APTAMER BIOSENSORS USEFUL FOR DETECTING HORMONES, HORMONE MIMICS, AND METABOLITES THEREOF

FIELD OF THE INVENTION

This present invention relates generally to polynucleotides and salts thereof, uses of the polynucleotides and salts thereof, e.g., for detecting a target molecule, such as a hormone, hormone mimic, or metabolite thereof, in a sample. In some embodiments, the polynucleotide or salt thereof forms a nanoparticle complex with an agent.

BACKGROUND OF THE INVENTION

There is an on-going need for methods for rapidly detecting and quantifying the presence of target molecules, for example, as part of environmental testing. For example, target biological molecules such as endocrine-disrupting compounds and hormones are often found as contaminants in the environment. Such contaminants can be found in waterways, soils, biological samples, of both plant and animals, as environmental pollutants from residential, agricultural, commercial and/or industrial applications.

In some cases, these molecules, together with their metabolites and/or synthetically modified variants pose a threat to the health of human, domestic-animal and wildlife populations by mimicking the activity of endogenous hormones, such as estrogens. These molecules may act by blocking, mimicking, stimulating or inhibiting the production and function of natural hormones. The compounds or their residues that mimic these endogenous steroidal hormones, and their metabolites, are typically lipid-soluble and thus, have the ability to bio-accumulate in living systems of mammals and marine species. Evidence of this has been identified in human blood plasma, breast milk, fetal tissues and biological fluids (Allmyr et al., Anal. Chem., 78: 6542-6546, 2006; Hileman, Chemical and Engineering News, 85: 31-33, 2007; Van-Pelt et al. Endocrinology, 140: 478-483, 2001; Skakkebaek et al., Human Reproduction 16: 972-978, 2001; Vandenberg et al., Endocrine Reviews, 33(3): 2012).

More conventional methodologies and techniques that are often used for the detection of small compounds include high performance liquid chromatography (HPLC) or gas chromatography coupled with mass spectrometry (GCMS). Although these techniques can be useful for this purpose, the accompanying analyses can be complicated to perform and can take a long period of time to complete. Furthermore, sample concentration is often required for standard chromatographic techniques and cannot be carried out in situ (Campbell, C. G., et al., 2006, Chemosphere, 65, 1265-80). Consequently, these techniques often cannot be performed on site, often require specialised equipment and trained operators, and often do not provide for a rapid assessment of the sample.

Accordingly, there is therefore a need for compositions and methods for the detection of target molecules, such as hormones, hormone mimics, metabolites of hormones and metabolites of hormone mimics, such as in the area of environmental and contaminant testing as in human veterinary medicine, and that can be provided by colorimetric assays which can provide visual indication of the presence of target molecules.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions useful for detecting a target molecule, such as a hormone, hormone mimic, or metabolite thereof, in a sample.

The present invention provides a polynucleotide or salt thereof comprising a sequence that has at least 70% sequence identity to the sequence of SEQ ID NO: 1-15, 20-128 or 129. The present invention also provides herein a polynucleotide or salt thereof consisting essentially, or consisting of the sequence of SEQ ID NO: 1-15, 17, 19-128 or 129. The present invention also provides an aptamer comprising, consisting essentially of or consisting of a polynucleotide of the invention, a functional fragment thereof, or a salt of either. The present invention also provides herein polynucleotide-agent complexes comprising (i) a polynucleotide disclosed herein, or functional fragment thereof, or a salt thereof, and (ii) an agent. Methods for detecting a target molecule in a sample comprising: (i) contacting a sample with a polynucleotide disclosed herein, or functional fragment thereof or a salt thereof, and (ii) measuring a change in a property of the sample, wherein the change in the property indicates the presence of the target molecule are also provide by the invention. The invention also provides herein methods for detecting a target molecule in a sample, comprising: (i) contacting a sample with a polynucleotide-agent complex disclosed herein, and (ii) measuring a change in a property of the sample, wherein a change in the property is indicative of the presence of the target molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of 17β-estradiol (E2)

FIG. 2 depicts the structure of bisphenol-A (BPA).

FIG. 3 depicts the structure of 17α-ethynylestradiol.

FIG. 4 is a schematic depiction of aggregation in an illustrative colorimetric assay.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in medicine, molecular biology and biochemistry a can be found in Dictionary of Microbiology and Molecular Biology, Singleton et al, (2d ed. (1994); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995); The Dictionary of Cell & Molecular Biology, 4th Edition. Lackie. J (Ed.), Academic Press Inc. (2007), and The Oxford Dictionary of Biochemistry and Molecular Biology, 2nd edition, Cammack et al. (Eds.), Oxford University Press Inc. (2006).

It is also believed that practice of various embodiments of, encompassed by, and falling within the scope of the present invention can be performed using standard molecular biology and biochemistry protocols and procedures as known in the art, and as described, for example in “Current Protocols in Nucleic Acid Chemistry, Wiley Online Library, Various; Molecular Cloning: A Laboratory Manual, Maniatis et al., Cold Spring Harbor Laboratory Press, (1982); Molecular Cloning: A Laboratory Manual (2 ed.), Sambrook et al., Cold Spring Harbor Laboratory Press, (1989); Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (Eds.), Academic Press Inc., (1987); Protein Synthesis and Ribosome Structure: Translating the Genome. Nierhaus, K and Wilson D (eds.), Wiley-VCH Inc. (2004); Synthetic Peptides: A User's Guide (Advances in Molecular Biology) 2nd edition, Grant G. (Ed.), Oxford University Press (2002); Remington: The Science and Practice of Pharmacy 21st edition, Beringer, P (Ed.), Lippincott Williams & Wilkins, (2005), pp. 2393; and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.

DEFINITIONS

The term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, “about 100” means from 90 to 110 and “about six” means from 5.4 to 6.6.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

The term “fragment” as used herein is used interchangeably with the term “functional fragment” and means the same thing. The term “functional fragment” as used herein means a part of a polynucleotide of the invention as disclosed herein that specifically binds to a target molecule.

Polynucleotides

The present invention provides polynucleotides and salts thereof. In some embodiments, a polynucleotide is an oligonucleotide or a single strand of RNA or DNA. In some embodiments, the polynucleotide or functional fragment thereof, or salt of either is an aptamer. The term “aptamer” refers to a polynucleotide or functional fragment thereof, or salt of either that specifically binds a target molecule. The term “specifically binds” is used interchangeably herein with “selectively binds” and means the same thing. As used herein the terms “specifically binds” and “selectively binds” in reference to an aptamer, describe the binding of an aptamer to a target molecule and mean that aptamer binding to the target molecule does not involve the formation of nucleotide base pairs between the aptamer and the target molecule. A person of skill in the art recognizes that it is well-known in the art that the polynucleotide sequence of an aptamer may include base pairs that are not required for specific binding of the aptamer to a given target molecule, and that smaller fragments of an aptamer, even fragments having below 50% sequence identity may still be capable of effectively binding to a target molecule (Alsager, Omar A., et al. “Ultrasensitive Colorimetric Detection of 17β-Estradiol: The Effect of Shortening DNA Aptamer Sequences.” Analytical chemistry 87.8 (2015): 4201-4209). In some embodiments, an aptamer exerts an inhibitory effect on a target, e.g., by binding of the target, by catalytically altering the target, by reacting with the target in a way which modifies the target or the functional activity of the target, by ionically or covalently attaching to the target or by facilitating the reaction between the target and another molecule. The aptamer can comprise a ribonucleotide, deoxyribonucleotide, or other type of nucleic acid, or two or more different types of nucleic acids. An aptamer can also comprise one or more modified bases, sugars, polyethylene glycol spacers or phosphate backbone units. In some embodiments, the aptamer comprises one or more 2′ sugar modifications, such as a 2′-O-alkyl (e.g., 2′-O-methyl or 2′-O-methoxyethyl) or a 2′-fluoro modification.

In some embodiments, an aptamer is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length.

In some embodiments, an aptamer is a polynucleotide of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15 nucleotides in length.

In some embodiments an aptamer is a polynucleotide of about 70 to 80, is about 60 to 70, is about 50 to 60, is about 40 to 50, is about 30 to 40, is about 20 to 30, is about 10 to 20 nucleotides in length.

In some embodiments an aptamer is a polynucleotide of about 75 to 85, is about 65 to 75, is about 55 to 65, is about 45 to 55, is about 35 to 45, is about 25 to 35, is about 15 to 25 nucleotides in length.

In some embodiments an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length.

In some embodiments an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length. In some embodiments the aptamer is about 73 to about 77 or about 74 to about 76 nucleotides in length.

A person of skill in the art will appreciate that an aptamer may be any length polynucleotide that falls within the size parameters set out herein. By way of non-limiting example an aptamer may be about 76, about 61, about 54, about 43, about 29 or about 27 nucleotides in length or may be 77, 62, 55, 44, 28 or 26 nucleotides in length. What is important is that the aptamer specifically binds the target molecule.

In some embodiments an aptamer of the invention is a polynucleotide of about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15 nucleotides in length that selectively binds a target molecule as described herein.

In some embodiments an aptamer is a polynucleotide of 70 to 80, is 60 to 70, is 50 to 60, is 40 to 50, is 30 to 40, is 20 to 30, is 10 to 20 nucleotides in length that selectively binds a target molecule as described herein.

In some embodiments an aptamer is a polynucleotide of 75 to 85, is 65 to 75, is 55 to 65, is 45 to 55, is 35 to 45, is 25 to 35, is 15 to 25 nucleotides in length that selectively binds a target molecule as described herein.

The term “salt” includes a non-toxic salt of an inorganic or organic acid, including, but not limited to, the following salts: halide (chloride, bromide, iodide, fluoride), acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, nitrate, oxalate, persulfate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.

The term “salt” also includes a non-toxic salt of an organic or inorganic base, including, but not limited to, the following salts: Na⁺, K⁺, Ca²⁺, Mg²⁺, and Li⁺.

In some embodiments, the target molecule is a small molecule. In some embodiments, the small molecule has a molecular weight (MW) of from about 60 to about 2000 g mol⁻¹. In some embodiments, the small molecule has a MW of from about 100 to about 500 g mol⁻¹. In some embodiments, the small molecule has a MW of from about 150 to about 350 g mol⁻¹. The molecular weight of such small molecules and the calculation of their molecular weight are well known to those of skill in the art.

In some embodiments, a small molecule is a naturally occurring or synthetic hormone, naturally occurring or synthetic hormone mimic, naturally occurring or synthetic phytoestrogen, naturally occurring or synthetic opiate or opioid, or derivative or metabolite thereof (e.g., morphine, oxycodone, hydromorphone, propoxyphene, nicomorphine, dihydrocodeine, diamorphine, papavereturn, codeine, ethylmorphine, phenylpiperidine and derivates thereof, methadone, dextropropoxyphene, buprenorphine, pentazocine, tilidine, tramadol, hydrocodone, meperidine, oxymorphone, alphaprodine, anileridine, dextromoramide, metopone, levorphanol, phenazocine, etoheptazine, propiram, profadol, phenampromide, thiambuten, pholcodeine, codeine, dihydrocodeinon, fentanyl, 3-trans-dimethylamino-4-phenyl-4-trans-carbethoxy-Λ′-cyclohexen, 3-dimethylamino-O-(4-methoxyphenyl-carbamoyl)-propiophenone oxime, (−)β-2′-hydroxy-2,9-dimethyl-5-phenyl-6,7-benzomorphane, (−)2′-hydroxy-2-(3-methyl-2-butenyl)-9-methyl-5-phenyl-6,7-benzomorphane, pirinitramide, (−)α-5,9-diethyl-2′hydroxy-2-methyl-6,7-benzomorphane, ethyl 1-(2-dimethylaminoethyl)-4,5,6,7-tetrahydro-3-methyl-4-oxo-6-phenyl-indol-2-carboxylate, 1-benzoylmethyl-2,3-dimethyl-3-(m-hydroxy-phenyl)-piperidine, N-allyl-7α(1-R-hydroxy-1-methylbutyl)-6,14-endo-ethanotetrahydronororipavine, (−) 2′-hydroxy-2-methyl-6,7-benzomorphane, noracylmethadol, phenoperidine, α-d1-methadol, α-1-methadol, β-d1-acetylmethadol, α-1-acetylmethadol and β-1-acetylmethadol), or a naturally occurring or synthetic organohalide.

In some embodiments, the small molecule is an endocrine-disrupting compound, a steroidal or non-steroidal sex hormone, a metabolite or synthetic variant thereof. In some embodiments, the small molecule is an estrogenic compound, for example, a compound that is chemically related to estrogen, a compound that mimics the effect of endogenous estrogen, a compound that exerts an estrogenic effect on a living organism (even though the compound might differ chemically from an estrogenic substance produced endogenously by the endocrine system of an organism), and a compound that is structurally similar to estrogen. Estrogenic compounds may be natural or synthetic, steroidal or non-steroidal, and include a metabolite of such compounds. Examples of estrogenic compounds include estrone (E1), estradiaol (also known as 17β-estradiol or E2; see FIG. 1), estriol (E3), estetrol (E4), a xenoestrogen, a phytoestrogen, and a mycoestrogen. Examples of xenoestrogens include polychlorinated biphenols (also referred to as polychlorobiphenyl or PCBs), bisphenol-A (BPA; FIG. 2), and phthalates. Examples of phytoestrogens include daidzein, formononetin, genistein, bichanin A, coumestrol, 4′-methoxycoumestrol, repensol, and trifoliol. Also included are derivatives of these compounds, such as 17α-ethynylestradiol (FIG. 3).

