNF-p, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF- PL TGF-P2, TGF-P3, EPO, TPO, FLT-3L, SCF, M-CSF and MSP (see, e.g., Cameron et al. Cytokines, Chemokines and their Receptors. Madame Curie Bioscience Database. Austin: Landes Bioscience 2000-2013).

[0083] Target analytes comprise binding locations that can be bound to by components of the devices described herein. In some embodiments, the target analytes comprise two locations (i.e., a first location and a second location) that can be bound to by components of the devices described herein. In embodiments wherein the target analyte is an oligonucleotide, the target analyte comprises two or more unique nucleic acid sequences that can be bound to by devices described herein. In embodiments wherein the target analyte is a cell, sub-cellular structure or protein, the target analyte comprises two or more unique antigens that can be bound to by devices described herein. In embodiments wherein the target analyte is spore (e.g., a fungi spore or a plant spore), the target analyte comprises two or more unique antigens that can be bound to by devices described herein.

[0084] Components of the devices described herein comprise regions that bind to a specific location on the target analyte. In some embodiments, a device for detecting presence or absence of a target analyte comprises a first component that comprises a region which binds to a first location on the target analyte and a second component that comprises a region which binds to a second location the target analyte.

[0085] In some embodiments, a region (e.g., a second region) of the first component comprises an oligonucleotide that is complementary to the first location on the target analyte. In other words, the oligonucleotide hybridizes with the first location on the target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g., a second region) of the first component comprises an oligonucleotide that is an aptamer that binds to the first location on the target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a second region) of the first component comprises a binding moiety that binds to the first location on the target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the target analyte.

[0086] In some embodiments, a region (e.g., a first region) of the second component comprises an oligonucleotide that is complementary to the second location on the target analyte. In other words, the oligonucleotide hybridizes with the second location on the target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g., a first region) of the second component comprises an oligonucleotide that is an aptamer that binds to the second location on the target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a first region) of the second component comprises a binding moiety that binds to the second location on the target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the target analyte.

[0087] In some aspects, the device is a device for detecting presence or absence of a first target analyte and a second target analyte. In such aspects, the device comprises (a) a substrate, (b) a first component that comprises (i) a first region attached to the substrate, (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected, and (iii) a second region which binds to a first location on the first target analyte, (c) a second component that comprises (i) a first region which binds to a second location on the first target analyte and (ii) a second region which binds to a first location on the second target analyte, and (d) a third component that comprises (i) a first region which binds to a second location on the second target analyte and (ii) a second region attached to a force transducer, wherein the force transducer is positionable in contact with the substrate, positioned relative to the first and second components to facilitate binding of the first and second components to the target analyte, and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

[0088] FIG. 7 shows a first component 202, a second component 250, a third component 270, a substrate 206, a first target analyte 240 and a second target analyte 260. First component 202 comprises a force sensor 204, wherein the force sensor 204 is a micelle. For example, as shown in FIG. 7, the micelle comprises multiple fluorophores and one quencher. In some cases, as shown in FIG. 7, the fluorophores and quencher are located in the hydrophobic center of the micelle, such that no fluorescent signal is detected. First component 202 further comprises a first region 212 attached to the substrate 206 and a second region 214 that binds to a first location 242 on the first target analyte 240. Second component 250 comprises a first region 252 that binds to a second location 244 on the first target analyte 240 and a second region 254 that binds to a first location 262 on the second target analyte 260. Third component 270 comprises a first region 272 that binds to a second location 264 on the second target analyte 260 and a second region 278 attached to a force transducer 274. Force transducer 274 can be positioned to be in contact with the substrate 206. In some cases, the first target analyte 240 and the second target analyte 260 may be oligonucleotides, as depicted in FIG. 7, such that the second region 214 of the first component hybridizes to first location 242 on the first target analyte, the first region 252 of the second component hybridizes to second location 244 on the first target analyte, the second region 254 hybridizes to the first location 262 on the second target analyte and the first region 272 of the third component hybridizes to the second location 264 on the second target analyte.

[0089] FIG. 8 shows the detection of a first target analyte and a second target analyte using the components described in FIG. 7. The top panel of FIG. 8 shows the first component 202 attached to the substrate 206, the second component 250 and the third component 270, wherein the third component 270 is positioned to be in contact with the substrate 206. A sample comprising the first target analyte 240 and second target analyte 260 is added. The middle panel of FIG. 8 shows the first and second components binding to the first target analyte, and the second and third components binding to the second target analyte. Next, a force is applied to the force transducer, such that the force transducer moves away from the first component. For example, as shown in the bottom panel of FIG. 8, the force is a magnetic force 300 and the force transducer is a magnetic nanoparticle. When the force transducer moves away from the first component, the quencher is removed from the hydrophobic center of the micelle. As such, the quencher is no longer within a proximal distance to the fluorophores to quench their fluorescent emission, and a fluorescent signal can now be detected.

[0090] In such embodiments wherein two target analytes are detected (i.e., a first target analyte and a second target analyte), both target analytes must be present in order for a fluorescent signal to be detected. In the case where only one target analyte is present (i.e., only the first target analyte is present or only the second target analyte is present), no fluorescent signal will be detected.

