Biosensors for hormone measurement

Abstract

A biosensor for detecting a hormone is provided. The biosensor for detecting a hormone can include a core component and a biological molecule (such as a receptor for the hormone or an antibody against the hormone) immobilized on the biosensor. Also provided are methods for detecting or quantitating a hormone in a sample using the biosensor provided herein. The biosensor can be a bio-layer interferometry (BLI) biosensor, and the signals generated by binding of a hormone to the biosensor can be indicative of spectral shift, which can be measured using BLI. Additional signals such as UV absorbance of the hormone or signals of a detectable label may be measured. The biosensor can detect the presence or amount of the hormone in a sample accurately without interference by the presence of biotin, hemolysis, or lipid in a sample.

Description

Patent Application BA2408-WOS116321 1090WO (116321.00030)BIOSENSORS FOR HORMONE MEASUREMENTBackground

[0001] The present disclosure relates to apparatus and methods for detecting and / or quantitating a hormone in a sample.

[0002] Measurement of hormones such as progesterone and estradiol provides important information regarding the subject’s health status that can be used for monitoring health (including pregnancy) or diagnosis, treatment, or prevention of a variety of diseases or conditions. However, accurate and efficient measurement of hormones in samples (such as blood, plasma, serum, or urine samples) is often challenging. Competitive immunoassays are commonly used in laboratories to measure hormone levels in biological samples. Because biotinylated antibodies competes with nanoprobes to bind to an analyte (e.g., a hormone), these immunoassays are sensitive to biotin in the samples, which often impacts the accuracy of the assays. Given that the majority of patients (reportedly about 64% of women) take dietary supplement / multivitamin that contains high levels of biotin, hormone measurements in biological samples using competitive immunoassays are often inaccurate. To improve accuracy, patients are usually requested to abstain from taking dietary supplements at least 3 days prior to the hormone measurement, making these time sensitive tests difficult to take and unsuitable for immediate testing. In addition, competitive immunoassays are sensitive to hemolysis and lipemia, rendering the hormone measurement inaccurate.

[0003] Other assays for hormone measurements also have limitations. For example, electrochemiluminescence immunoassay (ECLIA) is also rendered inaccurate by the presence of biotin, hemolysis, or lipemia in the sample. Liquid chromatography-tandem mass spectrometryWBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)(LC / MS-MS) is time consuming, with turnaround time of about 8-12 days. Lateral flow assay / test only provides qualitative results without accurate measurement.Summary

[0004] In view of the foregoing, there is a need for methods that efficiently, accurately, and rapidly detect the presence or amount of a hormone (such as a steroid hormone, such as progesterone or estradiol), including methods for efficient, accurate, and rapid hormone testing for pharmaceutical quality control. This disclosure is directed generally to systems and methods to address these shortcomings of the art and provide other additional or alternative advantages. The disclosure herein provides embodiments of biosensors, and methods using the biosensors, for detecting a hormone (such as a steroid hormone, e.g., progesterone and estradiol). The biosensor for detecting a hormone includes a core component and a receptor for the hormone or an antibody against the hormone immobilized on the core component. The biosensor can be a bio-layer interferometry (BLI) biosensor, and the optical signals generated by binding of a hormone to the biosensor can be indicative of spectral shift, which can be measured using BLI. The systems and methods provided herein can provide in vitro diagnostics, by providing tests on biological samples taken from the human body and detecting diseases or other conditions, monitoring the subject’s overall health to help cure, treat, or prevent diseases, and identifying patients who are likely to benefit from specific treatments or therapies.

[0005] In certain aspects of the present disclosure, a method for detecting the presence or amount of a hormone in a sample is provided. The method includes contacting the sample with a biosensor, and detecting optical signals generated by binding of the hormone to the biosensor, thereby detecting the presence or amount of a hormone. The biosensor includes a core component and a biological molecule, which is a receptor for the hormone or an antibody against the hormone,2WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) immobilized on the biosensor (e.g., the core component) such that the hormone in the sample binds to the hormone receptor / antibody of the biosensor.

[0006] In embodiments, contacting the sample with the biosensor includes contacting the sample with a biosensor having a biotinylated hormone receptor / antibody bound to streptavidin bound to the biosensor (e.g., streptavidin-coated biosensor). The biotinylated hormone receptor / antibody is immobilized on the biosensor via streptavidin-biotin interaction.

[0007] In embodiments, contacting the sample with the biosensor includes contacting the sample with a biosensor having the hormone receptor / antibody bound to the biosensor via then- terminus of the hormone receptor / antibody. The hormone receptor / antibody is immobilized on the biosensor via standard N-(3-(dimethylamino)propyl)-N’ -ethylcarbodiimide (EDC)-catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl-terminated biosensor surface.

[0008] In embodiments, contacting the sample with the biosensor includes contacting the sample with a bio-layer interferometry (BLI) biosensor as the biosensor.

[0009] In certain embodiments, the signals generated by binding of the hormone to the biosensor are indicative of spectral shift measured using BLI. In further embodiments, a positive spectral shift indicates binding of the hormone to the biosensor.

[0010] In embodiments, the signals generated by binding of the hormone to the biosensor are indicative of UV absorbance of the hormone bound to the biosensor, The method includes detecting IV absorbance of the hormone bound to the biosensor for example using UV-Vis spectrophotometry.3WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0011] In embodiments, the method further includes contacting the biosensor-bound hormone with a targeting molecule attached to a detectable label after contacting the sample with the biosensor such that the targeting molecule binds the biosensor-bound hormone; and detecting signals of the detectable label attached to the targeting molecule bound to the biosensor-bound hormone.

[0012] In some embodiments, the detectable label is a fluorescent label, and fluorescence signals are detected.

[0013] In some embodiments, the detectable label is a horseradish peroxidase (HRP). The method further includes contacting the HRP-labeled targeting molecule bound to the biosensorbound hormone with an HRP substrate to generate the signals of the detectable label. The signals of the detectable label are detected for example by colorimetric assays or absorbance measurement.

[0014] In some embodiments, detecting the signals includes detecting signals in a plurality of samples simultaneously. In further embodiments, detecting the signals includes detecting signals in the plurality of samples, each sample placed in a plurality of wells in a plate, using a plate reader.