In some embodiments, the small molecule is androstenedione, androstenediol, testosterone, dihydrotestosterone, pregnenolone, progesterone (P4), 17α-hydroxyprogesterone, Bis (4-hydroxyphenyl) methane (also referred to as bisphenol-F or BPF), cholesterol, adenosine, triclosan, a synthetic steroid (e.g., diethylstilboestrol (DES)), cocaine, heroin, tetrahydrocannabinol (THC), or any derivative or metabolite thereof. In other embodiments, the small molecule is an isoflavone, lignan, coumestan, organohalide (e.g., organochloride), polychlorinated organic compound, alkylphenol, or alkylphenol ethoxylate.

In some embodiments, the small molecule is a hormone or a marker of a condition of disease in a body. In some embodiments, the aptamer selectively binds a hormone and/or metabolite thereof. In some embodiments, the aptamer is useful to determine a particular status of an animal (e.g., a human or non-human animal, such as a domesticated animal (e.g., a cow, sheep, goat, horse, or pig); domesticated pet (e.g., a cat or dog), a wild animal (e.g., a monkey, bird, amphibian, rodent, rabbit, marsupial, or reptile), or an aquatic life form (e.g., fish)), such as the fertility status of the animal. In some embodiments, the aptamer selectively binds a known or an unknown marker of disease, for example a cancer-causing gene, for the detection of cancer or other disease, identification of a patient population that would be favourably disposed to therapy, detection of one or more molecules associated with disease or infection, or to determine a level of one or more specific metabolites associated with a particular condition.

In some embodiments, an aptamer binds 17β-estradiol (E2).

In some embodiments, an aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGCCTTTAAACTTGTATGGGGATTTAGAATTCTTTCCTCCCT GATAGTAAGAGCAATC (SEQ ID NO: 1) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GCCTTTAAACTTGTATGGGGATTTAGAATTCTTTCCTCCC (SEQ ID NO: 2) or TAGCCTTTAAACTTGTATGGGGATTTA (SEQ ID NO: 3).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATATGTTTCTTTTTCCATGCTAGTTTTTTAGTCTTTTTTACAATG ATAGTAAGAGCAATC (SEQ ID NO: 4) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TGTTTCTTTTTCCATGCTAGTTTTTTAGTCTTTTTTACAA (SEQ ID NO: 5).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATATAGTAGAGATTCACATTTGTGCCTATGATCTATTTCCGGAT GATAGTAAGAGCAATC (SEQ ID NO: 6) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TAGTAGAGATTCACATTTGTGCCTATGATCTATTTCCGGA (SEQ ID NO: 7), or CATTTGTGCCTATGATCTATTTCCGGAT (SEQ ID NO: 8). In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGTGTTGAATAGTGCCTTTTTGTCTTATTTTTATTTCTCCCT GATAGTAAGAGCAATC (SEQ ID NO: 9) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GTGTTGAATAGTGCCTTTTTGTCTTATTTTTATTTCTCCC (SEQ ID NO: 10).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATACATATTCCTGTCTCTTTTGTGATTTGCCATCCTTTTTTTCT GATAGTAAGAGCAATC (SEQ ID NO: 11) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CATATTCCTGTCTCTTTTGTGATTTGCCATCCTTTTTTTC (SEQ ID NO: 12).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATATGTGCATGTTTTTTTGTTTGATCATCACTTTCCCTTTACTT GATAGTAAGAGCAATC (SEQ ID NO: 13) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TGTGCATGTTTTTTTGTTTGATCATCACTTTCCCTTTACT (SEQ ID NO: 14) or CAATATGTGCATGTTTTTTTGTTTGAT (SEQ ID NO: 15).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 16) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 17), AAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTG (SEQ ID NO: 18) or GGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGGTG (SEQ ID NO: 19).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 20) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 21) or GGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 22).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCACCTGCTGTTGTAACTTGTGGAATGTGGGTCCCCTTC GTGATAGTAAGAGCAATC (SEQ ID NO: 23) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCACCTGCTGTTGTAACTTGTGGAATGTGGGTCCCCTTCG (SEQ ID NO: 24).

In some embodiments, the aptamer that binds E2 comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCCAAGTTCGGCATAGTGT GTGATAGTAAGAGCAATC (SEQ ID NO: 25) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGAAGGGATGCCGTTTGGGCCCCAAGTTCGGCATAGTGTG (SEQ ID NO: 26).

In some embodiments, an aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGCCTTTAAACTTGTATGGGGATTTAGAATTCTTTCCTCCCT GATAGTAAGAGCAATC (SEQ ID NO: 1) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GCCTTTAAACTTGTATGGGGATTTAGAATTCTTTCCTCCC (SEQ ID NO: 2) or TAGCCTTTAAACTTGTATGGGGATTTA (SEQ ID NO: 3).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATGTTTCTTTTTCCATGCTAGTTTTTTAGTCTTTTTTACAATG ATAGTAAGAGCAATC (SEQ ID NO: 4) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TGTTTCTTTTTCCATGCTAGTTTTTTAGTCTTTTTTACAA (SEQ ID NO: 5).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATAGTAGAGATTCACATTTGTGCCTATGATCTATTTCCGGAT GATAGTAAGAGCAATC (SEQ ID NO: 6) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TAGTAGAGATTCACATTTGTGCCTATGATCTATTTCCGGA (SEQ ID NO: 7), or CATTTGTGCCTATGATCTATTTCCGGAT (SEQ ID NO: 8).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGTGTTGAATAGTGCCTTTTTGTCTTATTTTTATTTCTCCCT GATAGTAAGAGCAATC (SEQ ID NO: 9) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GTGTTGAATAGTGCCTTTTTGTCTTATTTTTATTTCTCCC (SEQ ID NO: 10).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACATATTCCTGTCTCTTTTGTGATTTGCCATCCTTTTTTTCT GATAGTAAGAGCAATC (SEQ ID NO: 11) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CATATTCCTGTCTCTTTTGTGATTTGCCATCCTTTTTTTC (SEQ ID NO: 12).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATGTGCATGTTTTTTTGTTTGATCATCACTTTCCCTTTACTT GATAGTAAGAGCAATC (SEQ ID NO: 13) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TGTGCATGTTTTTTTGTTTGATCATCACTTTCCCTTTACT (SEQ ID NO: 14) or CAATATGTGCATGTTTTTTTGTTTGAT (SEQ ID NO: 15).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 16) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 17), AAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTG (SEQ ID NO: 18) or GGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGGTG (SEQ ID NO: 19).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 20) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 21) or GGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 22).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCACCTGCTGTTGTAACTTGTGGAATGTGGGTCCCCTTC GTGATAGTAAGAGCAATC (SEQ ID NO: 23) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCACCTGCTGTTGTAACTTGTGGAATGTGGGTCCCCTTCG (SEQ ID NO: 24).

In some embodiments, the aptamer that binds E2 consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCCAAGTTCGGCATAGTGT GTGATAGTAAGAGCAATC (SEQ ID NO: 25) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGAAGGGATGCCGTTTGGGCCCCAAGTTCGGCATAGTGTG (SEQ ID NO: 26).

In some embodiments, the aptamer binds E2; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 1-26, or from SEQ ID NOs: 1-15 and 20-26; and in some embodiments, selectively binds E2. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 1-26 or from SEQ ID NOs: 1-15, 17 and 19-26.

In some embodiments, the aptamer binds E2; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26; and in some embodiments, selectively binds E2. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26.

In some embodiments the aptamer that binds E2 is a polynucleotide of less than 80 nucleotides in length. In some embodiments the aptamer that binds E2 is less than 30 nucleotides in length.

In some embodiments the aptamer that binds E2 is a polynucleotide of about 25 or about 30 nucleotides in length, or of about 25 to 30 nucleotides in length, or of 25 to 30 nucleotides in length less and comprises or consists essentially of, or consists of SEQ ID NO: 3.

In some embodiments the aptamer that binds E2 is a polynucleotide comprising a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3.

In some embodiments the aptamer that binds E2 is a polynucleotide consisting essentially of, or consisting of a sequence that has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3.

In some embodiments, the aptamer binds androstenedione. In some embodiments, the aptamer that binds androstenedione comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 27) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 28).

In some embodiments, the aptamer that binds androstenedione comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGCCTCAGGGGCCAAAGTGAGTGTGGC TGGTGATAGTAAGAGCAATC (SEQ ID NO: 29) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGCCTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 30).

In some embodiments, the aptamer that binds androstenedione comprises the nucleotide sequence of ATACGAGCTTGTTCAATATTCATCTCAAATTAAAAAGTTGAGAAGTCCATTCTTCACAT GATAGTAAGAGCAATC (SEQ ID NO: 31) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TTCATCTCAAATTAAAAAGTTGAGAAGTCCATTCTTCACA (SEQ ID NO: 32).

In some embodiments, the aptamer that binds androstenedione comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGCAACAAGGCGAGTGTTGGCGGTTCGGTTGTGTGGGGT GGTGATAGTAAGAGCAATC (SEQ ID NO: 130) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GCAACAAGGCGAGTGTTGGCGGTTCGGTTGTGTGGGGTGG (SEQ ID NO: 131).

In some embodiments, the aptamer that binds androstenedione comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGAGTTGATAGAGTATTTTAACCGGGCTGTCGTGTTGGTTC TGATAGTAAGAGCAATC (SEQ ID NO: 132) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GAGTTGATAGAGTATTTTAACCGGGCTGTCGTGTTGGTTC (SEQ ID NO: 133).

In some embodiments, the aptamer that binds androstenedione consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 27) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 28).

In some embodiments, the aptamer that binds androstenedione consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGCCTCAGGGGCCAAAGTGAGTGTGGC TGGTGATAGTAAGAGCAATC (SEQ ID NO: 29) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGCCTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 30).

In some embodiments, the aptamer that binds androstenedione consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATTCATCTCAAATTAAAAAGTTGAGAAGTCCATTCTTCACAT GATAGTAAGAGCAATC (SEQ ID NO: 31) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TTCATCTCAAATTAAAAAGTTGAGAAGTCCATTCTTCACA (SEQ ID NO: 32).

In some embodiments, the aptamer that binds androstenedione consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGCAACAAGGCGAGTGTTGGCGGTTCGGTTGTGTGGGGT GGTGATAGTAAGAGCAATC (SEQ ID NO: 130) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GCAACAAGGCGAGTGTTGGCGGTTCGGTTGTGTGGGGTGG (SEQ ID NO: 131).

In some embodiments, the aptamer that binds androstenedione consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGAGTTGATAGAGTATTTTAACCGGGCTGTCGTGTTGGTTC TGATAGTAAGAGCAATC (SEQ ID NO: 132) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GAGTTGATAGAGTATTTTAACCGGGCTGTCGTGTTGGTTC (SEQ ID NO: 133).

In some embodiments, the aptamer binds androstenedione comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 27-32 and 130-133; and in some embodiments, selectively binds androstenedione. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 27-32 and 130-133.

In some embodiments, the aptamer binds androstenedione comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133; and in some embodiments, selectively binds androstenedione. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133.

In some embodiments the aptamer that binds androstenedione is a polynucleotide of less than 80 nucleotides in length.

In some embodiments the aptamer that binds androstenedione is a polynucleotide of about 73 or about 77 nucleotides in length, or of about 74 to 76 nucleotides in length, or of 70 to 80 nucleotides in length and comprises or consists essentially of, or consists of SEQ ID NO: 29 or SEQ ID NO: 27.

In some embodiments the aptamer that binds androstenedione is a polynucleotide comprising a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOs: 29 or 27.

In some embodiments the aptamer that binds androstenedione is a polynucleotide that consists essentially of, or consists of a sequence that has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOs: 29 or 27.