[0091] Components of the devices described herein for detecting two target analytes comprise regions that bind to specific locations on the two target analytes. In some embodiments, a device for detecting presence or absence of a first target analyte and a second target analyte comprises a first component that comprises a region which binds to a first location on the first target analyte, a second component that comprises a first region which binds to a second location the first target analyte and a second region which binds to a first location on the second target analyte, and a third component that comprises a region which binds to a second location on the second target analyte.

[0092] In some embodiments, a region (e.g., a second region) of the first component comprises an oligonucleotide that is complementary to the first location on the first target analyte. In other words, the oligonucleotide hybridizes with the first location on the first target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g. a second region) of the first component comprises an oligonucleotide that is an aptamer that binds to the first location on the first target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a second region) of the first component comprises a binding moiety that binds to the first location on the first target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the first target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the first target analyte.

[0093] In some embodiments, a region (e.g., a first region) of the second component comprises an oligonucleotide that is complementary to the second location on the first target analyte. In other words, the oligonucleotide hybridizes with the second location on the first target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g., a first region) of the second component comprises an oligonucleotide that is an aptamer that binds to the second location on the first target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a first region) of the second component comprises a binding moiety that binds to the second location on the first target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the first target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the first target analyte.

[0094] In some embodiments, a region (e.g., a second region) of the second component comprises an oligonucleotide that is complementary to the first location on the second target analyte. In other words, the oligonucleotide hybridizes with the first location on the second target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g., a second region) of the second component comprises an oligonucleotide that is an aptamer that binds to the first location on the second target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a second region) of the second component comprises a binding moiety that binds to the first location on the second target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the second target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the second target analyte.

[0095] In some embodiments, a region (e.g., a first region) of the third component comprises an oligonucleotide that is complementary to the second location on the second target analyte. In other words, the oligonucleotide hybridizes with the second location on the second target analyte. The oligonucleotide may be DNA, RNA or a combination thereof. In some embodiments, a region (e.g., a first region) of the third component comprises an oligonucleotide that is an aptamer that binds to the second location on the second target analyte. The aptamer may comprise DNA, RNA, peptides or combinations thereof. In some embodiments, a region (e.g., a first region) of the third component comprises a binding moiety that binds to the second location on the second target analyte. In some embodiments, the binding moiety is a protein. In some embodiments, the binding moiety is a protein that is an oligonucleotide binding protein. The oligonucleotide binding protein may be any protein that binds to a specific oligonucleotide sequence including, but not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas-based nucleases. The nucleases may or may not have nuclease cleavage activity. The oligonucleotide binding protein may be programmed to specifically bind to a specific oligonucleotide sequence of the second target analyte. In certain embodiments, the binding moiety is a protein that is an antibody, nanobody or antigen-binding protein. In such embodiments, the binding moiety binds to a specific antigen on the second target analyte.

[0096] In such embodiments wherein two target analytes are detected, the force transducer is positioned relative to the first component to facilitate binding of the first, second and third components to the first and second target analytes; and the force transducer is configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected. As such, the force transducer is able to be moved to a different position after binding to the first and second target analytes.

[0097] In such embodiments wherein two target analytes are detected, both the first and second target analytes may be oligonucleotides. In such embodiments wherein two target analytes are detected, both the first and second target analytes may be cells, sub-cellular structures, proteins or combinations thereof. In such embodiments wherein two target analytes are detected, the first target analyte may be an oligonucleotide and the second target analytes may be a cell, sub-cellular structure or protein. In such embodiments wherein two target analytes are detected, the first target analyte may be a cell, sub-cellular structure or protein and the second target analytes may be an oligonucleotide.

[0098] Aspects of the present disclosure include devices for detecting one or more target analytes (e.g., one target analyte, two target analytes, three target analytes, four target analytes, five target analytes, six target analytes, seven target analytes, eight target analytes, nine target analytes or ten target analytes) including, e.g., two or more target analytes, three or more target analytes, four or more target analytes, five or more target analytes, six or more target analytes, seven or more target analytes, eight or more target analytes, nine or more target analytes and ten or more target analytes. In such embodiments, the device comprises a substrate, a component comprising a force sensor, a component comprising a force transducer and the components needed to bind to the desired number of target analytes.

[0099] In some embodiments, the binding force between a component of the system (e.g., the first, second or third component) and a target analyte (e.g., the first or second target analyte) is stronger than the force sensed by the force sensor. For example, the binding force between the second region of the first component and the first location on the target analyte and the binding force between the first region of the second component and the second location on the target analyte are each stronger than force that is sensed by the force sensor. As such, the components of the device will stay bound to the target analyte even when a force is applied to the force transducer.

