Wearable device for continuous monitoring of target hormones and / or analytes, and methods of using thereof

Abstract

Provided herein are wearable devices and methods for continuous monitoring of one or more target hormones and / or analytes in a subject.

Description

23234-20010.40WEARABLE DEVICE FOR CONTINUOUS MONITORING OF TARGET HORMONES AND / OR ANALYTES, AND METHODS OF USING THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 748,627, filed January 23, 2025, the entire content of which is incorporated herein by reference for all purposes.FIELD

[0002] The present disclosure relates generally to wearable devices, and more specifically to a wearable device for continuous monitoring of one or more target hormones and / or analytes in a subject. For example, the device may be configured for continuous monitoring of luteinizing hormone and progesterone, or a metabolite thereof, as part of an ovulation test.BACKGROUND

[0003] A variety of devices attempt to detect hormone levels to allow for better understanding of the menstrual cycle. For example, it is well known in the art to utilize urine-based tests to detect luteinizing hormone (LH) in urine to assess the fertility status of a subject woman. In another example, blood testing has been utilized to evaluate for the presence of progesterone to confirm ovulation. In association with such tests, the timing of the testing remains critically important. If the subject woman neglects to time the test to correlate with a LH surge, for example, the subject woman may miss the signal that she is in a state of elevated fertility and may therefore miss a chance to conceive. As the various hormones associated with the menstrual cycle vary on different days and times within the menstrual cycle, it remains a challenge to estimate when testing should precisely take place.

[0004] A variety of other challenges remain in association with hormone testing. In the context of urine testing, in a situation where a subject woman is over-hydrated, her urine may become diluted. In such case, the woman’s hormonal concentration in urine may fall below levels perceptible to a urine test. Furthermore, collecting a sample of blood or urine to evaluate hormone levels is inconvenient and sometimes not accessible to women. It therefore remains desirable to have an alternative test that does not require a woman to estimate the proper time for or undergo the inconvenience of urine and blood testing. Ovulation-related hormones like LH can be very transient. A woman desiring to detect her LH surge could miss an ovulation signal if she doesn’t collect her sample at the right time or doesn’t sample 1MF-36617507223234-20010.40it enough times per day. In other contexts such as drug testing, the user could over hydrate and cause a false negative result, causing a potentially dangerous situation.BRIEF SUMMARY

[0005] In some aspects, provided is device, comprising: a cartridge configured to insert into a holder. In some embodiments, the holder is configured to affix onto skin of a subject, and the holder comprises a detection window. In some embodiments, the cartridge comprises: a cartridge body; a needle element, positioned at the bottom of the cartridge body, configured to pierce the skin when the cartridge is inserted into the holder; a nitrocellulose membrane having a bottom portion and a top portion separated by a space, wherein the bottom portion has a binding zone in contact with the microneedle array, wherein a low affinity binding partner is bound to the nitrocellulose membrane in the binding zone and does not move, wherein an antibody directed to a target hormone and / or analyte conjugated to a detection label forms a conjugate complex that is positioned on top of the low affinity binding partner and is not bound to the nitrocellulose membrane, wherein the top portion has a detection zone visible to the subject, positioned within the detection window when the cartridge is inserted into the holder, wherein a secondary antibody directed to the target hormone and / or analyte is positioned in the detection zone, and wherein the binding zone is offset from the detection zone; and an absorbent pad positioned at the end of the cartridge away from the holder, and is contact with the top portion of the nitrocellulose membrane.

[0006] In some variations, the needle element is a plurality of microneedles arranged in an array. In certain variations, the microneedle is capable of collecting interstitial fluid and / or skin capillary blood. In one variation, when the cartridge is inserted into the holder, the plurality of microneedles pierces the skin and hits interstitial fluid, and interstitial fluid enters the device and passes through the binding zone. In another variation, when the cartridge is inserted into the holder, the plurality of microneedles pierces the skin and hits skin capillaries, and skin capillary blood flows next to the microneedles such that the microneedles create a fluid flow path into the device.

[0007] In other variations, the needle element comprises a single needle or a lancet. In one variation, when the cartridge is inserted into the holder, the needle or lancet pierces the skin and hits interstitial fluid or skin capillaries, and interstitial fluid or blood flows through the needle or lancet into the device.2MF-36617507223234-20010.40

[0008] When the interstitial fluid does not have the target hormone and / or analyte, the interstitial fluid will not bind the conjugate complex and the interstitial fluid will flow through the device and will be soaked up by the absorbent pad. When the interstitial fluid contains the target hormone and / or analyte, the interstitial fluid binds to the conjugate complex and frees the complex from the low affinity binding partner, and the freed complex travels through the device, and the freed complex binds to the secondary antibody directed to a target hormone and / or analyte in the detection zone to create a color.

[0009] In some of the foregoing aspects, the device provided herein is configured to operate as an ovulation test for the subject. In some embodiments, the device is a wearable, configured to produce a visibly perceptible color upon detection of one or more target hormones and / or analytes in the bodily fluid. In some variations, the target hormone and / or analyte is luteinizing hormone.

[0010] In other aspects, provided is a method of continuously monitoring a target hormone and / or analyte in a bodily fluid of a subject using the device described herein. In some embodiments, the method comprises: affixing the device onto the skin of the subject; and visualizing a change responsive to the presence or increase of the target hormone and / or analyte in the bodily fluid. In some variations, the visualization step comprises detecting a color change responsive to the presence or increase of one or more target hormones and / or analytes. Other methods to visualize the change may be employed, including use of fluorescent dyes, photography, fluorometer, or electrochemical techniques.