In some embodiments, the aptamer binds bisphenol-A (BPA).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATCGCCGGCGCCGGCCTAGTCTCAAAAAGGGCACTCCCCT GTGATAGTAAGAGCAATC (SEQ ID NO: 33) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TCGCCGGCGCCGGCCTAGTCTCAAAAAGGGCACTCCCCTG (SEQ ID NO: 34) or AAGGGCACTCCCCTGTGAT (SEQ ID NO: 35).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATATACGTGTGTATGTGGCTTTGTATATACATCTGTGGGGG TGATAGTAAGAGCAATC (SEQ ID NO: 36) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TATACGTGTGTATGTGGCTTTGTATATACATCTGTGGGGG (SEQ ID NO: 37).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATGTACTTTCGTGTTTTGGGTTTTCGTTTGTTCTGTAGTGCT GATAGTAAGAGCAATC (SEQ ID NO: 38) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TGTACTTTCGTGTTTTGGGTTTTCGTTTGTTCTGTAGTGC (SEQ ID NO: 39). In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGTTCGGTTGTAAACTTGAGTCATGAGCCCGCTTCCCCG GTGATAGTAAGAGCAATC (SEQ ID NO: 40) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGTTCGGTTGTAAACTTGAGTCATGAGCCCGCTTCCCCGG (SEQ ID NO: 41) or TCATGAGCCCGCTTCCCC (SEQ ID NO: 42).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGAAATCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 43) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGAAATCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTGG (SEQ ID NO: 44).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATCCACACGTAACAATCATCGGCAATAACTAGTTCACGTGG TGATAGTAAGAGCAATC (SEQ ID NO: 45) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TCCACACGTAACAATCATCGGCAATAACTAGTTCACGTGG (SEQ ID NO: 46).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGTGTTGTCGGACTGGGATTAGGTGGGCATCAGCCTGGCT GTGATAGTAAGAGCAATC (SEQ ID NO: 47) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GTGTTGTCGGACTGGGATTAGGTGGGCATCAGCCTGGCTG (SEQ ID NO: 48).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATTTGTTCTTTATGTGATGTTCGAGTTTGTTGGTGTTGTTTT GATAGTAAGAGCAATC (SEQ ID NO: 49) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TTTGTTCTTTATGTGATGTTCGAGTTTGTTGGTGTTGTTT (SEQ ID NO: 50). In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATGAATTTTATTTTTTGTTGGTTTTAGGATATTTTGGTTTGTG ATAGTAAGAGCAATC (SEQ ID NO: 51) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TGAATTTTATTTTTTGTTGGTTTTAGGATATTTTGGTTTG (SEQ ID NO: 52). In some embodiments, the aptamer or salt thereof that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATA GTGATAGTAAGAGCAATC (SEQ ID NO: 53) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATAG (SEQ ID NO: 54) or TACAGTTCATTTCACCCTGAGAGTGGGCT (SEQ ID NO: 55).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 56) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 57).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGG GGTGATAGTAAGAGCAATC (SEQ ID NO: 58) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGGGG (SEQ ID NO: 59).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCCGGGAGTTGTCACCACATAAGTGTATGTTGAATTTTGG TGATAGTAAGAGCAATC (SEQ ID NO: 60) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCCGGGAGTTGTCACCACATAAGTGTATGTTGAATTTTGG (SEQ ID NO: 61).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 62) or a fragment thereof. In some embodiments, fragment comprises the sequence of CGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTGG (SEQ ID NO: 63).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGAAGTCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 64) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGAAGTCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTGG (SEQ ID NO: 65).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAAACCCACTGACACGAAAAACCGCCGACGCTCGAGCCGCC ATGATAGTAAGAGCAATC (SEQ ID NO: 66) or a fragment thereof. In some embodiments, the fragment comprises a sequence of AACCCACTGACACGAAAAACCGCCGACGCTCGAGCCGCCA (SEQ ID NO: 67).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATA GTGATAGTAAGAGCAATC (SEQ ID NO: 68) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATAG (SEQ ID NO: 69).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 70) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 71).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGCGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 72) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGAAGGGATGCCGTTTGCGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 73).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGAACCAAGCCTTGTTATCAAATAGCGGGAGGCCGGCTC CTGATAGTAAGAGCAATC (SEQ ID NO: 74) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGAACCAAGCCTTGTTATCAAATAGCGGGAGGCCGGCTCC (SEQ ID NO: 75).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGCAACAAGGCGAGTGTTGGTGGTTCGGTTGTGTGGGGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 134) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GCAACAAGGCGAGTGTTGGTGGTTCGGTTGTGTGGGGTGG (SEQ ID NO: 135).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATACTTTATCATTATATTGATATCCGCTAGTTAATCCGTGAGCT GATAGTAAGAGCAATC (SEQ ID NO: 136) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CTTTATCATTATATTGATATCCGCTAGTTAATCCGTGAGC (SEQ ID NO: 137).

In some embodiments, the aptamer that binds BPA comprises the nucleotide sequence of ATACGAGCTTGTTCAATATCTCATTACCCCTACCTCACCCCCCCTGTTTAACGACCCG TGATAGTAAGAGCAATC (SEQ ID NO: 138) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TCTCATTACCCCTACCTCACCCCCCCTGTTTAACGACCCG (SEQ ID NO: 139).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATCGCCGGCGCCGGCCTAGTCTCAAAAAGGGCACTCCCCT GTGATAGTAAGAGCAATC (SEQ ID NO: 33) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TCGCCGGCGCCGGCCTAGTCTCAAAAAGGGCACTCCCCTG (SEQ ID NO: 34) or AAGGGCACTCCCCTGTGAT (SEQ ID NO: 35).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATATACGTGTGTATGTGGCTTTGTATATACATCTGTGGGGG TGATAGTAAGAGCAATC (SEQ ID NO: 36) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TATACGTGTGTATGTGGCTTTGTATATACATCTGTGGGGG (SEQ ID NO: 37).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATGTACTTTCGTGTTTTGGGTTTTCGTTTGTTCTGTAGTGCT GATAGTAAGAGCAATC (SEQ ID NO: 38) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TGTACTTTCGTGTTTTGGGTTTTCGTTTGTTCTGTAGTGC (SEQ ID NO: 39).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGTTCGGTTGTAAACTTGAGTCATGAGCCCGCTTCCCCG GTGATAGTAAGAGCAATC (SEQ ID NO: 40) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGTTCGGTTGTAAACTTGAGTCATGAGCCCGCTTCCCCGG (SEQ ID NO: 41) or TCATGAGCCCGCTTCCCC (SEQ ID NO: 42).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGAAATCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 43) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGAAATCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTGG (SEQ ID NO: 44).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATCCACACGTAACAATCATCGGCAATAACTAGTTCACGTGG TGATAGTAAGAGCAATC (SEQ ID NO: 45) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TCCACACGTAACAATCATCGGCAATAACTAGTTCACGTGG (SEQ ID NO: 46).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGTGTTGTCGGACTGGGATTAGGTGGGCATCAGCCTGGCT GTGATAGTAAGAGCAATC (SEQ ID NO: 47) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GTGTTGTCGGACTGGGATTAGGTGGGCATCAGCCTGGCTG (SEQ ID NO: 48).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATTTGTTCTTTATGTGATGTTCGAGTTTGTTGGTGTTGTTTT GATAGTAAGAGCAATC (SEQ ID NO: 49) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TTTGTTCTTTATGTGATGTTCGAGTTTGTTGGTGTTGTTT (SEQ ID NO: 50). In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATGAATTTTATTTTTTGTTGGTTTTAGGATATTTTGGTTTGTG ATAGTAAGAGCAATC (SEQ ID NO: 51) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TGAATTTTATTTTTTGTTGGTTTTAGGATATTTTGGTTTG (SEQ ID NO: 52).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATA GTGATAGTAAGAGCAATC (SEQ ID NO: 53) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATAG (SEQ ID NO: 54) or TACAGTTCATTTCACCCTGAGAGTGGGCT (SEQ ID NO: 55).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 56) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 57).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGG GGTGATAGTAAGAGCAATC (SEQ ID NO: 58) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGGGG (SEQ ID NO: 59).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCCGGGAGTTGTCACCACATAAGTGTATGTTGAATTTTGG TGATAGTAAGAGCAATC (SEQ ID NO: 60) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCCGGGAGTTGTCACCACATAAGTGTATGTTGAATTTTGG (SEQ ID NO: 61).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 62) or a fragment thereof. In some embodiments, fragment consists essentially of, or consists of the sequence of CGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTGG (SEQ ID NO: 63).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGAAGTCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 64) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGAAGTCACGATTAGGTCCTCCGTCTGTGTGCGGTTGTGG (SEQ ID NO: 65).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAAACCCACTGACACGAAAAACCGCCGACGCTCGAGCCGCC ATGATAGTAAGAGCAATC (SEQ ID NO: 66) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of a sequence of AACCCACTGACACGAAAAACCGCCGACGCTCGAGCCGCCA (SEQ ID NO: 67).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATA GTGATAGTAAGAGCAATC (SEQ ID NO: 68) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CAGTTCATTTCACCCTGAGAGTGGGCTAAGTTGGGCATAG (SEQ ID NO: 69).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 70) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 71).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTTGCGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 72) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGAAGGGATGCCGTTTGCGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 73).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGAACCAAGCCTTGTTATCAAATAGCGGGAGGCCGGCTC CTGATAGTAAGAGCAATC (SEQ ID NO: 74) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGAACCAAGCCTTGTTATCAAATAGCGGGAGGCCGGCTCC (SEQ ID NO: 75).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGCAACAAGGCGAGTGTTGGTGGTTCGGTTGTGTGGGGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 134) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GCAACAAGGCGAGTGTTGGTGGTTCGGTTGTGTGGGGTGG (SEQ ID NO: 135).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACTTTATCATTATATTGATATCCGCTAGTTAATCCGTGAGCT GATAGTAAGAGCAATC (SEQ ID NO: 136) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CTTTATCATTATATTGATATCCGCTAGTTAATCCGTGAGC (SEQ ID NO: 137).

In some embodiments, the aptamer that binds BPA consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATCTCATTACCCCTACCTCACCCCCCCTGTTTAACGACCCG TGATAGTAAGAGCAATC (SEQ ID NO: 138) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TCTCATTACCCCTACCTCACCCCCCCTGTTTAACGACCCG (SEQ ID NO: 139).

In some embodiments, the aptamer binds BPA; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 33-75 and 134-139; and in some embodiments, selectively binds BPA. In some embodiments, the aptamer or salt thereof consists essentially of, or consists of a sequence selected from SEQ ID NOs: 33-75 and 134-139.

In some embodiments, the aptamer binds BPA; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139; and in some embodiments, selectively binds BPA. In some embodiments, the aptamer or salt thereof consists essentially of, or consists of a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139.

In some embodiments the aptamer that binds BPA is a polynucleotide of less than 80 nucleotides in length or less than 30 nucleotides in length.

In some embodiments the aptamer that binds BPA is a polynucleotide of about 73 or about 77 nucleotides in length, or of about 74 to 76 nucleotides in length, or of 70 to 80 nucleotides in length and comprises or consists essentially of, or consists of SEQ ID NO: 53 or SEQ ID NO: 56.

In some embodiments the aptamer that binds BPA is a polynucleotide comprising a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOs: 53 or 56.

In some embodiments the aptamer that binds BPA is a polynucleotide of about 27 or about nucleotides in length, or is 25 to 30 nucleotides in length, or is 26 to 29 nucleotides in length and comprises or consists essentially of, or consists of SEQ ID NO: 55.

In some embodiments the aptamer that binds BPA is a polynucleotide consisting essentially of, or consisting of a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOs: 55.

In some embodiments, the aptamer binds progesterone.

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTGCGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 76) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTGCGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 77).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 78) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTGG (SEQ ID NO: 79).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTCGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 80) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGAAGGGATGCCGTTCGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 81).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGACGTAAAACATCTTGGCCCGCTGGTCGGCCCATCTC GTGATAGTAAGAGCAATC (SEQ ID NO: 82) or a fragment thereof. In some embodiments, the fragment comprises a sequence of CCGACGTAAAACATCTTGGCCCGCTGGTCGGCCCATCTCG (SEQ ID NO: 83).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAATTATATTGTCATGATGCCCCCATCGTCCTTCCCAATGCG TGATAGTAAGAGCAATC (SEQ ID NO: 84) or a fragment thereof. In some embodiments, the fragment comprises the sequence of ATTATATTGTCATGATGCCCCCATCGTCCTTCCCAATGCG (SEQ ID NO: 85).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCCCCCCATAGCAGCCAAGCTTTTAACCGACCTGGGATA TTGATAGTAAGAGCAATC (SEQ ID NO: 86) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCCCCCCATAGCAGCCAAGCTTTTAACCGACCTGGGATAT (SEQ ID NO: 87).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATATCCCCTCCATGTTTTGCAAGGATCCCTCTGGATAGGCGC GTGATAGTAAGAGCAATC (SEQ ID NO: 88) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TCCCCTCCATGTTTTGCAAGGATCCCTCTGGATAGGCGCG (SEQ ID NO: 89).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTAGGGGAAAAAGTGAGTGTGGCTG GTGATAGTAAGAGCAATC (SEQ ID NO: 90) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTAGGGGAAAAAGTGAGTGTGGCTGG (SEQ ID NO: 91).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGGAGCCATTTAGTCTACCCGATCCTCAGGATACCGAAG CTGATAGTAAGAGCAATC (SEQ ID NO: 92) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CGGAGCCATTTAGTCTACCCGATCCTCAGGATACCGAAGC (SEQ ID NO: 93).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACTGCAACAGCGGTCCCGGGTGGGTGGTATGCACATCGA CGTGATAGTAAGAGCAATC (SEQ ID NO: 94) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CTGCAACAGCGGTCCCGGGTGGGTGGTATGCACATCGACG (SEQ ID NO: 95).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCTGATGGACTTTAAAACCTTTGACGTTCTTTTGGCTTCG TGATAGTAAGAGCAATC (SEQ ID NO: 96) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCTGATGGACTTTAAAACCTTTGACGTTCTTTTGGCTTCG (SEQ ID NO: 97).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCAGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 98) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCAGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 99).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATATTCGGTGGTGGTAGTCCTCTGTTCGATTGGTGTCGTGGG TTGATAGTAAGAGCAATC (SEQ ID NO: 100) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TTCGGTGGTGGTAGTCCTCTGTTCGATTGGTGTCGTGGGT (SEQ ID NO: 101).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGTCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 102) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTCAGGGTCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 103).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGCTCCCCGAGTTACAAATAAGCGCGGTCCAACGTCTTC GTGATAGTAAGAGCAATC (SEQ ID NO: 104) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGCTCCCCGAGTTACAAATAAGCGCGGTCCAACGTCTTCG (SEQ ID NO: 105).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAATAGCATATCCTAACCGGCCACCCCCATGCGTATCCCTTC TGATAGTAAGAGCAATC (SEQ ID NO: 106) or a fragment thereof. In some embodiments, the fragment comprises the sequence of ATAGCATATCCTAACCGGCCACCCCCATGCGTATCCCTTC (SEQ ID NO: 107).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACGTTCGGCTGTAAACTTGAGTCATGAGCCCGCTTCCCCG GTGATAGTAAGAGCAATC (SEQ ID NO: 108) or a fragment thereof. In some embodiments, the fragment comprises a sequence of CGTTCGGCTGTAAACTTGAGTCATGAGCCCGCTTCCCCGG (SEQ ID NO: 109).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 110) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 111).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACCCATTCCGCTCTTAGGCGGTCTCCCATTTATCTCCGTGG TGATAGTAAGAGCAATC (SEQ ID NO: 140) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CCCATTCCGCTCTTAGGCGGTCTCCCATTTATCTCCGTGG (SEQ ID NO: 141).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGATTGGGAATTGCACCTTTACCTTGATGGTCGTTGTGTTG TGATAGTAAGAGCAATC (SEQ ID NO: 142) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GATTGGGAATTGCACCTTTACCTTGATGGTCGTTGTGTTG (SEQ ID NO: 143).