[0015] In embodiments, the method further includes detecting the amount of a hormone in the sample based on the signals generated by binding of the hormone in the sample to the biosensor and a standard curve of association between known amounts of a hormone and signals generated by each known amount of a hormone. In certain embodiments, the signals are spectral shift measured by BLI and / or UV absorbance of the hormone measured by UV-Vis spectrophotometry. In certain embodiments, the signals are signals of a detectable label attached to a targeting molecule bound to the hormone bound to the biosensor. For example, the signals are fluorescence4WBD (US) 4936-9869-2469vlPatent Application BA2408-WO S116321 1090WO (116321.00030) of a fluorescent label, or color intensity or absorbance generated by a reaction between HRP as the detectable label and a HRP substrate.

[0016] In some embodiments, the presence or amount of a hormone in the sample is detected within 5 minutes of contacting the sample with the biosensor. In specific embodiments, the presence or amount of a hormone in 8 samples are detected within 5 minutes, and the presence or amount of a hormone in 96 samples are detected within 1 hour.

[0017] In other embodiments, the sample is a biological sample obtained from a subject.

[0018] In embodiments, the hormone is a steroid hormone, including but not limited to progestins (e.g., progesterone), estrogens (e.g., estradiol, estrone, estriol), androgens (e.g., dehydroepiandrosterone, testosterone, dihydrotestosterone, androstenedione), glucocorticoids (e.g., cortisol, 11-deoxycortisol, corticosterone, 11 -deoxy-corticosterone), mineralcorticoids (e.g., aldosterone).

[0019] In some embodiments of the present disclosure, a biosensor for detecting the presence or amount of a hormone in a sample is provided. The biosensor includes a core component and a biological molecule immobilized on the core component. The biological molecule is a receptor for the hormone or an antibody for the hormone. The biosensor is configured to detect the presence or amount of the hormone in the sample.

[0020] In embodiments, a biotinylated hormone receptor / antibody is bound to streptavidin bound to the biosensor (e.g., streptavidin-coated biosensor, Octet® SSA Biosensor) such that biotinylated hormone receptor / antibody is immobilized on the biosensor via streptavidin-biotin interaction.5WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0021] In embodiments, the hormone receptor / antibody is bound, at its N-terminus, to the biosensor (e.g., Amine Reactive 2nd Gen (AR2G) biosensor, e.g., Octet® AR2G Biosensor) such that the hormone receptor / antibody is immobilized on the biosensor through standard EDC- catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl-terminated biosensor surface. Covalent immobilization fastens the hormone receptor / antibody to the biosensor surface for analysis of binding events and kinetic characterization.

[0022] In certain embodiments, the biosensor is a bio-layer interferometry (BLI) biosensor.

[0023] In some embodiments of the present disclosure, a method for detecting the presence or amount of a hormone in a sample is provided. The method contains contacting the sample with a bio-layer interferometry (BLI) biosensor such that the hormone binds to the biosensor, and detecting the signals generated by binding of the hormone to the biosensor, thereby detecting the presence or amount of the hormone in the sample. The BLI biosensor contains a streptavidin- bound core component, and a biotinylated hormone receptor / antibody immobilized on the streptavidin-bound core component. The hormone in the sample binds to the hormone receptor / antibody of the biosensor, and generates signals such as a spectral shift that can be measured by BLI, and UV absorbance that can be measured for example by UV-Vis spectrophotometry or UV spectroscopy. In embodiments, the method further includes signal amplification, such as contacting the biosensor-bound hormone with a targeting molecule attached to a detectable label after contacting the sample with the biosensor such that the targeting molecule binds the biosensor-bound hormone; and detecting signals of the detectable label (such as a fluorescent label or an HRP label) attached to the targeting molecule bound to the biosensor-bound hormone.6WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)Brief Description of the Drawings

[0024] Various aspects of the present disclosure are described hereinbelow with reference to the drawings, which are incorporated in and constitute a part of this specification.

[0025] FIG. 1 is a schematic representation of a process of binding a hormone in a sample to a biosensor according to embodiments of the present disclosure.

[0026] FIG. 2 is a schematic representation of a process of detecting the presence or amount of a hormone in a sample according to embodiments of the present disclosure.

[0027] FIG. 3 is a flowchart of a process of detecting the presence or amount of a hormone in a sample according to embodiments of the present disclosure.Detailed Description

[0028] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof and with reference to the drawings. These example embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Features from one embodiment or aspect can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa. The disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.7WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0029] As used herein, the term “biosensor” refers to a device that detects the presence, physical characteristics, or amount of a substance of interest (an analyte) using a biological molecule (such as a receptor, an enzyme, an antibody, an aptamer, DNA), a living organism, or a cell or tissue thereof. In embodiments, the biosensor has a distal end and a proximal end. The distal end of the biosensor can have a surface coated with a thin layer of analyte-binding molecules (such as a receptor for or an antibody against the analyte, e.g., a hormone). The proximal end of the biosensor can be connected to a transducer that converts bio-recognition events into a measurable signal and / or a display that displays the signals.

[0030] As used herein, the term “proximal” refers to the portion of the device or component thereof that is closer to the light source in the device, and the term “distal” refers to the portion of the device or component thereof that is farther from the light source in the device and closer to the subject sample.

[0031] Biosensors can be used to detect the presence or amount of analytes in a sample and facilitate, for instance, the diagnosis and treatment of disease or scientific research and development. Biosensors can employ a solid surface having immobilized anti-analyte molecules to which analyte molecules bind specifically and with high affinity at a defined detection zone. The binding event can be detected directly, for example by a change in the mass, reflectivity, UV absorbance, thickness, color, or other characteristics indicative of a binding event. The analyte can be pre-labeled, for example with a chromophore, a fluorophore, or a radiolabel. Alternatively, the analyte can be labeled after it is bound at the detection zone, for example with a secondary, labeled anti-analyte antibody.8WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0032] An “analyte-binding” molecule refers to any molecule or ligand capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to, receptor-ligand binding reactions and antibody-antigen binding reactions.

[0033] As used herein, the term “bio-layer interferometry (BLI) biosensor” refers to a biosensor that uses BLI for detection of the presence, physical characteristics, or amount of a substance of interest (an analyte). BLI is an optical technique for measuring biomolecular interactions by analyzing interference patterns of light reflected from the surface of a biosensor tip. BLI allows for label-free optical analysis for real-time monitoring of biomolecular interactions.