[0011] In some embodiments of the foregoing aspects, the target hormone and / or analyte is luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), progesterone, estrogen, cortisol, or testosterone, or a metabolite of any of the foregoing; or a drug (e.g., cocaine, tetrahydrocannabinol, opioids); or a virus or bacteria.DESCRIPTION OF THE FIGURES

[0012] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0013] FIG. 1 depicts an exemplary lateral flow assay (LFA).

[0014] FIGS. 2A and 2B depict the detection of a positive result (FIG. 2A) and a negative result (FIG. 2B) using the lateral flow assay in FIG. 1.3MF-36617507223234-20010.40

[0015] FIG. 3 depicts an exemplary device incorporating the lateral flow assay described herein.

[0016] FIGS. 4A-4C depicts an exemplary device that includes a durable (circular) component and a consumable component that is replaced by the user as needed. As shown in these figures, as the consumable is inserted, the needles are pushed into place to contact the skin without damaging the needles.

[0017] FIG. 5 depicts an exemplary microneedle device configured to incorporate a lateral flow assay.

[0018] FIG. 6 depicts an exemplary process to produce a nitrocellulose membrane as described herein.

[0019] FIG. 7 depicts an exemplary process for continuous hormone monitoring.

[0020] FIG. 8 depicts an exemplary graphical user interface for monitoring changes in hormone levels over a period of time.

[0021] FIG. 9 depicts an exemplary graphical user interface for providing hormone test results to a user.

[0022] FIG. 10 depicts the results of the lateral flow assay to detect LH over a period of time.

[0023] FIG. 11 depicts an exemplary system in which the device is multiplexed such that a plurality of analytes can be analyzed within a single device.DETAILED DESCRIPTION

[0024] The following description sets forth exemplary systems, devices, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0025] In some aspects, provided are devices configured to affix to the skin of a subject and continually monitor bodily fluid collected through the subject’s skin to detect the presence of at least one target hormone and / or analyte, or detect an increase of at least one target hormone and / or analyte. In some embodiments, the device may be configured to target 4MF-36617507223234-20010.40different hormones and analytes including, for example, luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), progesterone, estrogen, cortisol, and testosterone, or a metabolite of any of the foregoing. In such embodiment, the device incorporates a suitable low affinity binding protein that binds to a particular antibody directed to that hormone or analyte. In other embodiments, the device may be configured to target drugs, such as cocaine, tetrahydrocannabinol (THC), opioids, or other recreational drugs or drugs of abuse.

[0026] In yet other embodiments, the device may be configured to target virus or bacteria. In such embodiment, the device incorporates a suitable low affinity binding protein that binds to that drug, virus or bacteria.

[0027] In some embodiments, at least one target hormone and / or analyte is luteinizing hormone (LH). A continuously monitoring solution for a hormone or analyte, and in a particular example LH, provides advantages over other single point in time solutions (such as urine and blood tests). A wearable continuously monitoring test can be regularly checked with minimal inconvenience by the subject woman wearing the device over the course of a day. Thus, the devices and methods provided herein can improve the manner by which a subject detects when a LH surge occurs. In one variation, the device is particularly well-suited to detect LH because during ovulation LH is low, but a spike in LH (generally referred to as a “LH surge”) causes the follicle to rupture and the release of an egg from the follicle, indicating high fertility. In one preferred configuration, the device provides more precise timing for the LH surge of its wearer.

[0028] In other embodiments, at least one target hormone and / or analyte is progesterone, or a metabolite thereof. In yet other embodiments, the device is configured to detect the presence of both LH and progesterone, or a metabolite thereof. In other embodiments, the device is configured to independently or additionally detect the presence of estrogen, or a metabolite thereof. In such embodiments, the device provided herein is particularly well-suited to evaluate for progesterone because after ovulation, a second hormone (following LH), progesterone, is produced which confirms that ovulation has occurred. Therefore, a configuration of the device to monitor both LH to detect the LH surge and progesterone to detect subsequent increase in progesterone after the surge could be useful for monitoring ovulation in a single menstrual cycle of a subject woman.5MF-36617507223234-20010.40

[0029] Exemplary devices and components and configurations thereof, as well as methods of using such devices, are described in further detail below.Wearable Device

[0030] In some aspects, provided is a device configured to affix onto skin of a subject and to continuously monitor bodily fluid of the subject for an increase in the presence of one or more target hormones and / or analytes.

[0031] The device described herein provides an alternative design to the use of stacked membranes that employ a magnetic force to draw the primary membrane away from the skin to the secondary membrane. The solution provided by the device herein preserves the binding of a low affinity binding partner to the primary membrane when the target hormone and / or analyte is present. The device described herein further provides an alternative design that allows the subject using the device to visually observe the signal properly, avoiding potential confusion in the result observed where a signal on the primary membrane is improperly observed through the secondary membrane stacked on top.

[0032] With reference to FIG. 1, an exemplary lateral flow assay (LFA) is depicted. In the presence of the target hormone and / or analyte in the interstitial fluid (ISF), the target hormone and / or analyte flows through the needle element (e.g., the microneedles) and binds to the target hormone and / or analyte antibody that is bound to the colloidal gold color, displacing the target hormone and / or analyte antibody from the low affinity binding protein that is particular for the target hormone and / or analyte antibody used. A large sample pad within the device draws interstitial fluid and maintains continual flow of interstitial fluid from the skin of the subject through the device. This LFA design uses lateral flow due to capillary action.