In some embodiments, the aptamer that binds progesterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGG GGTGATAGTAAGAGCAATC (SEQ ID NO: 146) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGGGG (SEQ ID NO: 147).

In some embodiments, the aptamer that binds progesterone consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTGCGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 76) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTGCGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 77).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 78) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGGGTGTGGTGTACGGCGTTGATGTTTTGGTGGACCGTGG (SEQ ID NO: 79).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGAAGGGATGCCGTTCGGGCCCAAGTTCGGCATAGTGTG GTGATAGTAAGAGCAATC (SEQ ID NO: 80) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGAAGGGATGCCGTTCGGGCCCAAGTTCGGCATAGTGTGG (SEQ ID NO: 81).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGACGTAAAACATCTTGGCCCGCTGGTCGGCCCATCTC GTGATAGTAAGAGCAATC (SEQ ID NO: 82) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of a sequence of CCGACGTAAAACATCTTGGCCCGCTGGTCGGCCCATCTCG (SEQ ID NO: 83).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAATTATATTGTCATGATGCCCCCATCGTCCTTCCCAATGCG TGATAGTAAGAGCAATC (SEQ ID NO: 84) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of ATTATATTGTCATGATGCCCCCATCGTCCTTCCCAATGCG (SEQ ID NO: 85).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCCCCCCATAGCAGCCAAGCTTTTAACCGACCTGGGATA TTGATAGTAAGAGCAATC (SEQ ID NO: 86) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCCCCCCATAGCAGCCAAGCTTTTAACCGACCTGGGATAT (SEQ ID NO: 87).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATCCCCTCCATGTTTTGCAAGGATCCCTCTGGATAGGCGC GTGATAGTAAGAGCAATC (SEQ ID NO: 88) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TCCCCTCCATGTTTTGCAAGGATCCCTCTGGATAGGCGCG (SEQ ID NO: 89).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTAGGGGAAAAAGTGAGTGTGGCTG GTGATAGTAAGAGCAATC (SEQ ID NO: 90) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTAGGGGAAAAAGTGAGTGTGGCTGG (SEQ ID NO: 91).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGGAGCCATTTAGTCTACCCGATCCTCAGGATACCGAAG CTGATAGTAAGAGCAATC (SEQ ID NO: 92) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CGGAGCCATTTAGTCTACCCGATCCTCAGGATACCGAAGC (SEQ ID NO: 93).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACTGCAACAGCGGTCCCGGGTGGGTGGTATGCACATCGA CGTGATAGTAAGAGCAATC (SEQ ID NO: 94) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CTGCAACAGCGGTCCCGGGTGGGTGGTATGCACATCGACG (SEQ ID NO: 95).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCTGATGGACTTTAAAACCTTTGACGTTCTTTTGGCTTCG TGATAGTAAGAGCAATC (SEQ ID NO: 96) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCTGATGGACTTTAAAACCTTTGACGTTCTTTTGGCTTCG (SEQ ID NO: 97).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCAGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 98) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCAGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 99).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATTCGGTGGTGGTAGTCCTCTGTTCGATTGGTGTCGTGGG TTGATAGTAAGAGCAATC (SEQ ID NO: 100) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TTCGGTGGTGGTAGTCCTCTGTTCGATTGGTGTCGTGGGT (SEQ ID NO: 101).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGTCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 102) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTCAGGGTCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 103).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGCTCCCCGAGTTACAAATAAGCGCGGTCCAACGTCTTC GTGATAGTAAGAGCAATC (SEQ ID NO: 104) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGCTCCCCGAGTTACAAATAAGCGCGGTCCAACGTCTTCG (SEQ ID NO: 105).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAATAGCATATCCTAACCGGCCACCCCCATGCGTATCCCTTC TGATAGTAAGAGCAATC (SEQ ID NO: 106) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of ATAGCATATCCTAACCGGCCACCCCCATGCGTATCCCTTC (SEQ ID NO: 107).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACGTTCGGCTGTAAACTTGAGTCATGAGCCCGCTTCCCCG GTGATAGTAAGAGCAATC (SEQ ID NO: 108) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of a sequence of CGTTCGGCTGTAAACTTGAGTCATGAGCCCGCTTCCCCGG (SEQ ID NO: 109).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCT GGTGATAGTAAGAGCAATC (SEQ ID NO: 110) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CCGTACGGCGGCGGTCAGGGGCCAAAGTGAGTGTGGCTGG (SEQ ID NO: 111).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACCCATTCCGCTCTTAGGCGGTCTCCCATTTATCTCCGTGG TGATAGTAAGAGCAATC (SEQ ID NO: 140) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists the sequence of CCCATTCCGCTCTTAGGCGGTCTCCCATTTATCTCCGTGG (SEQ ID NO: 141).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists the nucleotide sequence of ATACGAGCTTGTTCAATAGATTGGGAATTGCACCTTTACCTTGATGGTCGTTGTGTTG TGATAGTAAGAGCAATC (SEQ ID NO: 142) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GATTGGGAATTGCACCTTTACCTTGATGGTCGTTGTGTTG (SEQ ID NO: 143).

In some embodiments, the aptamer that binds progesterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGG GGTGATAGTAAGAGCAATC (SEQ ID NO: 146) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GGCGAGATGGTGGAGCAGGTATGCCGTTGGCGGGTCGGGG (SEQ ID NO: 147).

In some embodiments, the aptamer binds progesterone; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 76-111, 140-143, 146 and 147; and in some embodiments, selectively binds progesterone. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 76-111, 140-143, 146 and 147.

In some embodiments, the aptamer binds progesterone; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147; and in some embodiments, selectively binds progesterone. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147.

In some embodiments the aptamer that binds progesterone is a polynucleotide of less than 80 nucleotides in length.

In some embodiments the aptamer that binds progesterone is a polynucleotide of about 73 or about 77 nucleotides in length, or of about 74 to 76 nucleotides in length, or of 70 to 80 nucleotides in length and comprises or consists essentially of, or consists of SEQ ID NO: 86 or SEQ ID NO: 102.

In some embodiments, the aptamer binds testosterone.

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAACGCTGTTACAATGGCAATATGACTCTTCCGGAAGGTGTA TGATAGTAAGAGCAATC (SEQ ID NO: 112) or a fragment thereof. In some embodiments, the fragment comprises the sequence of ACGCTGTTACAATGGCAATATGACTCTTCCGGAAGGTGTA (SEQ ID NO: 113).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACATAATGGCGTCCCCCTCAAGCTTGAACGGTACGGGGTG CTGATAGTAAGAGCAATC (SEQ ID NO: 114) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CATAATGGCGTCCCCCTCAAGCTTGAACGGTACGGGGTGC (SEQ ID NO: 115).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAATCCCTATCTTCACTACAGTTAGTGACAGGTGCATTGTCT TGATAGTAAGAGCAATC (SEQ ID NO: 116) or a fragment thereof. In some embodiments, the fragment comprises the sequence of ATCCCTATCTTCACTACAGTTAGTGACAGGTGCATTGTCT (SEQ ID NO: 117).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAACTGTCTCCCGTATCCCCTCCAAACGTCCAGGTTCGTCTT TGATAGTAAGAGCAATC (SEQ ID NO: 118) or a fragment thereof. In some embodiments, the fragment comprises the sequence of ACTGTCTCCCGTATCCCCTCCAAACGTCCAGGTTCGTCTT (SEQ ID NO: 119).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATATTACAACGTTTACTTCATCTTTAAGTCTACATGCGGCTAGT GATAGTAAGAGCAATC (SEQ ID NO: 120) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TTACAACGTTTACTTCATCTTTAAGTCTACATGCGGCTAG (SEQ ID NO: 121).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGCCCTTTACACCATTCATGCCGCTCTTATCGGTAGTCGCG TGATAGTAAGAGCAATC (SEQ ID NO: 122) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GCCCTTTACACCATTCATGCCGCTCTTATCGGTAGTCGCG (SEQ ID NO: 123).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACATATTTTCGTATCCTACAACGTTTAATTGTACGGCGGATT GATAGTAAGAGCAATC (SEQ ID NO: 124) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CATATTTTCGTATCCTACAACGTTTAATTGTACGGCGGAT (SEQ ID NO: 125).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATAGCGTTTAGCGTTCAATTCATCCCGCTATCTGGCTGTATCG TGATAGTAAGAGCAATC (SEQ ID NO: 126) or a fragment thereof. In some embodiments, the fragment comprises the sequence of GCGTTTAGCGTTCAATTCATCCCGCTATCTGGCTGTATCG (SEQ ID NO: 127).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATATGCGCAGAACATTCTCCGTATGAAGGTTTTCGATACGGGA TGATAGTAAGAGCAATC (SEQ ID NO: 128) or a fragment thereof. In some embodiments, the fragment comprises the sequence of TGCGCAGAACATTCTCCGTATGAAGGTTTTCGATACGGGA (SEQ ID NO: 129).

In some embodiments, the aptamer that binds testosterone comprises the nucleotide sequence of ATACGAGCTTGTTCAATACAATGTGCACCGGGAGACCTATTCCGCCCACCAGATCCT ATGATAGTAAGAGCAATC (SEQ ID NO: 144) or a fragment thereof. In some embodiments, the fragment comprises the sequence of CAATGTGCACCGGGAGACCTATTCCGCCCACCAGATCCTA (SEQ ID NO: 145).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAACGCTGTTACAATGGCAATATGACTCTTCCGGAAGGTGTA TGATAGTAAGAGCAATC (SEQ ID NO: 112) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of ACGCTGTTACAATGGCAATATGACTCTTCCGGAAGGTGTA (SEQ ID NO: 113).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACATAATGGCGTCCCCCTCAAGCTTGAACGGTACGGGGTG CTGATAGTAAGAGCAATC (SEQ ID NO: 114) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CATAATGGCGTCCCCCTCAAGCTTGAACGGTACGGGGTGC (SEQ ID NO: 115).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAATCCCTATCTTCACTACAGTTAGTGACAGGTGCATTGTCT TGATAGTAAGAGCAATC (SEQ ID NO: 116) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of ATCCCTATCTTCACTACAGTTAGTGACAGGTGCATTGTCT (SEQ ID NO: 117).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAACTGTCTCCCGTATCCCCTCCAAACGTCCAGGTTCGTCTT TGATAGTAAGAGCAATC (SEQ ID NO: 118) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of ACTGTCTCCCGTATCCCCTCCAAACGTCCAGGTTCGTCTT (SEQ ID NO: 119).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATTACAACGTTTACTTCATCTTTAAGTCTACATGCGGCTAGT GATAGTAAGAGCAATC (SEQ ID NO: 120) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TTACAACGTTTACTTCATCTTTAAGTCTACATGCGGCTAG (SEQ ID NO: 121).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGCCCTTTACACCATTCATGCCGCTCTTATCGGTAGTCGCG TGATAGTAAGAGCAATC (SEQ ID NO: 122) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GCCCTTTACACCATTCATGCCGCTCTTATCGGTAGTCGCG (SEQ ID NO: 123).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACATATTTTCGTATCCTACAACGTTTAATTGTACGGCGGATT GATAGTAAGAGCAATC (SEQ ID NO: 124) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CATATTTTCGTATCCTACAACGTTTAATTGTACGGCGGAT (SEQ ID NO: 125).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATAGCGTTTAGCGTTCAATTCATCCCGCTATCTGGCTGTATCG TGATAGTAAGAGCAATC (SEQ ID NO: 126) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of GCGTTTAGCGTTCAATTCATCCCGCTATCTGGCTGTATCG (SEQ ID NO: 127).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATATGCGCAGAACATTCTCCGTATGAAGGTTTTCGATACGGGA TGATAGTAAGAGCAATC (SEQ ID NO: 128) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of TGCGCAGAACATTCTCCGTATGAAGGTTTTCGATACGGGA (SEQ ID NO: 129).