[0034] An “antibody” refers to a peptide or polypeptide derived from, modeled after, or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. An antibody includes an immunoglobulin molecule having two heavy chains and two light chains, as well as an antibody fragment, such as an antigen binding site (e.g., a fragment, a subsequence, a complementarity determining region (CDR)) that retains capacity to bind antigen, including: a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment, which consists of a VH domain; and an isolated complementarity determining region (CDR).

[0035] Antibodies include polyclonal antibodies and monoclonal antibodies. A “polyclonal antibody” refers to a heterologous mixture of immunoglobulins against an antigen, and can be9WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) produced by multiple cells by inoculating a mammal such as a goat, a mouse, and a rabbit with an immunogen. A “monoclonal antibody” refers to an antibody produced by identical immune cells which are clones of a single cell. A polyclonal antibody can bind to different epitopes of the same antigen, whereas a monoclonal antibody binds to one epitope of the antigen.

[0036] A “subject sample” as used herein refers to a sample with an unknown presence or amount of a hormone to be detected. A “reference sample” as used herein refers to a sample with a known presence or amount of the hormone.

[0037] A “subject” refers to an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey) and a non-primate (such as a mouse). In some aspects of the disclosure, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0038] A “patient” refers to a subject who shows symptoms and / or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and / or is at risk of developing a disease. A “patient” can be a human or veterinary subject. Any reference to subjects in the present disclosure should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patient who is in need of measurement of a hormone.

[0039] As used herein, the singular forms “a,” “an,” “the,” and the like include plural referents unless the context clearly dictates otherwise. In addition, while reference may be made herein to quantitative measures, values, geometric relationships or the like, unless otherwise stated, any one or more if not all of these may be absolute or approximate to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances or the like. Reference to10WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se with in a range suitable in the context, for example ± 10%. For example, description referring to “X” includes description of “X” and extends to a suitable range.

[0040] Measurement of hormones provides important information regarding the subject’s health status that can be used for monitoring health (including pregnancy) or diagnosis, treatment, or prevention of a variety of diseases or conditions. For example, progesterone is a steroid hormone with a molecular weight of 314.5 Da, mainly formed in the cells of the corpus luteum and during pregnancy in the placenta. Progesterone is decreased in primary or secondary hypogonadism and short luteal phase syndrome. The progesterone concentration correlates with the development and regression of the corpus luteum. Whereas progesterone is barely detectable in the follicular phase of the female cycle, a rise in the progesterone level is observed one day prior to ovulation. Increased progesterone synthesis occurs during the luteal phase. Detection of progesterone can be utilized in the diagnosis and monitoring of infertility, hypogonadism, short luteal phase syndrome, premenstrual syndrome (PMS), and premenstrual dysphoric disorder (PMDD).

[0041] As another example, estrogens are steroid hormones. The three major naturally occurring estrogens in humans are estrone (El), estradiol (E2), and estriol (E3). Estradiol is the predominant estrogen during reproductive years, both in terms of absolute serum levels as well as in terms of estrogenic activity. Estradiol is responsible for the regulation of the estrous and menstrual female reproductive cycles and for the development and maintenance of female secondary sex characteristics. Estradiol plays a key role in germ cell maturation and numerous other, non-sex-specific processes, including growth, bone metabolism, nervous system maturation, and endothelial responsiveness. Measurement of serum estradiol serves an integral role in the11WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) assessment of reproductive function in females and in the assessment of infertility, oligomenorrhea, and menopausal status. Estradiol is commonly measured for monitoring ovulation induction, as well as during preparation for in vitro fertilization.

[0042] However, accurate and efficient measurement of hormones in biological samples (such as blood, plasma, serum, or urine samples) is often challenging. For example, competitive immunoassays are rendered inaccurate by the presence of biotin, hemolysis, or lipemia in the sample as described above. Other assays for hormone measurements (e.g., ECLIA, LC / MS-MS, lateral flow assay) also have limitations. To overcome the deficiencies in the currently available hormone detection methods, in certain aspects of the present disclosure, a biosensor for detecting the presence or amount of a hormone in a sample is provided. The biosensor contains a core component and a hormone receptor / antibody immobilized on the biosensor (e.g., core component), and is configured to detect the presence or amount of the hormone in the sample. The hormone antibody can be a monoclonal antibody against the hormone.

[0043] Any suitable material can be used for the core component. For example, the core component may comprise a silicon substrate, activated with silane group, such as aminopropyl silane (APS) and epoxypropylsilane (EPS). The core component (optionally with one or more additional components on the biosensor) may be capable of binding biotin, as further described below.

[0044] The term “immobilize” or “immobilized” or “immobilizing” in the context of immobilizing an analyte-binding molecule (such as receptor for or an antibody against the hormone) onto a biosensor or a structure thereof (such as a core component) refers to the process of attaching or binding the analyte-binding molecule (such as a hormone receptor / antibody) to the12WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) biosensor or the structure thereof (such as a core component). Analyte-binding molecules (such as a hormone receptor / antibody) may be immobilized (attached, bound) to specific zones of the biosensor either by conjugating directly to the biosensor surface, or by indirect binding.

[0045] Immobilization of an analyte-binding molecule (such as a hormone receptor / antibody) on a biosensor can be done by any methods known in the art. For example, binding of the antibody with the biosensor can be by biotin-streptavidin interaction. For example, the hormone receptor / antibody can be biotinylated, and can be bound to streptavidin that is bound to the biosensor. Non-exhaustive examples of a biosensor having a streptavidin moiety for binding a biotinylated target molecule (e g., biotinylated hormone receptor / antibody) include Octet® SSA Biosensors.

[0046] An analyte-binding molecule (such as a hormone receptor / antibody) can also be immobilized at its N-terminus on the biosensor via amide bond formation, e.g., standard EDC- catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl -terminated biosensor surface. Covalent immobilization fastens the hormone receptor / antibody to the biosensor surface for analysis of binding events and kinetic characterization. Non-exhaustive examples of a biosensor having amine reactive moiety for binding a hormone receptor / antibody at its N-terminus include AR2G biosensors, e g., Octet® AR2G Biosensors. A potential advantage of immobilization using amide bond (e g., onto an AR2G biosensor) includes no involvement of streptavidin, avoiding free biotin in the samples from binding to streptavidin of the biosensor..