[0100] SYSTEMS FOR DETECTING TARGET ANALYTES

[0101] Certain aspects of the disclosure include systems for detecting the presence or absence of a target analyte. In these aspects, the system comprises (a) a substrate comprising a first component that comprises (i) a first region attached to the substrate, (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected and (iii) a second region which binds to a first location on the target analyte, and (b) a second component that comprises (i) a first region which binds to a second location on the target analyte and (ii) a second region attached to a force transducer, wherein the force transducer is positionable in contact with the substrate and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, and wherein when the force transducer is moved away from the first component, in the absence of the analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected.

[0102] Certain aspects of the disclosure include systems for detecting presence or absence of a first target analyte and a second target analyte. In these aspects, the system comprises (a) a substrate comprising a first component that comprises (i) a first region attached to the substrate, (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected and (iii) a second region which binds to a first location on the first target analyte, (b) a second component that comprises (i) a first region which binds to a second location on the first target analyte and (ii) a second region which binds to a first location on the second target analyte, and (c) a third component that comprises (i) a first region which binds to a second location on the second target analyte and (ii) a second region attached to a force transducer, wherein the force transducer is positionable in contact with the substrate and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the first target analyte and the second target analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, and wherein when the force transducer is moved away from the first component, in the absence of the first target analyte and / or the second target analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected.

[0103] Such systems may comprise any of the features of the devices discussed above.

[0104] In some embodiments, the substrate (e.g., the substrate comprising the first component) is contained within a flow cell. In these embodiments, liquids, gasses or other media may be flowed over the substrate such that target analytes can bind to the components of the device. Such liquids may comprise a target analyte (e.g., including a first and / or second target analyte), components of the device (e.g., the second component and / or third component) or combinations thereof. Several liquids may be flowed through the flow cell sequentially or simultaneously. Each liquid may comprise a target analyte, component or combinations of such.

[0105] In some embodiments, multiple substrates may be placed within the same flow cell, such that the same liquids can be flowed over multiple substrates.

[0106] In some embodiments, a system comprises a first set of components required to detect a first analyte and a second set of components required to detect a second analyte. Each set of components may comprise a unique fluorophore. For example, a first set of components may comprise a fluorophore that emits green fluorescence when a first target analyte is bound and a second set of components may comprise a fluorophore that emits red fluorescence when a second target analyte is bound. In this embodiment, the first target analyte is independently detected from the second target analyte since each target analyte is correlated to a certain color (i.e., wavelength) of fluorescence. In some embodiments, multiple targets may be detected in parallel.

[0107] METHODS FOR DETECTING TARGET ANALYTES

[0108] Aspects of the present disclosure include methods of detecting a target analyte in a sample. In some embodiments, the method comprises combining the substrate (e.g., the substrate comprising the first component, wherein the first component comprises a first region attached to the substrate, a force sensor and a second region which binds to the target analyte) and the second component (e.g., wherein the second component comprises a first region that binds to the target analyte and a second region attached to a force transducer) with the sample under conditions suitable for binding of the target analyte to the first and second components; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte.

[0109] In some embodiments, the method of combining the substrate and the second component with the sample comprises adding the sample to the substrate followed by adding the second component. In such embodiments, it is not necessary to immobilize the second component on the substrate. In other embodiments, the method of combining the substrate and the second component with the sample comprises adding the second component to the substrate, immobilizing the second component on the substrate, followed by adding the sample. In other embodiments, the method of combining the substrate and the second component with the sample comprises adding the second component and the sample to the substrate at substantially the same time. In other embodiments, the method of combining the substrate and the second component with the sample comprises adding the sample to the second component followed by adding the combined sample and second component to the substrate. In other words, the sample and the second component may be combined before being added to the substrate. In such embodiments, if the target analyte is present, the target analyte may bind to the second component prior to being added to the substrate. In some embodiments, the method further comprises repeatedly moving the force transducer over the substrate (e.g., a planar substrate) to provide a longer dwell time for the target analyte to bind to the first component.

[0110] Aspects of the present disclosure include methods of detecting a first target analyte and a second target analyte in a sample. In some embodiments, the method comprises combining the substrate (e.g., the substrate comprising the first component, wherein the first component comprises a first region attached to the substrate, a force sensor and a second region which binds to the first target analyte), the second component (e.g., wherein the second component comprises a first region which binds to the first target analyte and a second region which binds to the second target analyte), and the third component (e.g., wherein the third component comprises a first region that binds to the second target analyte and a second region attached to a force transducer), with the sample under conditions suitable for binding of the first target analyte to the first and second components and the second target analyte to the second and third components; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the first target analyte and / or second target analyte and presence of fluorescence indicates presence of the first and second target analytes. In some embodiments, the method of combining the substrate, the second component and the third component with the sample comprises adding the sample comprising the two target analytes and the second component to the substrate, followed by adding the third component. In such embodiments it is not necessary to immobilize the third component on the substrate. In other embodiments, the method comprises adding the third component to the substrate, immobilizing the third component on the substrate, followed by adding the sample comprising the two target analytes and the second component. In other embodiments, the method comprises adding the third component, the sample comprising the two target analytes and the second component at substantially the same time. In other embodiments, the method of combining the substrate, the second component and the third component with the sample comprises adding the sample to the second component and the third component followed by adding the combined sample, second component and third component to the substrate. In other words, the sample, the second component and the third component may be combined before being added to the substrate. In such embodiments, if the first and second target analytes are present, the target analytes may bind to the second and third components prior to being added to the substrate. In some embodiments, the method further comprises repeatedly moving the force transducer over the substrate (e.g., a planar substrate) to provide a longer dwell time for the target analyte to bind to the first component.