In some embodiments, the aptamer that binds testosterone consists essentially of, or consists of the nucleotide sequence of ATACGAGCTTGTTCAATACAATGTGCACCGGGAGACCTATTCCGCCCACCAGATCCT ATGATAGTAAGAGCAATC (SEQ ID NO: 144) or a fragment thereof. In some embodiments, the fragment consists essentially of, or consists of the sequence of CAATGTGCACCGGGAGACCTATTCCGCCCACCAGATCCTA (SEQ ID NO: 145).

In some embodiments, the aptamer binds testosterone; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 112-129, 144 and 145; and in some embodiments, selectively binds testosterone. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 112-129, 144, and 145.

In some embodiments, the aptamer binds testosterone; comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145; and in some embodiments, selectively binds testosterone. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145.

In some embodiments the aptamer that binds testosterone is a polynucleotide of less than 80 nucleotides in length.

In some embodiments the aptamer that binds testosterone is a polynucleotide of about 73 or about 77 nucleotides in length, or of about 74 to 76 nucleotides in length, or of 70 to 80 nucleotides in length and comprises or consists essentially of, or consists of SEQ ID NO: 122, SEQ ID NO: 124 or SEQ ID NO: 126.

In some embodiments the aptamer that binds BPA is a polynucleotide consists essentially of, or consists of a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 122, SEQ ID NO: 124 or SEQ ID NO: 126.

A polynucleotide or salt thereof of the present invention can be prepared using any methodology useful for selecting sequences that bind to a specific target. For example, an aptamer of the present invention can be identified using standard methodologies commonly known to those in the art, for example, synthesis by System Evolution of Ligands by Exponential Enrichment (SELEX). SELEX is a method for producing single stranded DNA or RNA molecules that specially bind to a target (Ellington A D & Szostak J W (1990). Nature. 346:818-822 and Blackwell T K & Weintraub H (1990) Science 250:1104-1110). During the SELEX process, a library of randomly generated polynucleotide sequences is exposed to a target substrate. The randomly generated polynucleotide sequences that fail to bind to the target substrate are removed, usually using affinity separation, and the polynucleotide sequences that are bound to the target substrate are replicated and amplified by standard amplification techniques such as polymerase chain reaction (PCR). The amplified sequences can be subject to further SELEX rounds and elution conditions can be varied to identify polynucleotide sequences having the highest binding affinity for the target substrate. Once identified, an aptamer of the present invention can be synthesized readily by a person of skill in the art in accordance with known methods of synthesizing nucleic acids such as direct synthesis or through the use of recombinant DNA vectors.

Polynucleotide-Agent Complex

In some embodiments, a polynucleotide or salt thereof of the present invention (e.g., an aptamer of the present invention) forms a complex with one or more complex-forming agents and provides a polynucleotide-agent complex. A “polynucleotide-agent complex” includes a complex in which its polynucleotide component is in the form of a salt. Where the polynucleotide or salt thereof forms a complex with two or more agents, the agents can be of the same or different type. In some embodiments, the agent is a particle or non-particle. In some embodiments, the agent is a nanoparticle (NP) (e.g., a polymer NP), microparticle, semiconducting particle, quantum dot, radioactive substance (e.g., radioisotope, radionuclide, radiolabel or radiotracer), dye, contrast agent, fluorescent molecule, phosphorescent molecule, bioluminescent molecule, chemiluminescent molecule, chromophore, photoaffinity molecule, colored particle or ligand, enzyme, or enhancing agent (e.g., paramagnetic ion). In some embodiments, the agent comprises a noble metal (e.g., gold, ruthenium, rhodium, palladium, iridium, osmium, silver, or platinum) that is in salt form or has an oxidation state of zero.

Examples of fluorescent and luminescent molecules include, but are not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators,” such as fluorescein, rhodamine, an acridine dye, an Alexa dye, and a cyanine dye.

Examples of enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucoronidase or β-lactamase. Where the polynucleotide or salt thereof forms a complex with an enzyme, the complex can further comprise a chromogen, a fluorogenic label or compound or a luminogenic compound that generates a detectable signal.

Examples of radioactive substances include, but are not limited to, compounds comprising ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ⁹⁹Tc, 99Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra or ²²⁵Ac.

Examples of paramagnetic ions include, but are not limited to, ions of transition and lanthanide metals (e.g., metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

Examples of fluorescent labels or compounds include, but are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red.

In some embodiments, the complex comprises a polynucleotide or salt thereof that is conjugated to the agent. In other embodiments, the complex comprises a polynucleotide or salt thereof that is not conjugated to the agent. In some embodiments, the complex comprises a polynucleotide or salt thereof that is reversibly bound to the agent. In other embodiments, the complex comprises a polynucleotide or salt thereof that is irreversibly bound to the agent.

In some embodiments, the polynucleotide or salt thereof is an aptamer that adsorbs on at least a portion of the outer surface of the agent (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the surface, or at least about 20% to 100%, about 30% to 90% or about 40% to 85%), e.g., where the agent is a particle, such as an NP, a microparticle, or a quantum dot. In some embodiments, the polynucleotide or salt thereof is an aptamer that, when admixed with an NP, reversibly adsorbs on at least a portion of the outer surface of the nanoparticle to form an aptamer-coated NP. In some embodiments, the aptamer-coated particle is stable in solutions containing salt (e.g., the aptamer remains complexed with the particle in salt solutions), e.g., in salt (e.g., NaCl, MgCl₂, KCl, or CaCl₂) concentrations of about 5 mM to about 50 mM, of about 10 mM to about 40 mM, of about 20 mM to about 35 mM or of about 30 mM ionic strength. In some embodiments, there is an increase in the ζ-potential value of the aptamer-coated particle when compared to an uncoated particle (increasing the ζ-potential value means that the value becomes more negative, which is readily understood and appreciated by those of skill in the art). In some embodiments, there is an increase in the ζ-potential value of the particles, for example from about −23.5 mV (±0.28) to about −40.2 mV (±0.9), which confirms adsorption of the aptamer on the outer surface of the particles. In some embodiments, the aptamer-coated particles dispersible in solution and, in some embodiments, are stable (e.g., the aptamer remains complexed with the particle in salt solutions, e.g., in a NaCl, MgCl₂, KCl, or CaCl₂solution) in salt concentrations, e.g., in salt (e.g., NaCl, MgCl₂, KCl, or CaCl₂) concentrations of about 5 mM to about 50 mM, of about 10 mM to about 40 mM, of about 20 mM to about 35 mM or of about 30 mM ionic strength. In some embodiments, the electrostatic repulsion between an aptamer's negatively charged phosphate backbone and a particle's (e.g., an NP's) citrate surface is balanced by the attractive interaction of nitrogen bases, with the surface of the particle (Brown, K. A., et al, 2008, J. Phys. Chem. C, 112, 7517-7521).

In some embodiments, where the agent is a particle, e.g., an NP, the ratio of number of particles to aptamer ranges from about 1:100 to about 1:1; in another embodiment, from about 1:50 to about 1:1; and in another embodiment, from about 1:10 to about 1:3.

Methods for Making a Polynucleotide-Agent Complex

Also provided herein are methods for producing a polynucleotide-agent complex. In one embodiment, the method comprises contacting a polynucleotide or salt thereof with an agent. In one embodiment, the agent is a gold nanoparticle, and the method comprises adsorption of an aptamer onto the gold NP, such as described in Example 6.

Detection Methods

Also provided herein are methods for detecting a target molecule in a sample, comprising: contacting the sample with a polynucleotide or salt thereof and measuring a change in a property of the sample, wherein the change in property indicates the presence of the target molecule. In some embodiments, the target molecule is a small molecule. In some embodiments, the polynucleotide or salt thereof is an aptamer.

In some embodiments, the methods further comprise incubating the sample after contacting and before measuring. In some embodiments, the incubation time is from about 1 second to about 24 hours, about 6 hours to about 18 hours, about 8 hours to about 12 hours, about 1 second to about 12 hours, about 30 seconds to about 7 hours, about 1 minute to about 6 hours, about 1 minute to about 5 hours, about 1 minute to about 4 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 45 minutes to about 4 hours, about 1 hour to about 4 hours, about 2 hours to about 4 hours, about 1 minute, about 30 minutes, about 45 minutes, about an hour, about 90 minutes, about 2 hours, about 180 minutes, about 3 hours, about 270 minutes, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, or about 24 hours.

In some embodiments, the incubation is performed at room temperature, at about 15° C. to about 30° C., about 18° C. to about 27° C., or about 25° C. In some embodiments, incubation is performed at about 2° C. to about 10° C., about 2° C. to about 5° C., or about 4° C.

In some embodiments, incubation is performed at about 15° C. to about 30° C., about 18° C. to about 27° C., or about 25° C. for about 1 second to about 12 hours, about 30 seconds to about 7 hours, about 1 minute to about 6 hours, about 1 minute to about 5 hours, about 1 minute to about 4 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 45 minutes to about 4 hours, about 1 hour to about 4 hours, about 2 hours to about 4 hours, about 1 minute, about 30 minutes, about 45 minutes, about an hour, about 90 minutes, about 2 hours, about 180 minutes, about 3 hours, about 270 minutes, or about 4 hours. In some embodiments, incubation is performed at about 2° C. to about 10° C., about 2° C. to about 5° C., or about 4° C. for about 6 hours to about 18 hours, about 8 hours to about 12 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, or about 24 hours.

In some embodiments, the change in property is a change in colour. In some embodiments, the change in colour is colorimetric. A colorimetric change can be identified or quantified using techniques such as UV-Visible spectroscopy. In some embodiments, the colorimetric change is visible to the naked eye.

Also provided herein are methods for detecting a target molecule in a sample, comprising: contacting the sample with a polynucleotide-agent complex and measuring a change in a property of the sample, wherein a change in property indicates the presence of the target molecule. In some embodiments, the target molecule is a small molecule. In some embodiments, the agent of the complex is a NP. In some embodiments, the methods further comprise incubating the sample after contacting and before measuring a change in a property of the sample.

In some embodiments, the incubation time is from about 1 second to about 24 hours, about 6 hours to about 18 hours, about 8 hours to about 12 hours, about 1 second to about 12 hours, about 30 seconds to about 7 hours, about 1 minute to about 6 hours, about 1 minute to about 5 hours, about 1 minute to about 4 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 45 minutes to about 4 hours, about 1 hour to about 4 hours, about 2 hours to about 4 hours, about 1 minute, about 30 minutes, about 45 minutes, about an hour, about 90 minutes, about 2 hours, about 180 minutes, about 3 hours, about 270 minutes, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, or about 24 hours.

Also provided herein are methods for detecting a target molecule in a sample, comprising: contacting a polynucleotide or salt thereof with an agent to provide a polynucleotide agent complex, contacting the sample with the polynucleotide-agent complex and measuring a change in a property of the sample, wherein a change in property indicates the presence of the target molecule. In some embodiments, the target molecule is a small molecule. In some embodiments, the agent is an NP. In some embodiments, the methods further comprise incubating the sample after contacting the sample with the polynucleotide-agent complex and before measuring a change in a property of the sample.

In some embodiments, measuring a change in the property of a sample is performed using a colorimetric aggregation assay (FIG. 4). In some embodiments, the agent, apart from or as a component of the polynucleotide-agent complex, exhibits a change in its optical absorption when aggregation with, for example a target molecule, is induced. Where the agent is an NP that comprises a noble metal, the NP's surface plasmon resonance is very sensitive to aggregation, resulting in a strong visible signature. Other types of nanoparticles or microparticles can also provide a visible signature upon aggregation, including semiconducting particles, quantum dots or polymer NPs. In some embodiments, where the polynucleotide or salt component of a polynucleotide-agent complex is an aptamer that binds to a target molecule, a conformational change in the aptamer is induced, and the aptamer's affinity to the agent is reduced. The dispersion becomes destabilised by salt, e.g., the particles are dispersed in suspension and with the introduction of salt, the particles clump together and aggregate (e.g., McKeague, et al., 2012, J. Nucleic Acids, 748913 and Stoltenburg, R., et al., 2007, Biomol. Eng., 24, 381-403) and the aptamer bound to the target molecule dissociates from the agent's surface. At a particular ionic strength, the presence of the target molecule is indicated by a colour change that occurs when an agent whose aptamer has dissociated from the agent's surface aggregates with the target molecule, e.g., the ionic strength can be optimised by titrating various concentrations of salt (e.g., NaCl, MgCl₂, KCl, CaCl₂) with a constant aptamer, and the absorbance at e.g., 523 nm (typically a low value) indicates that the salt tolerance has been met and complete aggregation of the AuNP occurs.

The degree of aggregation of an agent to a target molecule can be quantified using UV-visible absorption and can be evident to the naked eye. The aggregation of an agent to the target molecule can be indicated by colour observed. The colour change can depend on the concentration of the target molecule in the sample. In some embodiments, the methods further comprise quantifying the amount of target molecule in the sample or determining the concentration of the target molecule in the sample. In some embodiments, the agent is an NP. In some embodiments, the NP comprises a noble metal. In some embodiments, the target molecule is a small molecule.