[0047] An analyte-binding molecule (such as a hormone receptor / antibody) can also be immobilized on a biosensor by binding a histidine tagged analyte-binding molecule (such as a13WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) hormone receptor / antibody) to the biosensor that is capable of binding to a histidine tag. The biosensor that is capable of binding to a histidine tag can contain the core component, or the core component with one or more additional components that enable binding to a histidine tag. Non- exhaustive examples of a biosensor capable of binding to a histidine tag include Octet® HIS IK Biosensors and Octet® NTA Biosensors. An analyte-binding molecule (such as a hormone receptor / antibody) can also be immobilized on a biosensor by binding a human Fc tagged analytebinding molecule (such as a hormone receptor / antibody) to Protein A bound to the core component of the biosensor; by passive adsorption of the analyte-binding molecule (such as a hormone receptor / antibody) onto the core component of the biosensor, for example by incubating the biosensor with a solution containing the analyte-binding molecule (such as a hormone receptor / antibody); or by using a crosslinker. Conventional immobilization chemistries can be used for chemically (such as covalently) attaching a layer of the analyte-binding molecule to the biosensor. For example, a variety of bifunctional reagents containing a siloxane group can be used for chemical attachment to SiCh, and a hydroxyl, amine, carboxyl or other reaction group can be used for attachment of biological molecules, such as proteins (such as antigens, antibodies). It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte-binding molecules (such as antibodies) can be bound. Where the core component of the biosensor is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups, for binding with analyte-binding molecules (such as antibodies).

[0048] In an example of indirect binding, analyte-binding molecules (such as a hormone receptor / antibody) may be immobilized on particles or other solid supports, and the solid supports may be immobilized onto the biosensor surface. Solid supports that may be used to immobilize14WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) an analyte-binding molecule (such as a hormone receptor / antibody) include membrane filters, cellulose-based papers, beads (including polymeric, latex, and paramagnetic particles), silicon wafers, nanoparticles, gels, and multi-well plates.

[0049] The presence or amount of any hormone can be measured using the biosensors and methods provided herein. In embodiments, the hormone is a steroid hormone, including but not limited to progestins (e.g., progesterone), estrogens (e.g., estradiol, estrone, estriol), androgens (e.g., dehydroepiandrosterone, testosterone, dihydrotestosterone, androstenedione), glucocorticoids (e.g., cortisol, 11 -deoxycortisol, corticosterone, 11 -deoxy-corticosterone), mineralcorticoids (e.g., aldosterone). In specific embodiments, the hormone is progesterone or estradiol. In embodiments, the hormone is one or more of adrenaline (epinephrine), noradrenaline (norepinephrine), triiodothyronine, thyroxine, melatonin, dopamine, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin (islet amyloid polypeptide), anti -Mullerian hormone (Mullerian-inhibiting factor / hormone), adiponectin, adrenocorticotropic hormone, (corticotropin), corticotropin-releasing hormone, angiotensinogen, angiotensin, antidiuretic hormone, (vasopressin, arginine vasopressin), atrial natriuretic peptide (atriopeptin), brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, luteinizing hormone, gonadotropinreleasing hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon- like peptide- 1, growth horm one-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin (hypoglycemic hormone, antiketogenic hormone), insulin-like growth factor (somatomedin), leptin, lipotropin, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, osteocalcin, oxytocin, (pitocin), pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide,15WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) prolactin (leuteotropic hormone), prolactin-releasing hormone, relaxin, renin, secretin, somatostatin (growth hormone-inhibiting hormone, growth hormone release-inhibiting hormone, somatotropin release-inhibiting factor, somatotropin release-inhibiting hormone), thrombopoietin, thyroid-stimulating hormone (thyrotropin), thyrotropin-releasing hormone, vasoactive intestinal peptide, guanylin, and uroguanylin.

[0050] FIG. 1 schematically depicts an embodiment of a biosensor and a process for detecting a hormone (such as a steroid hormone) in a sample. The biosensor includes a core component (optionally with one or more additional components) 102 and a hormone receptor / antibody 104 immobilized on the biosensor. In the panel 108, the core component (optionally with one or more additional components) 102 is coated with the hormone receptor / antibody 104. In specific embodiments, the hormone receptor / antibody 104 is biotinylated, and is bound to streptavidin bound to the core component (optionally with one or more additional components) 102, and thus is immobilized on the biosensor via streptavidin-biotin interaction. In panel 110, the biosensor having the hormone receptor / antibody 104 immobilized on the core component (optionally with one or more additional components) 102 is in the baseline state and configured to detect the presence or amount of a hormone in the sample. In panel 112, the hormone of interest 106 has been contacted by the biosensor according to the methods provided herein, and the hormone 106 is bound to the hormone receptor / antibody 104 of the biosensor.

[0051] The biosensor may be a BLI biosensor. The BLI biosensor uses BLI to measure the signal / wavelength / spectral shift (nm) over time generated by the biosensor and an analyte (such as a hormone), with a positive shift indicating binding of an analyte, such as a hormone, to the biosensor.16WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0052] A method of the present disclosure for detecting the presence or amount of a hormone in a sample can include the steps of contacting the sample with a biosensor, and detecting signals generated by binding of a hormone to the biosensor, thereby detecting the presence or amount of a hormone in the sample. The biosensor contains a core component and a hormone receptor / antibody immobilized on the biosensor (e.g., on the core component) such that a hormone in the sample binds the hormone receptor / antibody of the biosensor.

[0053] The hormone receptor / antibody may be immobilized on the biosensor (the core component thereof) by any methods known in the art. For example, the hormone receptor / antibody can be biotinylated, and can be bound to streptavidin that is bound to the biosensor. Non- exhaustive examples of a biosensor having a streptavidin moiety for binding a biotinylated target molecule (e.g., biotinylated hormone receptor / antibody) include Octet® SSA Biosensors. As another example, the hormone receptor / antibody can be immobilized at its N-terminus on the biosensor via amide bond formation, e.g., standard EDC-catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl- terminated biosensor surface. Non-exhaustive examples of a biosensor having amine reactive moiety for binding a hormone receptor / antibody at its N-terminus include AR2G biosensors, e.g., Octet® AR2G Biosensors. Other methods of immobilizing the hormone receptor / antibody on the biosensor are described in the present disclosure.