[0033] With reference to FIGS. 2A and 2B, depicted is the detection of a positive result and a negative result, respectively, using the LFA described herein. The design offsets the binding zone from the detection zone, and therefore allows for traditional lateral flow parallel to the surface of the skin away from the binding zone offset into the detection zone. In some embodiments, one feature of the wearable device described herein is the use of a square sample pad helps to create capillary action, which keeps the flow going to draw enough liquid to continually have flow in the system. In some embodiments, another feature of the6MF-36617507223234-20010.40wearable device is the chemistry in the LFA, which is configured to bend around the applicator.

[0034] In other variations, alternatives to the bending design are contemplated. It should be understood that the bend slows down the fluid path. Thus, in other variations, the device is configured to control the fluid flow rate, which is important for wear time. In certain variations, the device is configured to control the fluid flow rate so that the device can continually pull interstitial fluid for at least 12 hours and delay time from when surge in interstitial fluid happens to when it is reported on the device.

[0035] With reference to FIG. 3, depicted is an exemplary device that may be affix to the skin of a subject. The device is configured to continually monitors, or capable of continually monitoring, bodily fluid collected through the subject’s skin to detect the presence of at least one target hormone and / or analyte, or detect an increase of at least one target hormone and / or analyte.

[0036] With reference to FIG. 4A, the device includes a reusable holder (which is the durable component of the device) and a single use cartridge (which is the consumable component of the device). To use the device, the user (subject) places the reusable holder (durable component) on the body with an adhesive (e.g., a skin-safe adhesive). Each morning, with reference to FIGS. 4B and 4C, the user inserts a new cartridge into the device. The cartridge has microneedles that when “snapped” into place (see FIG. 4C) enter the skin layer to hit the interstitial fluid. As the consumable cartridge is inserted into the durable holder, the needles are pushed into place to contact the user’s skin without damaging the needles. The interstitial fluid will either flow through hollow needles, or flow next to needles where a needle creates a fluid flow path. The interstitial fluid then flows into the device and on the sensor. The interstitial fluid passes first through the binding zone, where the low affinity binding partner conjugated to the carrier protein is bound to a primary membrane and will not move.

[0037] Any suitable membranes may be used. For example, in some variations, the membrane is a nitrocellulose (NC) membrane. In some embodiments, the target hormone and / or analyte antibody conjugated to the detection label is sprayed on top of the low affinity binding partner but is not bound to the membrane. For instance, in one variation, a low-affinity binding partner solution is prepared. The low-affinity binding partner solution is then7MF-36617507223234-20010.40sprayed onto a section of a nitrocellulose strip. The nitrocellulose strip is then dried. An antitarget hormone and / or analyte antibody is conjugated to a detection label. The target hormone and / or analyte-detection label is then sprayed on top of the low-affinity binding partner section. The antibody is allowed to bind to exposed low-affinity binding partner and dried. An anti-target hormone and / or analyte antibody solution is prepared, and an anti-target hormone and / or analyte antibody line is striped upstream from the low-affinity binding partner-target hormone and / or analyte-antibody detection label section of the nitrocellulose. The nitrocellulose is then dried.

[0038] In other variation, a solution of the low affinity binding partner is incubated with the anti-target hormone and / or analyte antibody solution conjugated with the detection label, so that it pre-binds the antibody. Then, the target hormone and / or analyte antibody bound to the low affinity binding partner conjugated with the detection label solution is sprayed on a section of a nitrocellulose strip, which is then dried. An anti-target hormone and / or analyte antibody solution is prepared, and an anti-target hormone and / or analyte antibody line is striped upstream from the detection labeled section of the nitrocellulose, which is then dried.

[0039] If the interstitial fluid does not have the target hormone and / or analyte, the interstitial fluid will not bind the conjugate complex. Instead, the interstitial fluid will flow through the device, round the curve in the device and be soaked up by the sample pad at the end. The device will continually run the fluid through it until the sample pad becomes saturated. In some variations, the cartridge must be replaced, e.g., once every 12-24 hours. If the fluid has the target hormone and / or analyte, the target hormone and / or analyte will bind to the conjugate complex, freeing it from the low affinity binding partner. The conjugate complex will travel through the device, around the curve to the detection zone. The detection zone is where a secondary the target hormone and / or analyte antibody is striped on a secondary membrane. The conjugate complex will bind to the secondary the target hormone and / or analyte antibody, which will create a color. The detection zone is visible to the user. In some variations, the detection zone may be covered with a camera that will image the zone and see the color change. The color change may be fed to an electronic device, such as a mobile device with an app via Bluetooth. The rate at which the red change occurs is a direct correlation to the level of the target hormone and / or analyte in the interstitial fluid. Once the app detects a significant change, the app will send a push notification to alert the user or medical professional.8MF-36617507223234-20010.40

[0040] With reference again to FIGS. 4A-4C, it should be understood that, in other variations, the device may be configured to include other needle elements, such as a single needle or a lancet in lieu of the plurality of microneedles arranged in an array. In some variations, the needle element comprises a single needle. In one variation, the needle element comprises a single needle having a length between about 25 pm and about 2000 pm, between about 25 pm and about 1000 pm, between about 100 pm and about 2000 pm, between about 100 pm and about 1000 pm, between about 750 pm and about 2000 pm, or between about 750 pm and about 1000 pm. In other variations, the needle element comprises a lancet. In certain variations of the foregoing, when the cartridge is inserted into the holder, the needle or lancet pierces the skin and hits interstitial fluid or skin capillaries, and interstitial fluid or blood flows through the needle or lancet into the device.