In other embodiments, the change in property is a change in size, surface potential, or mobility of the agent (e.g., as disclosed in WO2014/123430, which is incorporated by reference in its entirety). In some embodiments, the methods comprise measuring a change in size of the agent, wherein a size change indicates the presence of the target molecule. In other embodiments, the methods comprise measuring a change in surface potential of the agent, wherein a surface potential change indicates the presence of the target molecule. In other embodiments, the methods comprise measuring a change in mobility of the agent, wherein a wherein a mobility change indicates the presence of the target molecule. In some embodiments, the change in size, surface potential or mobility is measured using Resistive Pulse Sensing (RPS), dynamic light scattering (DLS), circular dichroism (CD) or a combination thereof.

In some embodiments, the methods comprise contacting a sample with a polynucleotide or salt thereof that is an aptamer. In some embodiments, the methods comprise contacting a sample with an aptamer that binds, in some embodiments selectively, E2 and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26.

In some embodiments, the methods comprise contacting a sample with an aptamer that binds androstenedione, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133.

In some embodiments, the methods comprise contacting a sample with an aptamer that binds BPA, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139.

In some embodiments, the methods comprise contacting a sample with an aptamer that binds progesterone, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147.

In some embodiments, the methods comprise contacting a sample with an aptamer that binds testosterone, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145.

In other embodiments, the methods comprise contacting a sample with a polynucleotide-agent complex, wherein the polynucleotide or polynucleotide salt component of the complex is an aptamer that binds E2, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 3, 1-2, 4-15 or 20-26; or SEQ ID NOs: 3, 1-2, 4-15, 17, or 19-26.

In other embodiments, the methods comprise contacting a sample with a polynucleotide-agent complex, wherein the polynucleotide or polynucleotide salt component of the complex is an aptamer that binds androstenedione, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 29, 27, 28, 30-32 and 130-133.

In other embodiments, the methods comprise contacting a sample with a polynucleotide-agent complex, wherein the polynucleotide or polynucleotide salt component of the complex is an aptamer that binds BPA, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 53, 55, 56, 33-52, 54, 57-75 and 134-139.

In other embodiments, the methods comprise contacting a sample with a polynucleotide-agent complex, wherein the polynucleotide or polynucleotide salt component of the complex is an aptamer that binds progesterone, in some embodiments selectively, and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 86, 102, 76-85, 87-101, 103-111, 140-143, 146 and 147.

In other embodiments, the methods comprise contacting a sample with a polynucleotide-agent complex, wherein the polynucleotide or polynucleotide salt component of the complex is an aptamer that selectively binds testosterone and comprises a sequence that has at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145. In some embodiments, the aptamer consists essentially of, or consists of a sequence selected from SEQ ID NOs: 122, 124, 126, 112-121, 123, 125, 127-129, 144, and 145.

In some embodiments, the methods comprise detecting a small molecule in a sample, wherein the small molecule is a hormone mimic, hormone, naturally occurring phytoestrogen, narcotic and metabolites thereof, and organohalides. In some embodiments, the small molecule is an endocrine disrupting compound, a steroidal sex hormone, metabolites or synthetic variants thereof. In some embodiments, the small molecule belongs to the estrogenic family of compounds, e.g., estrone (E1), estradiaol (also known as 17β-estradiol or E2), estriol (E3), estetrol (E4), xenoestrogens, phytoestrogens, or mycoestrogens, such as polychlorinated biphenols (also referred to as polychlorobiphenyl or PCBs), bisphenol-A (BPA), and phthalates. In some embodiments, the small molecule is daidzein, formononetin, genistein, bichanin A, coumestrol, 4′-methoxycoumestrol, repensol, trifoliol, or derivatives there of (e.g., 17α-ethynylestradiol).

In some embodiments, the small molecule is androstenedione, androstenediol, testosterone, dihydrotestosterone, pregnenolone, progesterone (P4), 17α-hydroxyprogesterone, Bis (4-hydroxyphenyl) methane (also referred to as bisphenol-F or BPF), cholesterol, adenosine, triclosan, a synthetic steroid such as diethylstilboestrol (DES), cocaine, heroin, tetrahydrocannabinol (THC), or any derivative or metabolite thereof. In other embodiments, the small molecule is an isoflavone, lignan, coumestan, organohalide (e.g., organochlorine), polychlorinated organic compound, alkylphenol, or alkylphenol ethoxylate,

In some embodiments, the small molecule is a hormone or a marker of a condition of disease in a body. For example, in some embodiments, the method comprises detecting a hormone and/or metabolite that establishes a status in an animal, such as the fertility status of a subject. In some embodiments, the small molecule is a known marker of disease, for example a cancer gene. In some embodiments, the method comprises detecting a known marker of disease, e.g., cancer marker, a molecule associated with infection, or a metabolite associated with a particular condition.

In some embodiments, the sample isolated or collected from an environmental or biological source, e.g., located ex vivo. The sample can be of biological origin, isolated from an animal or be collected from the environment. Sources of samples may include without limitation, for example, soils, waterways, tissue, blood, serum, urine, saliva, faeces, hair and wool. The sample may be an environmental sample, for example a water sample, soil sample, or even a plant sample. Alternatively, the sample may be from an animal, for example a tissue sample, a hair or wool sample, a urine sample, a blood sample, a serum sample, a saliva sample or a fecal sample.

In some embodiments, the methods can detect a target molecule whose concentration in a sample ranges from about 1 pM to about 100 μM. In some embodiments, the methods can detects a target molecule whose concentration in the sample ranges from about 200 pM to about 400 nM. In some embodiments, the target molecule is a small molecule.

The examples described herein are for purposes of illustrating embodiments of the invention. Other embodiments, methods, and types of analyses are within the capabilities of persons of ordinary skill in the art and need not be described in detail herein. Other embodiments within the scope are considered to be part of this invention.

EXAMPLES

The aptamers were dissolved in Milli-Q water and stored at −5 ° C. prior to use. Milli-Q water was used in all experiments (unless stated), and all other chemicals are of analytical grades purchased from standard chemical suppliers.

Example 1
Dot Blot Assay for Aptamers that Bind 17β-estradiol (E2)

Nitrocellulose membranes were immersed in BWB (Binding and Washing Buffer: 2 mM TRIS-HCl, pH 7.5 containing 10 mM NaCl, 0.5 mM KCl, 0.2 mM MgCl₂, 0.1 mM CaCl₂, 5% v/v EtOH; 1% v/v IGEPAL® ((Octylphenoxy)polyethoxyethanol) non-ionic surfactant) for 10 minutes before drying. 17β-estradiol (E2), progesterone (P4), androstenedione (Andro), bisphenol-A (BPA), testosterone (T), and triclosan were each dissolved in ethanol (e.g., stock solution of 50 mg per millilitre of ethanol of E2, P4, Andro, BPA, T, and triclosan), spotted onto the membranes and then immersed in blotto blocking solution for 2 hours at room temperature.

The biotin-labeled aptamers (having sequences as referred to in Table 1) in 3.16 μM in BWB were incubated individually with the membranes containing different amounts (600 nmol, 300 nmol, 150 nmol, 75 nmol, 37.5 nmol, and 17.25 nmol) of E2, P4, T, Andro, BPA and triclosan, overnight at 4° C. on a rocking incubator (15 oscillations/min). Non-specifically bound aptamers were removed by rinsing the membranes three times with BWB (10 minutes on a rocking incubator 40 oscillations/min).

The bound substrate-aptamer conjugates were visualized using chemoluminescence by incubation with streptavidin-peroxidase conjugate (167 ng/mL in BWB). The incubation was left for 2 hours at room temperature. The washing of the aptamer was repeated using BWB to remove non-specifically bound streptavidin-peroxidase conjugate from the surface of the membranes. Chemoluminescence reaction solution for visualization (0.1 M Tris-HCl pH 8.5, 25 μM luminal, 396 μM p-coumaric acid, and 0.01% of hydrogen peroxide in deionized H₂O), was prepared immediately before use. Membranes were incubated in the chemoluminescence solution for 1 minute at room temperature in a darkroom and then immediately placed on a clear plastic backing, wrapped using clear plastic wrap and placed in an x-ray cassette. X-ray films were carefully placed on top of the membranes and exposed for 15 minutes. The films were developed by soaking twice in GBX developing solution for 3 minutes and rinsed with deionized H₂O between soaking.

The results of the E2 aptamer dot blot assay (average of 3 assays) are presented in Table 1, in which “Y” indicates detection of the aptamer bound to the immobilized small molecule target (i.e., E2, P4, T, Andro, BPA or triclosan), and “N” indicates no detection the aptamer bound to the immobilized small molecule target.

TABLE 1

SEQ
Amount of E2 (moles)

ID NO.
600
300
150
75
37.5
17.25
P4
T
Andro
BPA
Triclosan

1
Y
Y
Y
N
N
N
N
N
N
N
N

3
—
—
—
—
—
—
N
N
N
N
N

4
Y
Y
N
N
N
N
N
N
N
N
N

6
Y
Y
N
N
N
N
—
—
—
—
—

8
—
—
—
—
—
—
N
N
N
N
N

9
N
Y
Y
N
N
N
N
N
N
N
N

11
Y
Y
N
N
N
N
N
N
N
N
N

13
N
Y
Y
N
N
N
N
N
N
N
N

15
—
—
—
—
—
—
N
N
N
N
N

16
Y
Y
Y
Y
Y
N
N
N
N
N
N

19
—
Y
Y
Y
Y
Y
N
N
N
N
N

20
N
Y
Y
Y
N
N
N
N
N
N
N

22
—
—
—
—
—
—
—
—
—
—
—

23
N
Y
Y
Y
N
N
N
N
N
N
N

25
N
Y
Y
Y
Y
N
N
N
N
N
N

Example 2
Dot Blot Assay for Aptamers that Bind Androstenedione

Nitrocellulose membranes were immersed in BWB for 10 minutes before drying. Androstenedione (Andro), 17β-estradiol (E2), progesterone (P4), and testosterone (T) were each dissolved in ethanol (e.g., stock solution of 50 mg per millilitre of ethanol of Andro, E2, P4, and T), spotted onto the membranes and then immersed in blotto blocking solution for 2 hours at room temperature.

The biotin-labeled aptamers (having sequences as referred to in Table 2) (3.16 μM in BWB) were incubated individually with the membranes containing different amounts (600 nmol, 300 nmol, 150 nmol, 75 nmol, 37.5 nmol, and 17.25 nmol) of Andro, E2, P4, and T overnight at 4° C. on a rocking incubator (15 oscillations/min). Non-specifically bound aptamers were removed by rinsing the membranes three times with BWB (10 minutes on a rocking incubator 40 oscillations/min).

The results of the Andro aptamer dot blot assay (three assays for each aptamer) are presented in Table 2, in which “Y” indicates detection of the aptamer bound to the immobilized small molecule target (i.e., Andro, E2, P4, or T) in at least two of the three assays, and “N” indicates no detection the aptamer bound to the immobilized small molecule target in at least two of the three assays.

TABLE 2

SEQ
Amount of Andro (nmoles)

ID NO.
600
300
150
75
37.5
17.25
E2
P4
T

27
Y
—
Y
N
N
N
N
N
N

27
Y
Y
N
Y
N
N
N
N
N

27
Y
Y
N
Y
N
N
N
N
N

130
Y
Y
N
Y
N
N
N
N
N

27
Y
Y
N
Y
N
N
N
N
N

27
N
Y
N
Y
N
N
N
N
N

27
Y
Y
N
Y
N
N
N
N
N

29
Y
Y
N
Y
N
N
N
N
N

31
Y
Y
N
N
N
N
N
N
N

132
Y
Y
N
Y
N
N
N
N
N

Example 3
Dot Blot Assay for Aptamers that Bind Bisphenol-A

Nitrocellulose membranes were immersed in BWB for 10 minutes before drying. Bisphenol-A (BPA), 17β-estradiol (E2), Bisphenol P (BPP), and Bis(4-hydroxyphenyl methane) (BPF), were each dissolved in ethanol (e.g., stock solution of 50 mg per millilitre of ethanol of BPA, BPP, and BPF), spotted onto the membranes and then immersed in blotto blocking solution for 2 hours at room temperature.

The biotin-labeled aptamers (having sequences as referred to in Table 3) (3.16 μM in BWB) were incubated individually with the membranes containing different amounts (600 nmol, 300 nmol, 150 nmol, 75 nmol, 37.5 nmol, and 17.25 nmol) of BPA, E2, BPP, and BPF, overnight at 4° C. on a rocking incubator (15 oscillations/min). Non-specifically bound aptamers were removed by rinsing the membranes three times with BWB (10 minutes on a rocking incubator 40 oscillations/min).

The results of the BPA aptamer dot blot assay (three assays for each aptamer) are presented in Table 3, in which “Y” indicates detection of the aptamer bound to the immobilized small molecule target (i.e., BPA, E2, BPP or BPF) in at least two of the three assays; “N” indicates no detection the aptamer bound to the immobilized molecule in at least two of the three assays; and “300” and “600” indicates that the aptamer bound to the immobilized small molecule target when the target was immobilized at an amount of 300 nmoles and 600 nmoles, respectively.