[0111] Aspects of the present disclosure include methods of detecting a target analyte in a sample, wherein any of the above described devices are combined with a sample comprising or suspected of comprising the target analyte under conditions suitable for the first and second components to bind to the target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte.

[0112] Aspects of the present disclosure include methods of detecting a first target analyte and a second target analyte in a sample, wherein any of the above devices for detecting a first target analyte and a second target analyte are combined with a sample comprising the first and second target analytes or suspected of comprising the first and second target analytes under conditions suitable for the first and second components to bind to the first target analyte, if present, and the second and third components to bind to the second target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the first target analyte and / or second target analyte and presence of fluorescence indicates presence of the first and second target analytes.

[0113] The term “sample” refers to a liquid of biological origin, a liquid comprising components of biological origin, a gas of biological origin or a gas comprising components of biological origin (e.g., an aerosol). Liquids of biological origin or biological liquids include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, mucus, vaginal fluids, semen, saliva, interstitial fluids, milk, amniotic fluid, dialysate, tears, intestinal fluid, peritoneal fluid, ascetic fluid and pleural fluid. Liquids comprising components of biological origin include, but are not limited to, cells in culture, cell supernatants, cell lysates, liquids comprising tissue samples or tissue cultures, liquids comprising biopsy specimens, liquids comprising sub-cellular structures, liquids comprising proteins, liquids comprising nucleic acids or combinations thereof. The term “sample” also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, combination of several samples, dilution, washing or enrichment for certain cell populations. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, cytokines etc. Gasses comprising components of biological origin (e.g., aerosols) include, but are not limited to, aerosols comprising particles derived from biological organisms such as bacteria, fungi, protozoa, algae, spores (e.g., plant spores or fungi spores), pollen, lichen, archaea and viruses.

[0114] In some embodiments, the sample comprises a target analyte. In some embodiments, the sample comprises a first target analyte and a second target analyte.

[0115] By “conditions suitable for binding”, it is meant that any conditions that are amenable for binding. For example, such conditions include, but are not limited to, temperature, pH, solvent and concentration.

[0116] The force transducer is moved by applying a force. In some embodiments, moving the force transducer away from the first component comprises applying a magnetic force to the force transducer. In such embodiments, the force transducer is a magnetic nanoparticle (e.g., an iron oxide nanoparticle). The magnetic force may be applied by a magnetic field or any type of magnet.

[0117] In some embodiments, moving the force transducer away from the first component comprises applying a flow of liquid or gas. In such embodiments, the force transducer is a kite component. The flow of liquid or gas may be flowed through a flow cell over the substrate.

[0118] In some embodiments, the applied force can be reversed and / or stopped, allowing for the force sensors to return to their initial structural conformation, and thus resulting in the absence of a fluorescent signal. Fluorescence may be measured using any instruments used to detect fluorescence, including, but not limited to, fluorometers, plate readers and optical microscopes. Such methods to detect fluorescence using optical microscopy include, but are not limited to, confocal microscopy, epifluorescence microscopy, total internal reflection fluorescent (TIRF) microscopy and super resolution microscopy.

[0119] EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0120] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any one of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0121] 1. A system for detecting presence or absence of a target analyte, the system comprising:

[0122] (a) a substrate comprising a first component that comprises:

[0123] (i) a first region attached to the substrate;

[0124] (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and

[0125] (iii) a second region which binds to a first location on the target analyte; and

[0126] (b) a second component that comprises:

[0127] (i) a first region which binds to a second location on the target analyte; and

[0128] (ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, and wherein when the force transducer is moved away from the first component, in the absence of the analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected. 2. A device for detecting presence or absence of a target analyte, the device comprising:

[0129] (a) a substrate;

[0130] (b) a first component that comprises:

[0131] (i) a first region attached to the substrate;

[0132] (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and

[0133] (iii) a second region which binds to a first location on the target analyte; and

[0134] (c) a second component that comprises:

[0135] (i) a first region which binds to a second location on the target analyte; and

[0136] (ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate, positioned relative to the first component to facilitate binding of the first and second components to the target analyte; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

[0137] 3. The device according to aspect 2, wherein the first component comprises two or more force sensors.

[0138] 4. The device according to aspect 3, wherein the two or more force sensors are connected in series.

[0139] 5. The device according to aspect 3, wherein the two or more force sensors are connected in parallel.

[0140] 6. The device according to any of the aspects 2-5, wherein the fluorophore and quencher are connected via a linker.

[0141] 7. The device according to aspect 6, wherein the linker is an oligonucleotide linker.