[0041] In some embodiments, the devices described herein may be multiplexed to measure multiple analytes at once or have multiple detection zones for the same analyte to detect different levels of the analyte. For example, in one variation, the device is configured for longer wear or range, and would measure LH twice, and direct flow to the second LH line once the first LH line is saturated or the second LH line would only activate when concentrations were above a pre-defined threshold.

[0042] With reference to FIG. 11, in certain embodiments, the device is multiplexed such that a plurality of analytes can be analyzed within a single device. In certain embodiments, the device is multiplexed such that at least 2, at least 3, at least 4, or at least 5 analytes can be analyzed within a single device. In certain embodiments, the device is multiplexed such that up to 5 or up to 10 analytes can be analyzed within a single device. In one embodiment, the device is multiplexed such that between 2 and 10, or between 5 and 10 analytes can be analyzed within a single device. When the device is multiplexed such that a plurality of analytes can be analyzed within a single device, the fluid enters the device via the needle element and the fluid is channeled into one of the fluid paths. In one variation, each track runs in parallel. In other variations, there are multiple detection zones on each track. For example, a first track is configured to measure, or capable of measuring, two analyses at once and instead of a single visual line, the device would display two lines.

[0043] In some variations, the device includes a housing that fully encloses and incorporates a camera and Bluetooth to continuously read and deliver results. In other9MF-36617507223234-20010.40variations, the device includes an aperture for a smartphone to capture a photo of the results, which can then be interpreted and reported to the user.

[0044] The devices and lateral flow assays described herein may be used to detect any suitable target hormones and / or analytes. For example, in some variations, the target hormone and / or analyte is luteinizing hormone (LH). In one embodiment when the device is configured to monitor LH, the low affinity binding partner is CRK conjugated to the carrier protein. It should be understood, however, that other suitable low affinity binding partners may be used, even for different LH antibodies used in the device. Any suitable antibody binding assays may be used to identify suitable low affinity binding partners. For example, an antibody binding assay may be used to screen different proteins. The top hit expected to bind to the antibody directed to the target hormone and / or analyte is the target hormone and / or analyte itself. The next top hit(s) to bind to the antibody directed to the target hormone and / or analyte may be identified as a “low affinity binding partner”.

[0045] In certain embodiments, a low-affinity binding partner is a molecule capable of reversibly binding an antibody or antibody conjugate specific for a target hormone and / or analyte, including for example proteins, peptides, or other binding moieties.

[0046] In some variations, a low affinity binding partner may be identified using a ratio of: (1) a binding affinity of the target hormone and / or analyte to an antibody directed to the target hormone and / or analyte to (2) a binding affinity of a low affinity binding partner to the antibody directed to the target hormone and / or analyte. In some variations, the ratio is 1 or greater. In some variations, the ratio is less than 40. Any suitable measurement of binding affinity may be used to identify binding affinities of the target hormone and / or analyte or the low affinity binding partner to the antibody directed to the target hormone and / or analyte. For example, an antibody binding assay may be used to measure the binding affinity of different proteins. Any hit that when treated as a low affinity binding partner results in a ratio that is equal to or greater than 1 and less than 40 may be identified as a suitable low affinity binding partner.

[0047] In some variations, suitable low affinity binding partners for LH as the analyte include CRK, CRKL, and CSBP2. In some variations, suitable low affinity binding partners for LH as the analyte include CRK.10MF-36617507223234-20010.40

[0048] In some variations, suitable low affinity binding partners for HCG as the analyte include GDI2 and SIRT5.

[0049] Any suitable detection labels may also be used in the device. For example, in some variations, the detection label is colloidal gold, latex beads, or fluorescent dyes. In other variations, any combination of suitable detection labels may also be used. In one variation, when colloidal gold is used as the detection label, the gold conjugate complex will bind to the secondary the target hormone and / or analyte antibody and create a red color. In other variations, visualization of the results using the device may (1) be visual (e.g., via a visual red dye), (2) employ fluorescent dyes to increase sensitivity so that visualization is by photography, (3) employ fluorometer, or (4) employ other electrochemical techniques.

[0050] In other embodiments, the devices provided herein are configured to detect or capable of detecting multiple hormones and / or analytes at the same time. In some variations, the device comprises two or more membranes sitting side by side, with each membrane having a single detection zone. In certain variations, the device comprises two, three, four or five membranes sitting side by side, with each membrane having a single detection zone. In other variations, the device comprises one membrane with multiple detection zones, such as three or four lines of color. In certain variations, the lines of color may all be red (for example, when colloidal gold is used as the detection label). In other variations, the lines of color could be different colors (for example, when different latex beads are used). In certain variations, the device may use fluorescence of different wavelengths, leading to different colors. The use of different colors would allow for a single detection zone where the camera could measure multiple colors at once. It should be understood that while the aforementioned descriptions refer to visualization by color, other methods of visualization discussed herein may also be employed.