[0054] The biosensor can be a BLI biosensor. In some embodiments, the signals are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. The signal / wavelength / spectral (nm) shift over time can indicate binding of an analyte (such as a hormone) to the BLI biosensor. In some embodiments, a positive signal / wavelength / spectral shift indicates binding of a hormone to the biosensor, which can be measured using BLI.17WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0055] Additionally or alternatively, the methods include measuring UV absorbance at a specific wavelength using, for example, UV-Vis spectrophotometry or UV spectroscopy, as some hormones (such as steroid hormones) absorb UV light at a specific wavelength. For example, estradiol and progesterone absorbs UV light at 205 nm and 242 nm, respectively. The UV absorbance measured at 205 nm indicates the presence or amount of estradiol bound to the biosensor. The UV absorbance measured at 242 nm indicates the presence or amount of progesterone bound to the biosensor. The methods of detecting a hormone by measuring UV absorbance may be used independently from, or in conjunction with the methods of detecting a hormone measuring by BLI the spectral shift generated by binding of a hormone to the biosensor.

[0056] Signals generated by binding of the hormone to the biosensor may be amplified by using a targeting molecule and a detectable label. For example, the method can further include, after contacting the sample with the biosensor, contacting the biosensor-bound hormone with a targeting molecule attached to a detectable label such that the targeting molecule binds to the biosensor-bound hormone; and detecting the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound hormone. In specific embodiments, the detectable label is a horseradish peroxidase (HRP) label, and the method further includes contacting the HRP-labeled targeting molecule bound to the biosensor-bound hormone with an HRP substrate (e g., luminol, 3,3’,5,5’-Tetramethylbenzidine(TMB), diaminobenzidine (DAB), 2,2’-azino-bis(3ethylbenzothiazoline-6-sulfonic acid (ABTS)), thereby generating the signals of the detectable label. In one embodiment, the HRP substrate is luminol, and catalysis of oxidation of luminol by the HRP generates a chromogenic signal at 428 nm, which may be measured to detect the presence or amount of a hormone in the sample. The signal can be enhanced in the presence of an enhancer by methods well known in the art, and resulting enhanced18WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) chemiluminescence may be measured, to detect the presence or amount of a hormone in the sample. In one embodiment, the HPR substrate is metal enhanced DAB, which includes cobalt and nickel chloride in a formulation of DAB peroxidase substrate. Catalysis of the metal enhanced DAB by the HRP can produce intense color for detection, providing enhanced sensitivity, low background, and high intensity signals. Chromogenic signals or enhanced chemiluminescence can be measured using any methods known in the art, such as using a luminometer or spectrophotometer.

[0057] In other embodiments, the detectable label is a fluorescent label, and the fluorescent signals of fluorescence labeled targeting molecule bound to a hormone that is bound to the biosensor is measured to detect the presence or amount of a hormone in the sample. Fluorescence can be measured using any methods known in the art, such as using a fluorometer. The methods of detecting a hormone using a targeting molecule attached to a detectable label (such as an HRP label or a fluorescent label) may be used independently from, or in conjunction with the label free methods of detecting the presence or amount of a hormone based on the spectral shift (BLI signals) or UV absorbance generated by binding of a hormone to the biosensor.

[0058] The signals in a plurality of samples can be detected simultaneously, for example using BLI, UV absorbance, and / or a targeting molecule attached to a detectable label. To facilitate simultaneous or high throughput detection, the plurality of samples can be each placed in a plurality of wells in a plate (such as wells in a 96 well plate or a 384 well plate), and the signals in the plurality of wells can be detected by plate reader. Any other sample placement format may be used to facilitate simultaneous or high throughput detection. In particular, the method provided herein may be used in high throughput detection of the presence or amount of a hormones in19WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) multiple samples, such as multiple biological samples, which can streamline accurate and efficient monitoring, diagnosis, or treatment in subjects.

[0059] In some embodiments, the method includes quantitating the amount of a hormone in the sample. The amount of a hormone in the sample (subject sample) can be quantitated based on the signals generated by binding of the hormone in the sample (subject sample) to the biosensor and a standard curve (a calibration curve). The standard curve can represent an association between known amounts of a hormone and signals generated by each known amount of a hormone obtained for example by using reference samples.

[0060] The method provided herein can provide fast and accurate detection of the presence or amount of a hormone in a sample and in multiple samples. For example, the presence of a hormone in a sample can be detected within 1 hour, 30 minutes, 20 minutes, 10 minutes, or 5 minutes of contacting the sample with the biosensor. Likewise, the amount of a hormone in a sample can be quantitated within 1 hour, 30 minutes, 20 minutes, 10 minutes, or 5 minutes of contacting the sample with the biosensor. The method provided herein can detect the presence or amount of a hormone in multiple samples, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more samples simultaneously, and thus may be capable of high throughout detection. For example, the method can detect the presence or amount of a hormone in multiple samples in a plate reader format. In certain embodiments, the method can detect the presence or amount of a hormone in multiple samples in a 96 well, where a standard curve representing an association between known amounts of a hormone and signals generated by each known amount of a hormone is recorded from first column containing 8 reference samples within 5 minutes. The presence or amount of a hormone in the rest of the 88 samples (columns 2-12) can be measured within 55 minutes (5 minutes per column). Thus, the presence or amount of a hormone in 88 subject samples in a 9620WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) well plate can be measured within 1 hour, which indicates the capability to detect the presence or amount of a hormone in approximately 1.5 samples per minute. Such short time until detection (detection of the presence or amount of a hormone in a sample), the capability of high throughput detection, and the ability to accurately detect the amount of the hormone in samples even in the presence of biotin, lipemia, or hemolysis according to the methods provided herein may offer advantages over currently available methods of detecting a hormone, which are inaccurate in the presence of biotin, lipemia, or hemolysis, and are low throughput, inconsistent, time-consuming, and / or expensive.