[0142] 8. The device according to aspect 7, wherein at least a portion of the oligonucleotide linker hybridizes with itself. 9. The device according to any of aspects 2-5, wherein the force sensor is a micelle.

[0143] 10. The device according to aspect 9, wherein the micelle comprises two or more fluorophores.

[0144] 11. The device according to aspect 10, wherein the micelle has a hydrophobic center comprising the two or more fluorophores.

[0145] 12. The device according to aspect 11 , wherein the hydrophobic center contains the quencher.

[0146] 13. The device according to any of aspects 2-12, wherein the fluorophore is a small molecule, fluorescent protein or quantum dot.

[0147] 14. The device according to any of aspects 2-13, wherein the quencher is a nanoparticle.

[0148] 15. The device according to aspect 14, wherein the nanoparticle is a gold nanoparticle.

[0149] 16. The device according to any of aspects 2-15, wherein the force transducer is a magnetic nanoparticle.

[0150] 17. The device according to aspect 16, wherein the magnetic nanoparticle is an iron oxide nanoparticle.

[0151] 18. The device according to any of aspects 2-15, wherein the force transducer is a kite component.

[0152] 19. The device according to any of aspects 2-18, wherein the second region of the first component comprises an oligonucleotide that is complementary to the first location on the target analyte.

[0153] 20. The device according to any of aspects 2-18, wherein the second region of the first component comprises a binding moiety that binds to the first location on the target analyte.

[0154] 21. The device according to aspect 20, wherein the binding moiety is a protein. 22. The device according to aspect 21, wherein the protein is an oligonucleotide binding protein.

[0155] 23. The device according to aspect 21, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0156] 24. The device according to any of aspects 2-23, wherein the first region of the second component comprises an oligonucleotide that is complementary to the second location on the target analyte.

[0157] 25. The device according to any of aspects 2-23, wherein the first region of the second component comprises a binding moiety that binds to the second location on the target analyte.

[0158] 26. The device according to aspect 25, wherein the binding moiety is a protein.

[0159] 27. The device according to aspect 26, wherein the protein is an oligonucleotide binding protein.

[0160] 28. The device according to aspect 26, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0161] 29. The device according to any of aspects 2-28, wherein the target analyte is an oligonucleotide.

[0162] 30. The device according to any of aspects 2-28, wherein the target analyte is a cell, sub- cellular structure or protein.

[0163] 31. The device according to aspect 30, wherein the protein is an antibody or cytokine.

[0164] 32. A system for detecting presence or absence of a first target analyte and a second target analyte, the system comprising:

[0165] (a) a substrate comprising a first component that comprises:

[0166] (i) a first region attached to the substrate; (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and

[0167] (iii) a second region which binds to a first location on the first target analyte;

[0168] (b) a second component that comprises:

[0169] (i) a first region which binds to a second location on the first target analyte; and

[0170] (ii) a second region which binds to a first location on the second target analyte; and

[0171] (c) a third component that comprises:

[0172] (i) a first region which binds to a second location on the second target analyte; and

[0173] (ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the first target analyte and the second target analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, and wherein when the force transducer is moved away from the first component, in the absence of the first target analyte and / or the second target analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected.

[0174] 33. A device for detecting presence or absence of a first target analyte and a second target analyte, the device comprising:

[0175] (a) a substrate;

[0176] (b) a first component that comprises:

[0177] (i) a first region attached to the substrate;

[0178] (ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and

[0179] (iii) a second region which binds to a first location on the first target analyte; and

[0180] (c) a second component that comprises:

[0181] (i) a first region which binds to a second location on the first target analyte; and

[0182] (ii) a second region which binds to a first location on the second target analyte; and

[0183] (d) a third component that comprises:

[0184] (i) a first region which binds to a second location on the second target analyte; and

[0185] (ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate, positioned relative to the first and second components to facilitate binding of the first, second and third components to the first and second target analytes; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

[0186] 34. The device according to aspect 33, wherein the first component comprises two or more force sensors.

[0187] 35. The device according to aspect 34, wherein the two or more force sensors are connected in series.

[0188] 36. The device according to aspect 34, wherein the two or more force sensors are connected in parallel.

[0189] 37. The device according to any of aspects 33-36, wherein the fluorophore and quencher are connected via a linker.

[0190] 38. The device according to aspect 37, wherein the linker is an oligonucleotide linker.

[0191] 39. The device according to aspect 38, wherein at least a portion of the oligonucleotide linker hybridizes with itself.

[0192] 40. The device according to any of aspects 33-36, wherein the force sensor is a micelle.

[0193] 41. The device according to aspect 40, wherein the micelle comprises two or more fluorophores.

[0194] 42. The device according to aspect 41, wherein micelle has a hydrophobic center comprising the two or more fluorophores.

[0195] 43. The device according to aspect 42, wherein the hydrophobic center contains the quencher. 44. The device according to any of aspects 33-43, wherein the fluorophore is a small molecule, fluorescent protein or quantum dot.

[0196] 45. The device according to any of aspects 33-44, wherein the quencher is a nanoparticle.

[0197] 46. The device according to aspect 45, wherein the nanoparticle is a gold nanoparticle.