[0051] In the case of an ovulation test, such a wearable device could simultaneously detect for the presence of LH, estrogen and progesterone or a metabolite thereof in an applied bodily fluid. In an example of use of such configuration, the user could place the device on her skin, preferably on her torso near the belly button. She would wear the device started after the end of her menstrual cycle. Once she had a surge in LH, the color in the LH detection zone would increase. Then when her progesterone increased, the color in the progesterone detection zone would increase. So, a woman that has not ovulated would see no color in the detection zone(s). A woman that is about to ovulate and produced LH would be 1 line / area of 11MF-36617507223234-20010.40color. Then, in one example, once she ovulated, she would see two lines / areas of color to account for the presence of LH and progesterone. At that point, the woman has successfully ovulated and can remove the device. The device could be replaced once daily or ideal, up to 14 days. Typically, a woman would start using this device to track ovulation on the 5-10thday or her cycle and stop using it on the 14th-23rdday of her cycle if her cycle length is the average length of 25-35 days.

[0052] The devices provided herein can provide continuous real-time monitoring of target hormones and / or analytes. For example, the devices may be used to provide a full view of hormone patterns and key events in IVF treatment. The devices are designed for easy of application and compliance by user, and notifications can be sent directly to the patient and / or doctor.

[0053] In some embodiments, the devices provided herein are configured to have different disposable cartridges. The same reader may be used for different test suites. For example, in one variation, a first suite is configured for ovulation analysis, a second suite for pregnancy tracking, and a third suite for drugs of abuse.

[0054] In some aspects, provided is a method of continually monitoring hormone and / or analyte levels in a subject, with reference to FIG. 7, an exemplary process for continuous hormone and / or analyte monitoring is depicted. In Step 1, the device as described herein is affixed onto the subject’s body. In Step 2, the subject inserts the cartridge (e.g., single use cartridge) into the holder. In Step 3, hormones and / or analytes are continuously monitored using the device. In Step 4, the cartridge is replaced on a daily basis. In Step 5, when the target hormones and / or analytes surge, the device is configured to send a notification to the patient or the doctor. When such notification is received by the doctor, a patient can be immediately seen to prevent premature ovulation or better time egg retrievals.

[0055] In some aspects, provided is an ornamental design for a graphical user interface for a display screen or portion thereof. With reference to FIG. 8, depicted is an exemplary graphical user interface for monitoring changes in hormone and / or analyte levels over a period of time. With reference to FIG. 9, depicted is an exemplary graphical user interface for providing hormone / analyte test results to a user.12MF-36617507223234-20010.40EXAMPLES

[0056] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1: Microneedle Device Incorporating Lateral Flow Assay

[0057] This example demonstrates the establishment of a fluid path using the microneedle device depicted in FIG. 5. A treated sample pad wicked fluid from the needles to the sensor. A sponge soaked with liquid was used in this study to act as an analog to skin.Example 2: Identification of Low Affinity Binding Partners

[0058] This example describes the identification of suitable low affinity binding proteins using a human protein array and determining the binding partners of a specific antibody. The lower affinity binding protein is then used in a lateral flow assay.

[0059] Four antibody samples were analyzed for binding specificities on HuProt human proteome arrays, which contain the largest human protein collection on a single array.Exemplary target hormones and / or analytes, specifically LH and HCG hormones, were added to the arrays as positive controls. After blocking, the arrays were probed with the antibody samples (10 pg / ml) at room temperature for 1 hour, including LH antibodies or HCG antibodies. Then the arrays were washed with TBST for 5 min x 3 and probed with Cy3-anti-mouse IgG secondary antibody under conditions optimized by CDI Labs for signal detection.

[0060] Measurements were made to identify which proteins the each antibody would bind to. Data analysis was performed by first eliminating non-specific hits that directly bound to the secondary antibodies from the analysis of the samples. CDI software was used to quantify the specificity of each individual sample to specific proteins on the array based on Z scores. The Z score is the average Z score of the duplicate spots of a given protein (each protein is printed in duplicate on a HuProtTM array). For arrays testing LH antibodies, the Z score of each spot on a given array was calculated according to the algorithm Z = [F532 -F532 (avg)] / F532(std), where F532(avg) and F532(std) are the average and standard derivation of the F532 values of all spots on the array, respectively. For arrays testing HCG13MF-36617507223234-20010.40antibodies, the Z score of each spot on a given array was calculated according to the algorithm Z = [F635 - F635 (avg)] / F635(std), where F635(avg) and F635(std) are the average and standard derivation of the F635 values of all spots on the array, respectively. Additional scores for each array were S scores, F532 (for LH antibodies) or F635 (for HCG antibodies), and B532 (for LH antibodies) or B635 (for HCG antibodies). S score is the difference of the Z scores of a given protein and the one ranked next to it. If the S score of the top hit was > 3, the antibody was considered to be highly specific for the top hit. F532 and F635 is the average foreground signal intensity of 2 replicate spots of a given protein in the detection channel (532 nm or 635 nm, respectively). B532 or B635 is the average background signal intensity of 2 replicate spots of a given protein in the detection channel (532 nm or 635 nm, respectively).

[0061] Z score analysis was performed to identify the target of each sample, with nonspecific hits bound by the secondary antibody removed from the analysis of the sample. The results using a first tested LH antibody is shown in Table 1. The results indicate that the first tested LH antibody recognized the LH hormone control as the top hit with extreme high specificity.Table 1 : Z score distribution of a first tested LH antibody on a native HuProt Array.

[0062] The results using a second tested LH antibody is shown in Table 2. The results indicate that the second tested LH antibody also recognized the LH hormone control as the top hit, but with low specificity since it showed equal binding signals on CRK.14MF-36617507223234-20010.40Table 2: Z score distribution of a second tested LH antibody on a native HuProt Array.