[0061] In embodiments, the sample is a biological sample. A “biological sample” as used herein refers to any sample obtained from a living organism. A biological sample can be a tissue sample, a swab containing cells (such as nasal or throat swab), or a body fluid sample. A “body fluid sample” as used herein refers to a sample of bodily fluid obtained from a subject, such as a patient. A body fluid sample can be blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions obtained from a subject, such as a patient, for example who has or is suspected to have abnormal levels of estrogen / progesterone. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0062] Once the presence or amount of a hormone is detected in a biological sample obtained from a subject (for example by using the biosensor provided herein having a hormone receptor / antibody immobilized on the biosensor), one of ordinary skill in the art (such as a clinician) can readily select management, a procedure, or a treatment regimen that is compatible with the detection results. For example, the method provided herein may further include selecting the21WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) subject as a candidate for certain management, treatment, or procedure, or administering to the subject such management, treatment, or procedure. For example, upon detection of a high amount of progesterone and / or estradiol in a sample obtained from a subject (for example by using the biosensor provided herein to which a hormone receptor / antibody is immobilized on the biosensor), the method provided herein can further include selecting the subject as a candidate for treatment or procedure directed to infertility, or administering to the subject such treatment or procedure. Upon detection of a low amount of progesterone in a sample obtained from a subject, the method provided herein can further include selecting the subj ect as a candidate for management, treatment, or procedure directed to infertility, hypogonadism or short luteal phase syndrome, or administering to the subject such management, treatment, or procedure. Upon detection of a specific pattern of fluctuation of progesterone levels over time in samples obtained from a subject, the method provided herein can further include selecting the subject as a candidate for management, treatment, or procedure directed to PMS or PMDD, or administering to the subject such treatment or procedure. Upon detection of a specific level or a specific pattern of fluctuation of estradiol levels over time in samples obtained from a subject, the method provided herein can further include selecting the subject as a candidate for management, treatment, or procedure directed to oligomenorrhea, menopause, or ovulation induction, or administering to the subject such treatment or procedure. One of ordinary skill in the art is aware of appropriate management, procedures, or treatments for conditions discussed in relation the methods of detection described herein. In addition, because the methods and systems described herein can detect the amount of a hormone in a sample (including a sample with biotin, lipemia, or hemolysis), the methods and apparatus provided herein may be used to monitor a course of management, treatment, or procedure and adjust the treatment accordingly. For example, certain amount of a hormone in samples obtained22WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) from the subject over time may indicate that the particular treatment is effective and the condition is improving, and the clinician may continue or taper the management, treatment, or procedure. On the other hand, certain amount of a hormone in samples obtained from the subject over time may indicate that the particular treatment or procedure is not effective or the condition is worsening, and the clinician may consider modification or change of the management, treatment regimen, or procedure.

[0063] The systems and methods provided herein can be used for iti vitro diagnostics, by providing tests on biological samples taken from the human body and detecting diseases or other conditions, monitoring the subject’s overall health to help cure, treat, or prevent diseases, and identifying patients who are likely to benefit from specific treatments or therapies.

[0064] FIG. 2 schematically represents an embodiment of a process of detecting the presence or amount of a hormone in a sample, such as a biological sample that may contain biotin, lipemia, or hemolysis. Signals generated in panel 202 of coating (immobilizing) the hormone receptor / antibody onto the biosensor (left panel), equilibrating the biosensor (middle panel), and binding the hormone to the biosensor (right panel) is measured by an analyzer and displayed on the display as shown in panel 204. In panel 202, any suitable method of immobilization may be used for immobilizing the hormone receptor / antibody onto the biosensor. For example, the hormone receptor / antibody may be biotinylated and bound to the biosensor coated with streptavidin (e.g., Octet® SSA Biosensors). As another example, the hormone receptor / antibody is immobilized, at its N-terminus, onto the biosensor (e.g., Amine Reactive 2nd Gen (AR2G) biosensors, e.g., Octet® AR2G Biosensors) through standard EDC-catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl-terminated biosensor surface.23WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0065] Tn panel 204, a positive spectral shift may indicate binding of the hormone to the biosensor, and can be measured using BLI. In embodiments, the biosensor is a BLI biosensor. In some embodiments, optical signals are generated by binding of an analyte (such as a hormone) to the BLI biosensor, and are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. In some embodiments, a positive signal / wavelength / spectral shift may indicate binding of a hormone to the biosensor, which can be measured using BLI. As shown in panel 206, use of standard curve (calibration curve) may facilitate accurate quantitation of the amount of a hormone in the sample. The standard curve (calibration curve) may represent an association between known amounts of a hormone and signals generated by each known amount of a hormone obtained for example by using reference samples. The presence or amount of a hormone in each subject sample can be determined based on the signals generated by binding of a hormone in the sample to the biosensor, and the standard curve. The standard curve may be linear for certain ranges of steroid hormone.

[0066] FIG. 3 represents an embodiment of a process of detecting the presence or amount of a hormone in a sample, such as a biological sample. Process 300, for detecting the presence or amount of a hormone in a sample, begins with step 302 of immobilizing a hormone receptor / antibody onto the biosensor core component. Any suitable method of immobilization may be used. For example, the hormone receptor / antibody may be biotinylated and bound to the biosensor coated with streptavidin. As another example, the hormone receptor / antibody is immobilized, at its N-terminus, onto the biosensor through standard EDC-catalyzed amide bond formation to create a covalent bond between a reactive amine on the hormone receptor / antibody and the carboxyl-terminated biosensor surface. The hormone receptor / antibody may also be attached to a histidine tag such that the hormone receptor / antibody is immobilized via the histidine24WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) tag on the biosensor that is capable of binding to a histidine tag. Passive adsorption, or attachment using a crosslinker may also be used.

[0067] Process 300 continues to step 304, to provide the biosensor having a hormone receptor / antibody immobilized thereon. Process 300 continues to step 306, to contact the sample with the biosensor. Contacting the sample with the hormone receptor / antibody bound biosensor allows for binding of the hormone to the biosensor. Process 300 continues to step 308, to detect optical signals generated by binding of the hormone to the biosensor, thereby detecting the presence or amount of a hormone in the sample. In embodiments, the biosensor is a BLI biosensor. In some embodiments, the signals are optical signals generated by binding of an analyte (such as a hormone) to the BLI biosensor, and are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. In some embodiments, a positive signal / wavelength / spectral shift may indicate binding of a hormone to the biosensor, which can be measured using BLI. Additionally or alternatively, UV absorbance of the hormone bound to the biosensor can be measured at a specific wavelength, which is indicative of the amount of the hormone bound to the biosensor. For example, the UV absorbance measured at 205 nm indicates the presence or amount of estradiol bound to the biosensor. The UV absorbance measured at 242 nm indicates the presence or amount of progesterone bound to the biosensor. The step of detecting a hormone by measuring UV absorbance may be used independently from, or in conjunction with the step of detecting a hormone measuring by BLI the spectral shift (BLI signals) generated by binding of a hormone to the biosensor.