[0198] 47. The device according to any of aspects 33-46, wherein the force transducer is a magnetic nanoparticle.

[0199] 48. The device according to aspect 47, wherein the magnetic nanoparticle is an iron oxide nanoparticle.

[0200] 49. The device according to any of aspects 33-46, wherein the force transducer is a kite component.

[0201] 50. The device according to any of aspects 33-49, wherein the second region of the first component comprises an oligonucleotide that is complementary to the first location on the first target analyte.

[0202] 51. The device according to any of aspects 33-49, wherein the second region of the first component comprises a binding moiety that binds to the first location on the first target analyte.

[0203] 52. The device according to aspect 51, wherein the binding moiety is a protein.

[0204] 53. The device according to aspect 52, wherein the protein is an oligonucleotide binding protein.

[0205] 54. The device according to aspect 52, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0206] 55. The device according to any of aspects 33-54, wherein the first region of the second component comprises an oligonucleotide that is complementary to the second location on the first target analyte. 56. The device according to any of aspects 33-54, wherein the first region of the second component comprises a binding moiety that binds to the second location on the first target analyte.

[0207] 57. The device according to aspect 56, wherein the binding moiety is a protein.

[0208] 58. The device according to aspect 57, wherein the protein is an oligonucleotide binding protein.

[0209] 59. The device according to aspect 57, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0210] 60. The device according to any of aspects 33-59, wherein the second region of the second component comprises an oligonucleotide that is complementary to the first location on the second target analyte.

[0211] 61. The device according to any of aspects 33-59, wherein the second region of the second component comprises a binding moiety that binds to the first location on the second target analyte.

[0212] 62. The device according to aspect 61, wherein the binding moiety is a protein.

[0213] 63. The device according to aspect 62, wherein the protein is an oligonucleotide binding protein.

[0214] 64. The device according to aspect 62, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0215] 65. The device according to any of aspects 33-64, wherein the first region of the third component comprises an oligonucleotide that is complementary to the second location on the second target analyte.

[0216] 66. The device according to any of aspects 33-64, wherein the first region of the third component comprises a binding moiety that binds to the second location on the second target analyte. 67. The device according to aspect 66, wherein the binding moiety is a protein.

[0217] 68. The device according to aspect 67, wherein the protein is an oligonucleotide binding protein.

[0218] 69. The device according to aspect 67, wherein the protein is an antibody, nanobody or antigen-binding protein.

[0219] 70. The device according to any of aspects 33-69, wherein the first target analyte is an oligonucleotide.

[0220] 71. The device according to any of aspects 33-69, wherein the first target analyte is a cell, sub- cellular structure or protein.

[0221] 72. The device according to aspect 71, wherein the protein is an antibody or cytokine.

[0222] 73. The device according to any of aspects 33-72, wherein the second target analyte is an oligonucleotide.

[0223] 74. The device according to any of aspects 33-72, wherein the second target analyte is a cell, sub-cellular structure or protein.

[0224] 75. The device according to aspect 74, wherein the protein is an antibody or cytokine.

[0225] 76. A method of detecting a target analyte in a sample, the method comprising: combining the substrate and the second component of aspect 1 with the sample under conditions suitable for binding of the target analyte to the first and second components; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte. 77. The method according to aspect 76, wherein the combining comprises adding the sample to the substrate followed by adding the second component.

[0226] 78. The method according to aspect 76, wherein the combining comprises adding the second component to the substrate, immobilizing the second component on the substrate, followed by adding the sample.

[0227] 79. A method of detecting a target analyte in a sample, the method comprising: combining the device of any of aspects 2-31 with the sample comprising or suspected of comprising the target analyte under conditions suitable for the first and second components to bind to the target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte.

[0228] 80. The method according to aspect 79, wherein the moving comprises applying a magnetic force to the force transducer.

[0229] 81. The method according to aspect 79, wherein the moving comprises applying a flow of liquid or gas.

[0230] 82. A method of detecting a first and second target analyte in a sample, the method comprising: combining the device of any of aspects 33-75 with the sample comprising the first and second target analytes or suspected of comprising the first and second target analytes under conditions suitable for the first and second components to bind to the first target analyte, if present, and the second and third components to bind to the second target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the first target analyte and / or second target analyte and presence of fluorescence indicates presence of the first and second target analytes.

[0231] 83. The method according to aspect 82, wherein the moving comprises applying a magnetic force to the force transducer. 84. The method according to aspect 82, wherein the moving comprises applying a flow of liquid or gas.

Claims

CLAIMSWhat is claimed is:

1. A system for detecting presence or absence of a target analyte, the system comprising:(a) a substrate comprising a first component that comprises:(i) a first region attached to the substrate;(ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and(iii) a second region which binds to a first location on the target analyte; and(b) a second component that comprises:(i) a first region which binds to a second location on the target analyte; and(ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, and wherein when the force transducer is moved away from the first component, in the absence of the analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected.