[0063] The results using a first tested HCG antibody is shown in Table 3. The results indicate that the first tested HCG antibody recognized the HCG control as the top hit with extremely high specificity.Table 3: Z score distribution of a first tested HCG antibody on a native HuProt Array.

[0064] The results using a second tested HCG antibody is shown in Table 4. The results indicate that the second tested HCG antibody recognized CDI’s CGB2 and CGB proteins as the top hits with high specificity and the HCG control as the 8thhit.Table 4: Z score distribution of a second tested HCG antibody on a native HuProt Array.15MF-36617507223234-20010.40

[0065] To select a suitable low affinity binding partner, the ratio of: (1) the binding affinity of the target hormone and / or analyte to an antibody to (2) the binding affinity of the low affinity binding partner to the same antibody should be 1 or greater, as the antibody should have the same or higher binding affinity to the target hormone and / or analyte as compared to the low affinity binding partner in order to free the antibody from the low affinity binding partner to instead bind to the target hormone and / or analyte. Further, the ratio of (1) to (2) should be less than 40, as the low affinity binding partner needs to effectively bind to the antibody so that the antibody will not move until the target hormone and / or analyte is present. For the second tested LH antibody, the binding affinity (based on the F532) for LH was 65535 and the binding affinity for the second ranked hit CRK was 65535, resulting in a ratio of 1. For the second tested HCG antibody, the binding affinity (based on the F635) for HCG was 2988.5 and the binding affinity for the ninth ranked hit SIRT5 was 1829, resulting in a ratio of around 1.6. As all these ratios are equal or higher to 1 and less than 40, it was observed that a suitable low affinity binding partner for the second tested LH antibody was CRK, and a suitable low affinity binding partner for the second tested HCG antibody was SIRT2. This procedure may be repeated for other target hormones and / or analytes to identify suitable low affinity binding partners to the target hormone and / or analyte for a particular antibody directed to the target hormone and / or analyte.Example 3A: Membrane Preparation

[0066] This example describes the preparation of a membrane for use in the lateral flow assay as described herein. See FIG. 6. “CRK” refers adapter molecule crk, also known as proto-oncogene c-Crk, which is a protein that in humans is encoded by the CRK gene.

[0067] CRK protein was prepared in a suitable buffer. The CRK solution was sprayed on the bottom quarter section of a nitrocellulose strip. The nitrocellulose strip was dried. Anti LH antibody was conjugated to colloidal gold. The LH-gold conjugate was sprayed directly 16MF-36617507223234-20010.40on top of the CRK section. The nitrocellulose was placed onto the benchtop to allow the antibody to bind to exposed CRK and dry. An anti-LH antibody solution was prepared in buffer. An anti-LH antibody line was striped upstream from the CRK-LH-Ab gold section of the nitrocellulose. The nitrocellulose was dried.Example 3B: Membrane Preparation

[0068] This example describes another method for preparation of a membrane for use in the lateral flow assay as described herein. CRK protein was prepared in a suitable buffer. The CRKsolution was incubated with the anti-LH antibody-gold conjugate so that it pre-binds the antibody. Then, the CRK bound anti-LH antibody-gold conjugate solution was sprayed on the bottom quarter section of a nitrocellulose strip. The nitrocellulose strip was dried. An anti-LHantibody solution was prepared in a suitable buffer. An anti-LH antibody line was striped upstream from the CRK-LH-Ab gold section of the nitrocellulose. The nitrocellulose was dried.Example 4: Lateral Flow Assay

[0069] This example describes the use of the lateral flow assay as described herein to detect LH. A length of the nitrocellulose membrane prepared according to Example 3 was placed onto a backing card. Then, a length of the absorbent pad was placed onto the backing card, such as there was contact with the nitrocellulose membrane. More stacked pads were added for extended testing and held in place with a clip. The nitrocellulose-absorbent pad was cut into ~5mm strips.

[0070] Positive and negative control solutions were also created. The negative control solution was 1% TWEEN in PBS. The positive control solution contained 212 lU / mL LH in the negative control solution.

[0071] Methods of preparing the nitrocellulose strip were tested: no washing, washing 2x in 65 pL using a pipette containing the negative control solution, and washing by submerging the nitrocellulose strip in a beaker of BPS and mixing using a magnetic stir bar on low for 15 minutes.17MF-36617507223234-20010.40

[0072] Following preparation of the nitrocellulose strip, the CRK-LH-Ab gold section of the nitrocellulose strip was submerged into approximately 2 mL of the test solutions. Fluid was allowed to flow for 1 hour while recording the detection line. A video of the test was imported into a piece of software that showed the average luma, and RGB channels were plotted over time. Specifically, the raw video footage (Motorola G100) was exported into a tracker video analysis software. An area of interest was identified over the detection line. The software then output the average red channel intensity of the area of interest at a specific sampling rate of 1Hz (sample every 24 frames, recorded at 24 fps).