[0068] In step 308, use of standard curve (or calibration curve) may facilitate accurate quantitation of the amount of a hormone in the sample. The standard curve (or calibration curve) may represent an association between known amounts of a hormone and signals generated by each 25WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) known amount of a hormone obtained for example by using reference samples. The presence or amount of a hormone in each subject sample can be determined based on the signals generated by binding of a hormone in the sample to the biosensor, and the standard curve.

[0069] From step 306, process 300 may alternatively or additionally continue to step 310, to contact the biosensor-bound hormone with a targeting molecule attached to a detectable label such that the targeting molecule binds to the biosensor-bound hormone. The targeting molecule can be any molecule that binds the biosensor-bound hormone and can be used for its detection without affecting the binding of the hormone to the biosensor. In certain embodiments, the targeting molecule is an antibody against the hormone. In certain embodiments, the targeting molecule is a receptor for the hormone. The targeting molecule can be different from the hormone receptor / antibody immobilized on the biosensor to bind the hormone to the biosensor. The detectable label may be any detectable label, such as a fluorescent label or a HRP label. To generate the HRP signals, the HRP-labeled targeting molecule bound to the biosensor-bound hormone is contacted with an HRP substrate (such as luminol, TMB, DAB, metal enhanced DAB, or ABTS), and optionally with a chemiluminescence enhancer according to the procedures well known in the art. In one embodiment, catalysis of the metal enhanced DAB by the HRP produces enhanced color for detection. The signals of the detectable label may be detected using standard methods, such as fluorometry, luminometry, spectrophotometry, and colorimetry.

[0070] Process 300 continues to step 312, to detect the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound hormone, for example by measuring fluorescent signals, colorimetric signals, or chemiluminescence signals by standard methods in the art, thereby detecting the presence or amount of hormone. Use of standard curve (or calibration curve) may facilitate accurate quantitation of the amount of a hormone in the sample. The standard26WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) curve (or calibration curve) may represent an association between known amounts of the detectable label and signals generated by each known amount of the detectable label for example by using reference samples. Steps 310 and 312 for detecting hormone using targeting molecule attached to a detectable label (such as a fluorescent label or an HRP label) may be used independently from, or in conjunction with, step 308 for detecting hormone based on the signals generated by binding of hormone to the biosensor without using labels, for example by using BLI.

[0071] In some embodiments, the biosensor provided herein contains an optical fiber having a proximal end portion and a distal end portion, the proximal end portion configured to receive light from a light source and configured to deliver reflected light to a detector. The distal end portion configured to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion. In some embodiments, the biosensor further comprises an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.

[0072] In some embodiments, the biosensor provided herein contains an optical fiber having a proximal end portion and a distal end portion, the proximal end portion configured to receive light from a light source and configured to deliver reflected light to a detector. The distal end portion configured to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion. In some embodiments, the biosensor further comprises an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective27WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.

[0073] In some embodiments of the methods provided herein, the signals such as those generated by binding of a hormone to the biosensor (e.g., spectral shift, UV absorbance), or binding of a targeting molecule are measured by a detector. The signals can be detected based on any label-free technique for detecting a change in a property of a sensor surface, such as BLI, Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM), and Refl ectometric Interference Spectroscopy (RIfS). Additionally or alternatively, the signals of the detectable label, such as fluorescent label, can be detected by a detector. In specific embodiments, the signals are measured by an interferometer. The interferometer can comprise the biosensor, and can constitute a BLI sensor. In some embodiments, the interferometer further comprises: a first optical waveguide configured to receive light from a light source; a second optical waveguide configured to deliver reflected light to a detector; and an optical coupler spatially separates a distal portion of the first optical waveguide from a distal portion of the second optical waveguide, wherein the biosensor is attached to the optical coupler. In some embodiments, the interferometer further comprises a light source that is in optical communication with the first optical waveguide and configured to provide light to the first optical waveguide. In some embodiments, the interferometer further comprises a detector configured to receive light from the second optical waveguide. In some embodiments, the first optical waveguide and the second optical waveguide are disposed in a fiber optic bundle.

[0074] For example, an interferometer can include a light source, an optical assembly, and a detector unit. The BLI sensor or optical assembly functions as a sensing element or detector tip to28WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) detect analytes attached to an end thereof. The detector unit detects interference signals produced by interfering light waves reflected from the optical assembly. The light source directs light into the optical assembly, which is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or fiber that extends from the light source to the optical assembly, a second optical waveguide or fiber which carry reflected light from the optical assembly to the detector, and an optical coupler which optically couples the first optical waveguide and the second optical waveguide. In some embodiments, the coupling assembly includes a lens system constructed to focus a light beam on an upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector.

[0075] The light source can be a white light source, such as a light emitting diode (LED), that produces light over a broad spectrum, e.g., 400 nm or less to 700 nm or greater, typically over a spectral range of at least 100 nm. In some embodiments, the light source can be a plurality of sources each having a different characteristic wavelength, such as LEDs designed for light emission at different selected wavelengths in the visible light range. The same function can be achieved by a single light source, such as, white light source, with suitable fdters for directing light with different selected wavelengths onto the optical assembly.

[0076] The detector may be a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly. In some embodiments, where the light source operates to direct different selected wavelengths onto the optical assembly, the detector may be a simple photodetector for recording light intensity at each of the different irradiating wavelengths. In certain embodiments, the detector may include one or more filters which allows detection of light intensity, for instance from a white-light source, at each of a plurality of selected wavelengths of the interference reflectance wave.29WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0077] The first optical waveguide and / or the second optical waveguide may be in the form of a fiber optic bundle (FOB). As shown, the first optical waveguide includes several fiber optic elements surrounding a single fiber optic element of the second optical waveguide. This arrangement separates delivery of light from the light source from delivery of the reflected light from the optical assembly to the detector. It will be appreciated that other arrangements of the first optical waveguide and the second optical waveguide may allow for spatial separation of the light from the light source and the reflected light from the optical assembly. The separation of the light from the light source and the reflected light from the optical assembly may improve a signal to noise ratio (SNR) of the apparatus. In some embodiments, the first optical waveguide is a single fiber and the second fiber optical waveguide is formed of a plurality of fibers.