2. A device for detecting presence or absence of a target analyte, the device comprising:(a) a substrate;(b) a first component that comprises:(i) a first region attached to the substrate;(ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and(iii) a second region which binds to a first location on the target analyte; and(c) a second component that comprises:(i) a first region which binds to a second location on the target analyte; and(ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate, positioned relative to the first component to facilitate binding of the first and second components to the target analyte; andconfigured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

3. The device according to claim 2, wherein the first component comprises two or more force sensors.

4. The device according to claim 3, wherein the two or more force sensors are connected in series.

5. The device according to claim 3, wherein the two or more force sensors are connected in parallel.

6. The device according to any of the claims 2-5, wherein the fluorophore and quencher are connected via a linker.

7. The device according to claim 6, wherein the linker is an oligonucleotide linker.

8. The device according to claim 7, wherein at least a portion of the oligonucleotide linker hybridizes with itself.

9. The device according to any of claims 2-5, wherein the force sensor is a micelle.

10. The device according to claim 9, wherein the micelle comprises two or more fluorophores.

11. The device according to claim 10, wherein the micelle has a hydrophobic center comprising the two or more fluorophores.

12. The device according to claim 11, wherein the hydrophobic center contains the quencher.

13. The device according to any of claims 2-12, wherein the fluorophore is a small molecule, fluorescent protein or quantum dot.

14. The device according to any of claims 2-13, wherein the quencher is a nanoparticle.

15. The device according to claim 14, wherein the nanoparticle is a gold nanoparticle.

16. The device according to any of claims 2-15, wherein the force transducer is a magnetic nanoparticle.

17. The device according to claim 16, wherein the magnetic nanoparticle is an iron oxide nanoparticle.

18. The device according to any of claims 2-15, wherein the force transducer is a kite component.

19. The device according to any of claims 2-18, wherein the second region of the first component comprises an oligonucleotide that is complementary to the first location on the target analyte.

20. The device according to any of claims 2-18, wherein the second region of the first component comprises a binding moiety that binds to the first location on the target analyte.

21. The device according to claim 20, wherein the binding moiety is a protein.

22. The device according to claim 21, wherein the protein is an oligonucleotide binding protein.

23. The device according to claim 21 , wherein the protein is an antibody, nanobody or antigenbinding protein.

24. The device according to any of claims 2-23, wherein the first region of the second component comprises an oligonucleotide that is complementary to the second location on the target analyte.

25. The device according to any of claims 2-23, wherein the first region of the second component comprises a binding moiety that binds to the second location on the target analyte.

26. The device according to claim 25, wherein the binding moiety is a protein.

27. The device according to claim 26, wherein the protein is an oligonucleotide binding protein.

28. The device according to claim 26, wherein the protein is an antibody, nanobody or antigenbinding protein.

29. The device according to any of claims 2-28, wherein the target analyte is an oligonucleotide.

30. The device according to any of claims 2-28, wherein the target analyte is a cell, sub-cellular structure or protein.

31. The device according to claim 30, wherein the protein is an antibody or cytokine.

32. A system for detecting presence or absence of a first target analyte and a second target analyte, the system comprising:(a) a substrate comprising a first component that comprises:(i) a first region attached to the substrate;(ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and(iii) a second region which binds to a first location on the first target analyte;(b) a second component that comprises:(i) a first region which binds to a second location on the first target analyte; and(ii) a second region which binds to a first location on the second target analyte; and(c) a third component that comprises:(i) a first region which binds to a second location on the second target analyte; and(ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, in the presence of the first target analyte and the second target analyte, the fluorophore and quencher dissociate such that a fluorescent signal is detected, andwherein when the force transducer is moved away from the first component, in the absence of the first target analyte and / or the second target analyte, the fluorophore and quencher remain associated such that a fluorescent signal is not detected.

33. A device for detecting presence or absence of a first target analyte and a second target analyte, the device comprising:(a) a substrate;(b) a first component that comprises:(i) a first region attached to the substrate;(ii) a force sensor comprising a fluorophore and a quencher, wherein the fluorophore and quencher are associated such that no fluorescent signal is detected; and(iii) a second region which binds to a first location on the first target analyte; and(c) a second component that comprises:(i) a first region which binds to a second location on the first target analyte; and(ii) a second region which binds to a first location on the second target analyte; and(d) a third component that comprises:(i) a first region which binds to a second location on the second target analyte; and(ii) a second region attached to a force transducer, wherein the force transducer is: positionable in contact with the substrate, positioned relative to the first and second components to facilitate binding of the first, second and third components to the first and second target analytes; and configured to be movable relative to the first component, wherein when the force transducer is moved away from the first component, the fluorophore and quencher dissociate such that a fluorescent signal is detected.