[0073] As shown in FIG. 10, the red channel value of the detection line brightened during washing with a solution containing no LH. After the positive control solution containing LH was added, the detection line darkened over time. These results indicate that the lateral flow assay was able to continually monitor the present of LH in solution and only produced signal when LH was present.18MF-366175072

Claims

23234-20010.40CLAIMSWhat is claimed is:

1. A device, comprising: a cartridge configured to insert into a holder,wherein the holder is configured to affix onto skin of a subject, and the holder comprises a detection window; andwherein the cartridge comprises:a cartridge body;a needle element, positioned at the bottom of the cartridge body, configured to pierce the skin when the cartridge is inserted into the holder;a nitrocellulose membrane having a bottom portion and a top portion separated by a space,wherein the bottom portion has a binding zone in contact with the needle element,wherein a low affinity binding partner is bound to the nitrocellulose membrane in the binding zone and does not move,wherein an antibody directed to a target hormone and / or analyte conjugated to a detection label forms a conjugate complex that is positioned on top of the low affinity binding partner and is not bound to the nitrocellulose membrane, wherein the top portion has a detection zone visible to the subject, positioned within the detection window when the cartridge is inserted into the holder,wherein a secondary antibody directed to the target hormone and / or analyte is positioned in the detection zone, and wherein the binding zone is offset from the detection zone; and an absorbent pad positioned at the end of the cartridge away from the holder, and is contact with the top portion of the nitrocellulose membrane.

2. The device of claim 1, wherein when the cartridge is inserted into the holder, the needle element pierces the skin and hits interstitial fluid, and interstitial fluid enters the device and passes through the binding zone, and19MF-36617507223234-20010.40when the interstitial fluid does not have the target hormone and / or analyte, the interstitial fluid will not bind the conjugate complex and the interstitial fluid will flow through the device, and be soaked up by the absorbent pad,when the interstitial fluid contains the target hormone and / or analyte, the interstitial fluid binds to the conjugate complex and frees the complex from the low affinity binding partner, and the freed complex travels through the device, and the freed complex binds to the secondary antibody directed to a target hormone and / or analyte in the detection zone to create a color.

3. The device of claim 1 or 2, wherein the nitrocellulose membrane is curved around the cartridge body at the end of the cartridge that inserts into the holder.

4. The device of any one of the preceding claims, wherein a ratio of: (1) a binding affinity of the target hormone and / or analyte to the antibody directed to the target hormone and / or analyte to (2) a binding affinity of the low affinity binding partner to the antibody directed to the target hormone and / or analyte is equal to or greater than 1 and less than 40.

5. The device of any one of the preceding claims, wherein the target hormone and / or analyte is:luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), progesterone, estrogen, cortisol, or testosterone, or a metabolite of any of the foregoing; ora recreational drug or drug of abuse; ora virus or bacteria.

6. The device of any one of the preceding claims, wherein the target hormone and / or analyte is luteinizing hormone (LH), and wherein the antibody directed to the target hormone and / or analyte is a LH antibody.

7. The device of any one of the preceding claims, wherein the detection label comprises colloidal gold, latex beads, or fluorescent dyes, or any combination thereof.

8. A device, comprising: a cartridge configured to insert into a holder,wherein the holder is configured to affix onto skin of a subject, and the holder comprises a detection window; andwherein the cartridge comprises:20MF-36617507223234-20010.40a cartridge body;a needle element, positioned at the bottom of the cartridge body, configured to pierce the skin when the cartridge is inserted into the holder;a nitrocellulose membrane that is curved around the cartridge body at the end of the cartridge that inserts into the holder,wherein the curved nitrocellulose membrane has a bottom portion and a top portion,wherein the bottom portion has a binding zone in contact with the needle element,wherein a low affinity binding partner is bound to the nitrocellulose membrane in the binding zone and does not move,wherein luteinizing hormone (LH) antibody conjugated to colloidal gold forms a gold conjugate complex that is positioned on top of the low affinity binding partner and is not bound to the nitrocellulose membrane, wherein the top portion has a detection zone visible to the subject, positioned within the detection window when the cartridge is inserted into the holder,wherein a secondary LH antibody is positioned in the detection zone, andwherein the binding zone is offset from the detection zone; and an absorbent pad positioned at the end of the cartridge away from the holder, and is contact with the top portion of the nitrocellulose membrane.

9. The device of claim 8, wherein when the cartridge is inserted into the holder, the needle element pierces the skin and hits interstitial fluid, and interstitial fluid enters the device and passes through the binding zone, andwhen the interstitial fluid does not have LH, the interstitial fluid will not bind the gold conjugate complex and the interstitial fluid will flow through the device, round the curve in the device and be soaked up by the absorbent pad,when the interstitial fluid contains LH, the interstitial fluid binds to the gold conjugate complex and frees the complex from the low affinity binding partner, and the freed gold21MF-36617507223234-20010.40complex travels through the device, around the curve to the detection zone, and the freed gold complex binds to the secondary LH antibody in the detection zone to create a red color.

10. The device of any one of claims 6 to 9, wherein a ratio of: (1) a binding affinity of LH to the LH antibody to (2) a binding affinity of the low affinity binding partner to the LH antibody is equal to or greater than 1 and less than 40.

11. The device of any one of the preceding claims, wherein the low affinity binding partner is CRK.

12. The device of any one of the preceding claims, further comprising: a camera configured to image the detection zone and detect a color change.

13. The device of any one of the preceding claims, wherein the cartridge is a single use cartridge, and the holder is a reusable holder.

14. The device of any one of claims 1 to 13, wherein the needle element comprises a plurality of microneedles arranged in an array.

15. The device of any claim 14, wherein the microneedles are hollow.

16. The device of claim 14 or 15, wherein when the cartridge is inserted into the holder, the plurality of microneedles pierces the skin and hits interstitial fluid, and interstitial fluid flows through the hollow microneedles into the device.