TABLE 3

SEQ
Amount of BPA (nmoles)
E2

BPF

ID NO.
600
300
150
75
37.5
17.25
(nmoles)
BPP
(nmoles)

33
Y
Y
N
N
N
N
600 &
N
N

300

35
—
—
—
—
—
—
—
—
—

36
N
Y
Y
N
N
N
600
N
300

38
N
Y
Y
N
N
N
N
N
N

40
N
Y
Y
Y
N
N
N
N
N

42
Y
Y
Y
Y
Y
N
N
N
N

43
N
N
Y
N
N
N
N
N
N

45
N
N
Y
Y
Y
N
N
N
N

47
N
Y
Y
Y
N
N
N
N
N

49
N
N
Y
Y
Y
N
N
N
N

51
N
Y
Y
Y
Y
N
N
N
600

53
N
N
Y
Y
Y
N
N
N
N

55
Y
Y
Y
Y
Y
N
N
N
N

56
Y
Y
Y
N
N
N
N
N
N

56
Y
Y
Y
Y
Y
N
N
N
N

56
Y
Y
Y
N
N
N
N
Y
N

62
Y
Y
Y
Y
Y
N
N
N
N

58
Y
Y
Y
N
N
N
N
N
Y

56
Y
Y
Y
N
Y
N
N
N
N

56
N
Y
Y
Y
N
N
N
N
N

60
Y
N
Y
Y
Y
N
N
N
N

56
N
Y
Y
Y
Y
N
N
N
N

62
Y
Y
Y
Y
Y
N
N
N
N

64
Y
Y
Y
Y
N
N
N
N
N

56
N
Y
Y
Y
Y
N
N
N
N

66
N
Y
Y
N
N
N
N
N
N

68
Y
Y
Y
Y
Y
N
N
N
N

134
Y
Y
Y
Y
N
N
N
N
N

136
Y
Y
Y
Y
Y
N
N
N
N

138
Y
Y
Y
Y
N
N
N
N
N

70
Y
Y
Y
Y
Y
N
N
N
N

72
N
Y
Y
Y
Y
N
N
N
N

74
Y
Y
Y
Y
Y
N
N
N
N

Example 4
Dot Blot Assay for Aptamers that Bind Progesterone

Nitrocellulose membranes were immersed in BWB for 10 minutes before drying. Progesterone (P4), 17β-estradiol (E2), androstenedione (Andro), bisphenol-A (BPA), testosterone (T) and triclosan were each dissolved in ethanol (e.g., stock solution of 50 mg per millilitre of ethanol of E2, P4, Andro, BPA, T, and triclosan), spotted onto the membranes and then immersed in blotto blocking solution for 2 hours at room temperature.

The biotin-labeled aptamers (having sequences as referred to in Table 4) (3.16 μM in BWB) with different sequences were incubated individually with the membranes containing different amounts (600 nmol, 300 nmol, 150 nmol, 75 nmol, 37.5 nmol, and 17.25 nmol) of P4, E2, T, Andro, BPA and triclosan, overnight at 4 ° C. on a rocking incubator (15 oscillations/min). Non-specifically bound aptamers were removed by rinsing the membranes three times with BWB (10 minutes on a rocking incubator 40 oscillations/min).

The results of the P4 aptamer dot blot assay (three assays per aptamer) are presented in Table 4, in which “Y” indicates detection of the aptamer bound to the immobilized small molecule target (i.e., P4, E2, T, Andro, BPA, or triclosan) in at least two of the three assays; “N” indicates no detection the aptamer bound to the immobilized small molecule target in at least two of the three assays; and “NA” indicates data not available.

TABLE 4

SEQ
Amount of P4 (nmoles)

ID NO.
600
300
150
75
37.5
17.25
E2
T
Andro
BPA
Triclosan

76
Y
Y
Y
Y
N
N
N
Y
Y
N
N

78
Y
Y
Y
N
N
N
Y
Y
N
N
N

78
N
Y
Y
Y
N
N
Y
N
N
N
N

80
N
Y
Y
Y
N
N
N
Y
N
N
N

82
Y
Y
Y
Y
N
N
N
N
N
N
N

84
N
Y
Y
N
Y
N
N
Y
N
N
N

86
Y
Y
Y
Y
Y
N
N
N
N
N
N

88
Y
Y
Y
Y
Y
N
N
N
N
N
N

90
Y
Y
Y
Y
Y
N
N
N
N
N
N

140
N
Y
Y
Y
Y
N
N
N
N
N
N

92
N
Y
Y
Y
N
N
N
Y
N
N
N

94
N
Y
Y
Y
N
N
N
N
N
N
N

96
Y
Y
Y
N
N
N
N
Y
N
N
N

146
N
Y
Y
Y
N
N
N
N
N
N
N

98
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

78
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

110
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

100
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

102
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

104
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

106
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

108
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

142
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

110
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

Example 5
Dot Blot Assay for Aptamers that Bind Testosterone

Nitrocellulose membranes were immersed in BWB for 10 minutes before drying. Testosterone (T), 17β-estradiol (E2), progesterone (P4), androstenedione (Andro), bisphenol-A (BPA) and triclosan were each dissolved in ethanol (e.g., stock solution of 50 mg per millilitre of ethanol of E2, P4, Andro, BPA, T, and triclosan), spotted onto the membranes and then immersed in blotto blocking solution for 2 hours at room temperature.

The biotin-labeled aptamers (having sequences as referred to in Table 5) (3.16 μM in BWB) were incubated individually with the membranes containing different amounts (600 nmol, 300 nmol, 150 nmol, 75 nmol, 37.5 nmol, and 17.25 nmol) of P4, E2, T, Andro, BPA and triclosan, overnight at 4° C. on a rocking incubator (15 oscillations/min). Non-specifically bound aptamers were removed by rinsing the membranes three times with BWB (10 minutes on a rocking incubator 40 oscillations/min).

The results of the T aptamer dot blot assay (three assays per aptamer) are presented in Table 5 (average of 3 assays for each aptamer), in which “Y” indicates detection of the aptamer bound to the immobilized molecule (i.e., T, P4, E2, Andro, BPA, or triclosan) in at least two of the three assays; “N” indicates no detection the aptamer bound to the immobilized molecule in at least two of the three assays; and “NA” indicates data not available.

TABLE 5

SEQ
Amount of P4 (nmoles)

ID NO.
600
300
150
75
37.5
17.25
E2
T
Andro
BPA
Triclosan

144
N
Y
Y
Y
N
N
N
N
N
N
N

112
Y
Y
Y
N
Y
N
N
N
N
N
N

114
Y
Y
Y
N
Y
N
N
N
N
N
N

116
Y
Y
Y
Y
NA
N
N
N
N
N
N

118
N
Y
Y
Y
Y
N
N
N
N
N
N

120
Y
Y
N
Y
Y
N
N
N
N
N
N

122
Y
Y
Y
N
Y
N
N
N
N
N
N

124
N
Y
Y
Y
Y
N
N
N
N
N
N

126
Y
Y
Y
Y
Y
N
N
N
N
N
N

128
N
Y
Y
Y
Y
N
N
N
N
N
N

Example 6
Gold Nanoparticle Binding Assay

Gold Nanoparticle (AuNP) Synthesis and Adsorption of Aptamer onto AuNPs

AuNPs are synthesised using the traditional citrate reduction of chloroauric acid (HAuCl₄). Briefly, 10 nm diameter AuNPs are synthesised by prompt addition of sodium citrate to a boiling solution of 0.1% chloroauric acid with constant stirring. The reaction is monitored by observing the colour change from transparent to deep blue followed by red wine. Boiling of the solution is maintained for 5 further minutes before cooling to room temperature. The size of the AuNPs is determined by undertaking transmission electron microscopy (TEM) and the concentration of the generated AuNPs is calculated using the UV-visible spectroscopy measurements at 523 nm and the extension coefficient of 2.7×10⁸M⁻¹cm⁻¹at 523 nm.

Aptamer Co-Incubation

The AuNPs solution is centrifuged for 15 minutes at 12,500 rpm to remove excess citrate ions and subsequently re-suspended in Milli-Q water to achieve a final concentration of 14 nM. Immediately, various titrations of the aptamer (nmoles from 0.1-0.4) are added to 3 mL of AuNPs and periodically vortexed to allow for the adsorption of aptamers onto the AuNP surface. Maximum adsorption is accomplished from 15-60 minutes.

Preparation of Target Molecules

Appropriate volume of 100% analytical grade EtOH is added to the distilled water to achieve a final working solution with 5% (v/v) EtOH-distilled water solution. Appropriate volumes of the target molecule stock solution (prepared in 100% EtOH) are diluted using the 5% EtOH-dH₂O.

Detection of Small Molecules Using AuNPs-Aptamer Sensor

The sensing of small molecules is carried out in three independent experiments. Briefly, appropriate concentrations of the prepared target molecule working solutions are added to 100 μL of AuNP-aptamer solution and incubated for 10 minutes at room temperature. Subsequently, a volume of 0.5 M NaCl solution is added to induce the aggregation of AuNPs which are no longer protected by the adsorbed aptamer molecules. The microcentrifuge tubes containing the solutions are immediately vortexed for 10 sec before analysing for visible colour change and aggregation of AuNPs. The sensing is also confirmed by UV-visible spectrophotometer analyses of the solution (100-800 nm wavelength). For experimental control, 20 μL of 5% (v/v) EtOH-natural water is added to 100 μL of AuNP-aptamer complex and treated exactly as the samples.

The Kd of particular AuNPs-aptamer sensors contain the following aptamers: SEQ ID NOs: 3, 27, 29, 53, 55, 56, 86, 102, 122, 124 and 126 were determined using the protocols above.

The following table summarizes the binding constants determined in the various experiments.

Binding affinity

Target
Aptamer
(KD) in nM

Estradiol
SEQ ID NO: 3
3 nM

Androstenedione
SEQ ID NO: 27
359 nM

BPA
SEQ ID NO: 53
13 nM

BPA
SEQ ID NO: 55
6 nM

Progesterone
SEQ ID NO: 86
72.6 nM

Progesterone
SEQ ID NO: 102
1.3 μM

Testosterone
SEQ ID NO: 122
21.7 nM

Example 7
Aptamer Binding Assay Using Fluorescence Methodology
Gel Preparation

The conjugated target molecule gels (i.e., target conjugated to molecule gels) and unconjugated control gels are washed using 1×BWB and filter dried. The dry gel is then collected and weighed before addition of distilled water to make a 50 mg/mL solution.

Preparation of HEX-Labelled Aptamers

A serial titration of hexachloro-fluorescein (HEX)-labelled aptamer using 1×BWB containing 0.1% IGEPAL® is prepared. The samples are vortexed and spun shortly, then mixed and spun down. The aptamers are then incubated for at least 1 hour at 4° C. before each assay.

Blocking of Gel

The gels are washed by taking 20 μl (50 mg/ml) of unconjugated and conjugated beads that are individually resuspended in 200 μl 1×BWB with 0.1% IGEPAL®. The gels are then centrifuged for 2 min at a speed of 13 rpm, then 180 μl of solution is carefully pipetted out of each bead suspension.

The beads are blocked by adding 90 μl of 1×BWB with 0.1% IGEPAL® and 10 μl yeast tRNA (10 mg/ml) into each bead suspension, mixed, spun down, and then incubated for 60 minutes at room temperature with gentle shaking.

The beads are then centrifuged for 2 min at a speed of 13 rpm after blocking, then 100 μl of solution is carefully pipetted out of each bead suspension.

Co-incubation

Seventy-five μl of aptamer binding solution is added into each bead suspension and the beads are incubated at room temperature for 2 hours with gentle shaking.

Gel Washing

The beads are centrifuged for 2 min at a speed of 13 rpm, then 75 μl of solution is carefully pipetted out of each bead suspension. Each bead suspension is washed three times with 200 μl of 1×BWB 0.1% IGEPAL®, in which approximately 30 μl of solution is left in the final wash.

Each bead suspension is visualized under a fluorescent microscope. Images are captured under Bright Light (14.9 ms exposure), UV, blue and green light excitation with fixed parameters and the exposure time is determined. The captured images are analysed using IGOR pro 6 (WaveMetrics, Inc., Portland, Oreg.).

Example 8
General Procedure for K_DMeasurement

The procedure for estimating the K_Dof an aptamer is based on the procedure disclosed in Alsager, O. A; Kumar, S.; Willmott, G. R.; McNatty, K. P.; Hodgkiss, J. M. Biosens. Bioelectron. 2014, 57C, 262. The aptamer is immobilized on polystyrene nanoparticles, exposed to aliquots of the small molecule that the aptamer is intended to selectively bind, and the fraction bound small molecule is measured via UV fluorescence and fit to a binding isotherm after separation of the nanoparticles from the supernatant.

For aptamers that bind E2, 200 nmol of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) (20 μL of 0.01 M in 2-(N-morpholino)ethanesulfonic acid (MES)) is added to activate 400 μL of carboxylate polystyrene nanoparticles (NPs, 5.2×10¹⁰particle mL⁻¹) in MES for 40 min, followed by addition of 0.1 nmol of the aptamer and incubation overnight. The samples are centrifuged at 14000 rpm for 30 min and the supernatant is discharged. 1 mL of E2 with various concentrations in BWB containing 5% ethanol is added to the samples, sonicated for 10 mins, and incubated overnight. The samples are then centrifuged at 14000 rpm for 30 min, the supernatant is isolated, transferred to a 1 cm quartz cuvette, and the fluorescence of the unbound E2 is collected via 279 nm with a Shimadzu RF-5301PC spectrofluorophotometer. The measured fluorescence intensities at 310 nm are first converted to concentration via an E2 calibration curve. By expressing a measured E2 concentration as an unbound fraction, f_a, and plotting against total E2 concentration, the K_Dis determined by fitting to the binding isotherm in equation 1.