[0078] The distal tip of the fiber optic bundle can be aligned with a proximal end portion of the optical fiber when the BLI sensor is attached to the optical coupler. The BLI sensor may be fixedly attached to the optical coupler to align and maintain a position of the proximal end portion with respect to the tip.

[0079] The BLI sensor can include the optical fiber having a proximal end and a distal end. The proximal end and / or the distal end of the optical fiber may be polished ends. The BLI sensor can have an optical resonator having a first reflecting surface and a second reflecting surface distal of the first reflecting surface. The optical fiber is substantially transparent between the proximal end and distal end thereof. The optical resonator may be transparent between the first and second reflecting surfaces. The distance between the first and second reflecting surfaces defines a thickness of the optical resonator. The thickness of the optical resonator may be in a range of 50 nm to 5,000 nm, such as between 400 nm and 1,000 nm.30WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)

[0080] The second reflecting surface is formed of a layer of analyte-binding molecules which are effective to bind analyte molecules specifically and with high affinity. That is, the analyte and anti-analyte molecules are opposite members of a binding pair which can include, without limitations, antigen-antibody pairs, complementary nucleic acids, and receptor-binding agent pairs. In specific embodiments, the analyte molecule is a hormone, and the anti-analyte molecule is receptor for or an antibody against the hormone that binds to the hormone.

[0081] The index of refraction of the optical fiber may be similar to that of the second reflecting surface so that light reflected from the second reflecting surface occurs predominantly from the layer formed by the analyte-binding molecules, rather than from the interface between the optical fiber and the analyte-binding molecules. Similarly, as analyte molecules bind to distal end portion of the optical assembly, light reflected from the distal end portion of the assembly occurs predominantly from the layer formed by the analyte-binding molecules and bound analyte, rather than from the interface region.

[0082] The first reflecting surface of the optical assembly is formed as a layer of transparent material having an index of refraction that is substantially different from that of the optical fiber such that this layer functions to reflect a portion of the light directed onto the optical assembly.

[0083] The thickness of an analyte-binding layer disposed in the distal end portion of the optical element may be designed to optimize the overall sensitivity based on specific hardware and optical components. Conventional immobilization chemistries are used in chemically, such as covalently, attaching a layer of analyte-binding molecules to the lower surface of the optical element. For example, a variety of bifunctional reagents containing a siloxane group for chemical attachment to SiCh, and a hydroxyl, amine, carboxyl or other reaction group for attachment of31WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) biological molecules, such as proteins (such as antigens, antibodies), or nucleic acids. It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte-binding molecules can be bound. Where the optical fiber is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups.

[0084] In certain embodiments, the analyte-binding layer is formed under conditions in which a distal end surface of the optical fiber is densely coated, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer. The analytebinding layer can be either a monolayer or a multi-layer matrix.

[0085] The measurement of the presence or amount of analyte (such as a hormone) to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the second reflecting surfaces changes accordingly. Because the thickness of all other layers remains the same, the interference wave formed by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change.

[0086] Assuming that there are two reflected beams, the first beam is reflected from the first reflecting surface and the second beam is reflected from the analyte-binding molecules and bound analyte and the surrounding medium at the second reflecting surface. The conversion of the phase shifting to a thickness change of the bound analytes is well known in the art.

[0087] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Any combination of the32WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) above embodiments is also envisioned and is within the scope of the appended claims. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto.33WBD (US) 4936-9869-2469vl

Claims

Patent Application BA2408-WOS116321 1090WO (116321.00030)What is Claimed:

1. A method of detecting the presence or amount of a hormone in a sample, the method comprising: contacting the sample with a biosensor, the biosensor comprising a core component and a biological molecule immobilized thereon such that the hormone in the sample binds to the biological molecule, wherein the biological molecule is a receptor for the hormone or an antibody against the hormone; and detecting optical signals generated by binding of the hormone to the biosensor thereby detecting the presence or amount of the hormone in the sample.

2. The method of claim 1, wherein the signals are indicative of spectral shift measured using bio-layer interferometry (BLI).

3. The method of claim 2, wherein a positive spectral shift indicates binding of the hormone to the biosensor.

4. The method of claim 1, further comprising: detecting UV absorbance of the hormone bound to the biosensor.

5. The method of claim 1, further comprising: contacting the biosensor-bound hormone with a targeting molecule attached to a detectable label after contacting the sample with the biosensor such that the targeting molecule binds the biosensor-bound hormone; and34WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030) detecting signals of the detectable label attached to the targeting molecule bound to the biosensor-bound hormone.

6. The method of claim 5, wherein the detectable label is a fluorescent label.

7. The method of claim 5, wherein the detectable label is a horseradish peroxidase (HRP) label, and the method further comprises contacting the HRP-labeled targeting molecule bound to the biosensor with an HRP substrate to generate the signals of the detectable label.

8. The method of claim 1, wherein the hormone is a steroid hormone.

9. The method of claim 8, wherein the steroid hormone is progesterone or estradiol.

10. The method of claim 1, wherein the optical signals comprise light reflected from an interface internal to the biosensor and light reflected from an end of the biosensor.

11. A biosensor for detecting the presence or amount of a hormone in a sample, the biosensor comprising: a core component; and a biological molecule immobilized on the core component, wherein the biological molecule is a receptor for the hormone or an antibody against the hormone, and wherein the biosensor is configured to detect optical signals generated by binding of the hormone to the biosensor thereby to detect the presence or amount of the hormone in the sample.35WBD (US) 4936-9869-2469vlPatent Application BA2408-WOS116321 1090WO (116321.00030)12. The biosensor of claim 11, wherein the biosensor is a bio-layer interferometry (BLT) biosensor.

13. The biosensor of claim 11, wherein the hormone is a steroid hormone.

14. The biosensor of claim 13, wherein the steroid hormone is progesterone or estradiol.

15. The biosensor of claim 11, wherein the optical signals comprise light reflected from an interface internal to the biosensor and light reflected from an end of the biosensor.36WBD (US) 4936-9869-2469vl

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