17. The device of any one of claims 14 to 16, wherein when the cartridge is inserted into the holder, the plurality of microneedles pierces the skin and hits interstitial fluid or skin capillaries, and interstitial fluid or blood flows next to the microneedles such that the microneedles create a fluid flow path into the device.

18. The device of any one of claims 1 to 13, wherein the needle element comprises a single needle or a lancet.

19. The device of claim 18, wherein when the cartridge is inserted into the holder, the needle or lancet pierces the skin and hits interstitial fluid or skin capillaries, and interstitial fluid or blood flows through the needle or lancet into the device.22MF-36617507223234-20010.4020. The device of any one of the preceding claims, wherein the device is multiplexed such that a plurality of analytes can be analyzed within a single device.

21. The device of any one of the preceding claims, wherein the reusable holder further comprises an adhesive to affix the reusable holder onto the skin of the subject.

22. The device of any one of the preceding claims, wherein the device is configured to affix to the subject’s torso near the belly button.

23. A lateral flow assay, comprising:a nitrocellulose membrane having a bottom portion and a top portion separated by a space,wherein the bottom portion has a binding zone that is configured to come into contact with interstitial fluid,wherein a low affinity binding partner is bound to the nitrocellulose membrane in the binding zone,wherein an antibody directed to a target hormone and / or analyte conjugated to a detection label forms a conjugate complex that is positioned on top of the low affinity binding partner and is not bound to the nitrocellulose membrane,wherein the top portion has a detection zone visible to the subject, wherein a secondary antibody directed to the target hormone and / or analyte is positioned in the detection zone, andwherein the binding zone is offset from the detection zone.

24. The lateral flow assay of claim 23, wherein:when the interstitial fluid does not have the target hormone and / or analyte, the interstitial fluid will not bind the conjugate complex,when the interstitial fluid contains the target hormone and / or analyte, the interstitial fluid binds to the conjugate complex and frees the complex from the low affinity binding partner, and the freed complex binds to the secondary antibody directed to the target hormone and / or analyte in the detection zone to create a color.

25. The later flow assay of claim 23 or claim 24, wherein a ratio of: (1) a binding affinity of the target hormone and / or analyte to the antibody directed to the target hormone and / or23MF-36617507223234-20010.40analyte to (2) a binding affinity of the low affinity binding partner to the antibody directed to the target hormone and / or analyte is equal to or greater than 1 and less than 40.

26. The lateral flow assay of any one of claims 23 to 25, wherein the target hormone and / or analyte is:luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), progesterone, estrogen, cortisol, or testosterone, or a metabolite of any of the foregoing; ora recreational drug or drug of abuse; ora virus or bacteria.

27. The lateral flow assay of any one of claims 23 to 26, wherein the target hormone and / or analyte is luteinizing hormone (LH), and wherein the antibody directed to the target hormone and / or analyte is a LH antibody.

28. The lateral flow assay of any one of claims 23 to 27, wherein the detection label comprises colloidal gold, latex beads, or fluorescent dyes, or any combination thereof.

29. A lateral flow assay, comprising:a nitrocellulose membrane that is curved around the cartridge body at the end of the cartridge that inserts into the holder,wherein the curved nitrocellulose membrane has a bottom portion and a top portion,wherein the bottom portion has a binding zone that is configured to come into contact with interstitial fluid,wherein a low affinity binding partner is bound to the nitrocellulose membrane in the binding zone,wherein luteinizing hormone (LH) antibody conjugated to colloidal gold forms a gold conjugate complex that is positioned on top of the low affinity binding partner and is not bound to the nitrocellulose membrane, wherein the top portion has a detection zone visible to the subject, wherein a secondary LH antibody is positioned in the detection zone, andwherein the binding zone is offset from the detection zone.24MF-36617507223234-20010.4030. The lateral flow assay of claim 29, wherein:when the interstitial fluid does not have LH, the interstitial fluid will not bind the gold conjugate complex,when the interstitial fluid contains LH, the interstitial fluid binds to the gold conjugate complex and frees the complex from the low affinity binding partner, and the freed gold complex binds to the secondary LH antibody in the detection zone to create a red color.

31. The lateral flow assay of any one of claims 27 to 30, wherein a ratio of: (1) a binding affinity of LH to the LH antibody to (2) a binding affinity of the low affinity binding partner to the LH antibody is equal to or greater than 1 and less than 40.

32. The lateral flow assay of any one of claims 23 to 31, wherein the low affinity binding partner is CRK.

33. A method of continuously monitoring a target hormone and / or analyte in a bodily fluid of a subject, comprising:affixing the device of any one of claims 1 to 22 onto the skin of the subject; and visualizing a change responsive to the presence or increase of the target hormone and / or analyte in the bodily fluid.

34. The method of claim 33, wherein visualizing the change comprises detecting a color change responsive to the presence or increase of the target hormone and / or analyte in the bodily fluid.

35. The method of claim 33 or 34, wherein the target hormone and / or analyte is:luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), progesterone, estrogen, cortisol, or testosterone, or a metabolite of any of the foregoing; ora recreational drug or drug of abuse; ora virus or bacteria.

36. The method of any one of claims 33 to 35, further comprising:photographing the device;sweeping the photograph of the device, or a cropped area of the device;25MF-36617507223234-20010.40creating a vector of color values including the color values obtained from a plurality of horizontal pixel positions on the photograph of the device or cropped area of the photograph of the device;identifying the location of the perceptible color depicted within the photograph of the device; andcalibrating to determine a hormone or analyte level corresponding to a color value.26MF-366175072

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