$\begin{matrix} f_{a} = \frac{[E 2]}{K_{D} + [E 2]} & (1) \end{matrix}$

The Kd of particular aptamers of SEQ ID NOs: 3, 27, 29, 53, 55, 56, 86, 122, 124 and 126 were determined as follows:

The aptamers were used in this example are aptamers as disclosed herein of the above SEQ ID NOs:. Each of the aptamers was labelled with 5′Cy5. The target molecules were used unlabelled. A total of 5 different targets were studied (Estradiol, Testosterone, Progesterone, Androstenedione and Bisphenol A).

General Setup:
1. Aptamers:

The aptamers were purchased at Sigma Aldrich Germany and solved in PBS buffer to a final concentration of 100 micro M. Respective pre-dilutions (500 nM final in 2 mM Tris-HCL pH7.5, 10 mM NaCl, 0.5 mM KCl, 0.2 mM MgCl2, 0.1 mM CaCl2 and 0.05% Pluronic) were kept on 90° C. for 3 min and immediately store on ice for 5 min. The aptamers were diluted to 10 nM with reaction buffer prior used at RT.

Initial Tests:

- The aptamers were initially tested for aggregations (MST time traces). None of the aptamers showed precipitation or aggregation effects.
- Also photobleaching was tested not to have an influence on the MST measurements.
- MST time traces showed nicest separation at laser power between 40 and 80%.
  
  Therefore these two setting were chosen for the later experiments.

2. Targets:

1 mg/ml stock solution of each target (also BPA) in 100% EtOH were prepared. The targets were diluted in reaction buffer (without EtOH) to a final concentration of 5% EtOH (working solution). The working solution was then diluted with reaction buffer to the respective needed concentrations (to get optimal binding curves).

Technical Setup:

The aptamers were used at a final concentration of 5 nM in the experiments. The concentration of target varied depending on the experiment. In all capillaries the reaction buffer was used in a constant manner, to prevent dilution effects. The LED power was routinely used between 7 and 8%, whereas each technical run was performed at 40 and 80% laser power. The reaction temperature was set to 25° C.

Quality Statement:

While the experiments no sticking effects to the capillaries or aggregation effects were detected. Hence the experiments were performed at optimal technical conditions.

Data Analysis:

The MST time traces were transformed into Fnorm values, either using the Thermophoresis+TJump or TJump analysis method. The Fnorm values were plotted against the concentration of target and the data points were subsequently fitted using the Kd fit derived from the law of mass actions:

1:1 Binding Model

A+T⇔AT

F(c_T)=F_u+(F_b−F_u)*c_AT/c_A

c
_AT
/c
_a=fraction bound=1/2c_A*(c_T+c_A+K_d−√(c_T+c_A+K_d)²−4c_Tc_A)

F_ufluorescence in unbound state
F_bfluorescence in bound state
K_ddissociation constant, to be determined
c_ATconcentration of formed complex
c_Aconstant concentration of molecule A (fluorescent), known
c_Tconcentration of titrated molecule T

Results:

The following table summarizes the binding constants determined in the various experiments.

Binding affinity

Target
Aptamer
(KD) in nM

Estradiol
SEQ ID NO: 3
27.8 ± 10.3 nM

Androstenedione
SEQ ID NO: 27
191.6 ± 84.6 nM

Androstenedione
SEQ ID NO: 29
15.3 ± 6.5 nM

BPA
SEQ ID NO: 53
11.7 ± 5.4 nM

BPA
SEQ ID NO: 55
7.4 ± 2.6 nM

BPA
SEQ ID NO: 56
265.7 ± 123.3 nM

Progesterone
SEQ ID NO: 86
64.5 ± 31.2 nM

Testosterone
SEQ ID NO: 122
33.8 ± 14.5 nM

Testosterone
SEQ ID NO: 124
80.6 ± 17.6 nM

Testosterone
SEQ ID NO: 126
76.5 ± 32.3 nM

Example 9
Detection of Small Molecules with AuNPs

Sample water is collected from the Hutt River, Wellington, New Zealand and pre-treated by stirring 50 mL overnight at room temperature with 1 g of activated charcoal and filtering twice through 0.22 μm syringe-filters to provide treated water. The conductivity of the treated water is measured as 100 μs cm⁻¹(at 25° C. with the pH is 8). Alternatively, Milli-Q water is used instead of treated river water. Stock solutions of the target small molecules are made in ethanol before adding appropriate volumes to the treated water or Milli-Q water, and adjusting the final ethanol content to 5%, ensuring sufficient target small molecule solubility. 20 μL of the pre-treated test samples are added to 100 μL of AuNP-aptamer solution to obtain different E2 concentrations and provide a total reaction volume of 120 μL. Control samples are made up from blank water containing 5% ethanol. Samples are incubated for 10 minutes at room temperature to facilitate binding to the target. The optimized NaCl concentration determined from the salt titration experiments is added to the target detection solutions followed by gentle shaking, The samples are visually inspected after 15 minutes, and the UV-vis absorption of 5 μL aliquots is measured using a Thermo-Scientific NanoDrop™ 1000 Spectrophotometer.

Example 10
Animal Urine Study

Rat urine is collected from sexually mature ship rats (Rattus rattus), filtered with 0.22 μm syringe-filters, and spiked with target small molecules and interfering molecules after adjusting the content of ethanol to 5% (control rat urine sample comprised blank rat urine containing 5% ethanol). 5 μL of spiked urine is added to 100 μL AuNP-poly-T or AuNP-aptamer, incubated at 50° C. for 10 min, followed by addition of optimised NaCl (57.4 mM), gentle shaking, visual inspection after 15 min and measurement of UV-vis absorption as described above.

Example 11
ζ-Potential Measurements For Au Nanoparticles

120 μL samples of bare AuNPs (uncoated with aptamer), AuNP-aptamer, and AuNP-aptamer in the presence of 100 nM of a target small molecule in milli-Q water, are incubated at room temperature for 1 hour and are centrifuged at 12,500 rpm for 15 minutes. The excess aptamer is removed by decantation of the supernatant and the NPs are re-suspended in 1 mL Milli-Q water. Samples are loaded in a folded capillary cell, inserted into a Zetasizer Nano ZS equipped with a 633 nm laser (Malvern Instruments, UK) and equilibrated at 25° C. for 2 minutes prior to measurement. Measurements are made in triplicate, with fixed parameters of pH 7, viscosity 0.887 mPa s, and refractive index of 1.33. The measurements are reported as average value±standard deviation.

Example 12
CD Studies

1 mL solutions of the aptamers at 400 nM and 600 nM, respectively, are prepared in water containing 5% ethanol, 23.8 mM NaCl, and 0 or 10 μM of E2. Samples are measured in a 1 cm path length quartz cell. CD spectra are measured using a Chirascan CD spectrometer instrument over the wavelength range from 200 to 400 nm, scanned at 200 nm per minute.

All publications, patents and patent applications disclosed and cited herein are incorporated herein by reference in their entirety. While the compositions and methods of this invention have been described in terms of embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

REFERENCES

The following references are specifically incorporated herein by reference.

US 2012/0088232
Alsager, O. A, Kumar, S., Willmott, G. R., McNatty, K. P., Hodgkiss, J. M., 2014. Biosens. Bioelectron. 57C, 262-268.
Brown, K. A., Park, S., Hamad-Schifferli, K., 2008. J. Phys. Chem. C 112, 7517-7521.
Campbell, C. G., Borglin, S. E., Green, F. B., Grayson, A., Wozei, E., Stringfellow, W. T., 2006. Chemosphere 65, 1265-80.
Cekan, P., Jonsson, E. O., Sigurdsson, S. T., 2009. Nucleic Acids Res. 37, 3990-5.
Cho, E. J., Lee, J. W., Ellington, A. D., 2009, Ann. Rev. Anal. Chem., 2(1), 241-264.
Cruz-Aguado, J. and, Penner, G., 2008. J. Agric. Food Chem. 56, 10456-61.
Ellington, A. D., Szostak, J. W., 1990. Nature 346, 818-22.
Ge, J., Xing, W., Xue, X., Liu, C., Lu, T., Liao, J., 2007. J. Phys. Chem. C 111, 17305-17310.
Geyer, H. J., Rimkus, G. G., Scheunert, I., Kaune, A., Kettrup, K. S. A., G, M. Z. D. C., Larry, M., Donald, G. H., 2000. Bioaccumulation—New Aspects and Developments. Springer-Verlag, Berlin/Heidelberg.
Grabar, K. C., Freeman, R. G., Hommer, M. B., Natan, M. J., 1995. Anal. Chem. 67, 1217-1225.
Haiss, W., Thanh, N. T. K., Aveyard, J., Fernig, D. G., 2007. Anal. Chem. 79, 4215-21.
He, J., Liu, Y., Fan, M., Liu, X., 2011. J. Agric. Food Chem. 59, 1582-6.
Huizenga, D. E., Szostak, J. W., 1995. Biochemistry 34, 656-65.
Jana, N. R., Gearheart, L., Murphy, C. J., 2001. Adv. Mater. 13, 1389-1393.
Jin, R., Wu, G., Li, Z., Mirkin, C. A., Schatz, G. C., 2003. J. Am. Chem. Soc. 125, 1643-54.
Jo, M., Ahn, J.-Y., Lee, J., Lee, S., Hong, S. W., Yoo, J.-W., Kang, J., Dua, P., Lee, D.-K., Hong, S., Kim, S., 2011. Oligonucleotides 21, 85-91.
Karsisiotis, A. I., Hessari, N. M., Novellino, E., Spada, G. P., Randazzo, A., Webba da Silva, M., 2011. Angew. Chem. Int. Ed. Engl. 50, 10645-8.
Kim, Y. S., Jung, H. S., Matsuura, T., Lee, H. Y., Kawai, T., Gu, M. B., 2007. Biosens. Bioelectron. 22, 2525-31.
Kim, Y. S., Kim, J. H., Kim, I. A., Lee, S. J., Jurng, J., Gu, M. B., 2010. Bioelectron. 26, 1644-9.
Li, H., Rothberg, L., 2004. Proc. Natl. Acad. Sci. U. S. A. 101, 14036-9.
McKeague, M., Derosa, M. C., 2012. J. Nucleic Acids 2012, 748913.
McManus, S. a, Li, Y., 2013. PLoS One 8, e64131.
Mei, Z., Chu, H., Chen, W., Xue, F., Liu, J., Xu, H., Zhang, R., Zheng, L., 2013. Biosens. Bioelectron. 39, 26-30.
Olowu, R. A.; Arotiba, O.; Maliu, S. N.; Waryo, T. T.; Baker, P.; Iwouoha, E., 2010, Sensors, 10, 9872
Olowu, R. A., Ndangili, P. M., Baleg, A. A, Ikpo, C. O., Njomo, N., Baker, P, Iwuoha, E., 2011, Int. J. Electrochem. Sci., 6, 1686
Nonaka, Y., Sode, K., Ikebukuro, K., 2010. Molecules 15, 215-25.
Redel, E., Krämer, J., Thomann, R., Janiak, C., 2009. J. Organomet. Chem. 694, 1069-1075.
Shi, H., Zhao, G., Liu, M., Fan, L., Cao, T., 2013. J. Hazard. Mater. 260, 754-761.
Song, K.-M., Cho, M., Jo, H., Min, K., Jeon, S. H., Kim, T., Han, M. S., Ku, J. K., Ban, C., 2011. Anal. Biochem. 415, 175-81.
Song, S., et al., 2008, Trends in Analytical Chemistry, 27(2), 108-117.
Stoltenburg, R., Reinemann, C., Strehlitz, B., 2007. Biomol. Eng. 24, 381-403.
Teranishi, T., Hosoe, M., Tanaka, T., Miyake, M., 1999. J. Phys. Chem. B 103, 3818-3827.
Tuerk, C., Gold, L., 1990, Science (80), 249, 505-510.
Vorličková, M., Kejnovská, I., Bednářová, K., Renčiuk, D., Kypr, J., 2012. Chirality 24, 691-8.
Wu, J., Chu, H., Mei, Z., Deng, Y., Xue, F., Zheng, L., Chen, W., 2012. Anal. Chim. Acta 753, 27-31.
Wu, S.-H., Chen, D.-H., 2004. J. Colloid Interface Sci. 273, 165-9.
Xue, F., Wu, J., Chu, H., Mei, Z., Ye, Y., Liu, J., Zhang, R., Peng, C., Zheng, L., Chen, W., 2012. Microchim. Acta 180, 109-115.
Yang, C., Wang, Y., Marty, J.-L., Yang, X., 2011. Biosens. Bioelectron. 26, 2724-7.
Yin, Y., Li, Z.-Y., Zhong, Z., Gates, B., Xia, Y., Venkateswaran, S., 2002. J. Mater. Chem. 12, 522-527.
Zhao, W., Brook, M. a, Li, Y., 2008. Chembiochem 9, 2363-71.
Zheng, Y., Wang, Y., Yang, X., 2011. Sensors Actuators B Chem. 156, 95-99.

APTAMER BIOSENSORS USEFUL FOR DETECTING HORMONES, HORMONE MIMICS, AND METABOLITES THEREOF

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

PCT Information

Provisional Applications (1)