34. The device according to claim 33, wherein the first component comprises two or more force sensors.

35. The device according to claim 34, wherein the two or more force sensors are connected in series.

36. The device according to claim 34, wherein the two or more force sensors are connected in parallel.

37. The device according to any of claims 33-36, wherein the fluorophore and quencher are connected via a linker.

38. The device according to claim 37, wherein the linker is an oligonucleotide linker.

39. The device according to claim 38, wherein at least a portion of the oligonucleotide linker hybridizes with itself.

40. The device according to any of claims 33-36, wherein the force sensor is a micelle.

41. The device according to claim 40, wherein the micelle comprises two or more fluorophores.

42. The device according to claim 41, wherein micelle has a hydrophobic center comprising the two or more fluorophores.

43. The device according to claim 42, wherein the hydrophobic center contains the quencher.

44. The device according to any of claims 33-43, wherein the fluorophore is a small molecule, fluorescent protein or quantum dot.

45. The device according to any of claims 33-44, wherein the quencher is a nanoparticle.

46. The device according to claim 45, wherein the nanoparticle is a gold nanoparticle.

47. The device according to any of claims 33-46, wherein the force transducer is a magnetic nanoparticle.

48. The device according to claim 47, wherein the magnetic nanoparticle is an iron oxide nanoparticle.

49. The device according to any of claims 33-46, wherein the force transducer is a kite component.

50. The device according to any of claims 33-49, wherein the second region of the first component comprises an oligonucleotide that is complementary to the first location on the first target analyte.

51. The device according to any of claims 33-49, wherein the second region of the first component comprises a binding moiety that binds to the first location on the first target analyte.

52. The device according to claim 51, wherein the binding moiety is a protein.

53. The device according to claim 52, wherein the protein is an oligonucleotide binding protein.

54. The device according to claim 52, wherein the protein is an antibody, nanobody or antigenbinding protein.

55. The device according to any of claims 33-54, wherein the first region of the second component comprises an oligonucleotide that is complementary to the second location on the first target analyte.

56. The device according to any of claims 33-54, wherein the first region of the second component comprises a binding moiety that binds to the second location on the first target analyte.

57. The device according to claim 56, wherein the binding moiety is a protein.

58. The device according to claim 57, wherein the protein is an oligonucleotide binding protein.

59. The device according to claim 57, wherein the protein is an antibody, nanobody or antigenbinding protein.

60. The device according to any of claims 33-59, wherein the second region of the second component comprises an oligonucleotide that is complementary to the first location on the second target analyte.

61. The device according to any of claims 33-59, wherein the second region of the second component comprises a binding moiety that binds to the first location on the second target analyte.

62. The device according to claim 61, wherein the binding moiety is a protein.

63. The device according to claim 62, wherein the protein is an oligonucleotide binding protein.

64. The device according to claim 62, wherein the protein is an antibody, nanobody or antigenbinding protein.

65. The device according to any of claims 33-64, wherein the first region of the third component comprises an oligonucleotide that is complementary to the second location on the second target analyte.

66. The device according to any of claims 33-64, wherein the first region of the third component comprises a binding moiety that binds to the second location on the second target analyte.

67. The device according to claim 66, wherein the binding moiety is a protein.

68. The device according to claim 67, wherein the protein is an oligonucleotide binding protein.

69. The device according to claim 67, wherein the protein is an antibody, nanobody or antigenbinding protein.

70. The device according to any of claims 33-69, wherein the first target analyte is an oligonucleotide.

71. The device according to any of claims 33-69, wherein the first target analyte is a cell, sub- cellular structure or protein.

72. The device according to claim 71, wherein the protein is an antibody or cytokine.

73. The device according to any of claims 33-72, wherein the second target analyte is an oligonucleotide.

74. The device according to any of claims 33-72, wherein the second target analyte is a cell, sub-cellular structure or protein.

75. The device according to claim 74, wherein the protein is an antibody or cytokine.

76. A method of detecting a target analyte in a sample, the method comprising: combining the substrate and the second component of claim 1 with the sample under conditions suitable for binding of the target analyte to the first and second components; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte.

77. The method according to claim 76, wherein the combining comprises adding the sample to the substrate followed by adding the second component.

78. The method according to claim 76, wherein the combining comprises adding the second component to the substrate, immobilizing the second component on the substrate, followed by adding the sample.

79. A method of detecting a target analyte in a sample, the method comprising: combining the device of any of claims 2-31 with the sample comprising or suspected of comprising the target analyte under conditions suitable for the first and second components to bind to the target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the target analyte and presence of fluorescence indicates presence of the target analyte.

80. The method according to claim 79, wherein the moving comprises applying a magnetic force to the force transducer.

81. The method according to claim 79, wherein the moving comprises applying a flow of liquid or gas.

82. A method of detecting a first and second target analyte in a sample, the method comprising: combining the device of any of claims 33-75 with the sample comprising the first and second target analytes or suspected of comprising the first and second target analytes under conditions suitable for the first and second components to bind to the first target analyte, if present, and the second and third components to bind to the second target analyte, if present; moving the force transducer away from the first component; and measuring fluorescence at the location at which the first component is attached, wherein absence of fluorescence indicates absence of the first target analyte and / or second target analyte and presence of fluorescence indicates presence of the first and second target analytes.

83. The method according to claim 82, wherein the moving comprises applying a magnetic force to the force transducer.

84. The method according to claim 82, wherein the moving comprises applying a flow of liquid or gas.

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