Magnetic formulations for biomarker sampling and enhanced drug delivery

Abstract

This disclosure describes formulations, methods, and apparatus for biomarker sampling and therapeutic delivery using magnetic formulations. When combined with the application of an external magnetic field, the magnetic formulation moves within the nasal cavity. Magnetic formulations offer the benefits of targeting or guiding formulation placement via a magnetic field, enhancing formulation mixing via a magnetic field, enhancing biological material collection via antibody-coated magnetic beads, or enhancing sample retrieval via an inserter with a magnetic tip. Exemplary biological materials for collection include proteins, enzymes, neural stem cells, and other biomarkers.

Description

[0001] Cross-referencing This PCT application claims the benefit of U.S. Provisional Application No. 63 / 606,080, filed December 4, 2023, the contents of which are incorporated herein by reference.

[0002] background The human nasal cavity is a region of interest for both biomarker sampling and therapeutic delivery. Intranasal delivery of sampling and therapeutic formulations allows for interaction with target areas of the nasal cavity, such as the olfactory region. For certain applications, precise control of contact between the intranasally delivered formulation and the target area of ​​the nasal cavity is advantageous. There is a need for formulations, methods, and devices that enhance intranasal biomarker sampling and therapeutic delivery. Additionally, there is a need for formulations, methods, and devices that enhance biomarker sampling and therapeutic delivery targeting other body surfaces.

[0003] Overview The inventors recognize that challenges associated with intranasal biomarker sampling and therapeutic delivery include difficulties related to insufficient contact between the intranasally delivered formulation and the target region of the nasal cavity. In one aspect, this document provides a method for collecting biomaterial from an intranasal region of a subject, the method comprising: delivering a magnetic formulation comprising more than one magnetic particle to the intranasal region of the subject, wherein the magnetic formulation is configured to capture biomaterial; and retrieving at least a portion of the magnetic formulation from the intranasal region, thereby collecting any biomaterial captured by that portion of the magnetic formulation. In some embodiments, the intranasal region is the olfactory region of the subject or a target subregion of the nasal cavity. In some embodiments, the biomaterial is captured from cerebrospinal fluid (CSF). In some embodiments, analysis of the collected biomaterial provides quantitative values ​​of the concentration of tau protein, the concentration of β-amyloid protein, or a ratio thereof.

[0004] By incorporating via reference All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Brief description of the attached diagram The novel features of this disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings of illustrative embodiments utilizing the principles of this disclosure.

[0006] Figure 1 A flowchart illustrating the steps in a method for collecting biological materials according to one embodiment is depicted.

[0007] Figure 2AAn illustration depicts an embodiment for collecting biological material from a nasal cavity, wherein the embodiment includes a device comprising a cannula inserted into the nasal cavity.

[0008] Figure 2B Depicting according to Figure 2A An illustration of an implementation scheme for collecting biological materials, wherein a container holding the preparation is connected to a sheath.

[0009] Figure 2C Depicting according to Figure 2A An illustration of an implementation scheme for collecting biological materials, wherein the formulation is delivered into the nasal cavity.

[0010] Figure 2D Depicting according to Figure 2A An illustration of an implementation scheme for collecting biological materials, wherein the biological materials are captured by a formulation.

[0011] Figure 2E Depicting according to Figure 2A An illustration of an implementation scheme for collecting biological materials, wherein a recovery vessel is connected to a sleeve to remove and collect biological materials.

[0012] Figure 2F Depicting according to Figure 2A An illustration of an implementation scheme for collecting biological material, wherein the formulation and captured biological material are removed through a cannula and placed into a recovery vessel.

[0013] Figure 3A An illustration depicts another embodiment for collecting biological material from a nasal cavity, wherein the embodiment includes a device comprising a flexible sphere and a cannula inserted into the nasal cavity.

[0014] Figure 3B Depicting according to Figure 3A An illustration of an implementation scheme for collecting biological materials, wherein a formulation is delivered into the nasal cavity by pressing a flexible sphere.

[0015] Figure 3C Depicting according to Figure 3A An illustration of an implementation scheme for collecting biological materials, wherein the biological materials are captured by a formulation.

[0016] Figure 3D Depicting according to Figure 3A An illustration of an implementation scheme for collecting biological material, wherein a flexible sphere is allowed to relax to retrieve and capture the biological material.

[0017] Figure 4A An illustration depicts another embodiment for collecting biological material from the nasal cavity, wherein the embodiment includes a delivery device comprising a container for containing the preparation, a deployment mechanism, and a cannula inserted into the nasal cavity.

[0018] Figure 4B Depicting according to Figure 4A An illustration of an implementation scheme for collecting biological materials, wherein the preparation is delivered to the nasal cavity via a press-deployment mechanism.

[0019] Figure 4C Depicting according to Figure 4A An illustration of an embodiment for collecting biological materials, wherein the delivery device is removed from the nasal cavity.

[0020] Figure 4D Depicting according to Figure 4A An illustration of an implementation scheme for collecting biological materials, wherein the biological materials are captured by a formulation.

[0021] Figure 4E Depicting according to Figure 4A The illustration shows an embodiment for collecting biological material, in which a retrieval device filled with wicking material and a retrieval cannula are inserted into the nasal cavity.

[0022] Figure 4F Depicting according to Figure 4A The illustration shows an implementation scheme for collecting biological materials, wherein the recovery device uses wicking material to aspirate the preparation and biological materials through a cannula.

[0023] Figure 5 A simplified nose model constructed from three machined polycarbonate sheets is depicted.

[0024] Figure 6 A bead is depicted settling in water (1 cP) within the nasal model after a few seconds. The diagonal passages of the nasal model correspond to the human nasal passage. Because the nasal model is oriented inverted relative to the human nasal cavity, the flat bottom surface of the cavity within the nasal model corresponds to the olfactory region.

[0025] Figure 7 A bead was depicted settling in water (39 cP) after 30 seconds inside a nasal model.

[0026] Figure 8 The image depicts the retrieval of a bead using a small magnet inside the nasal passage of a model.

[0027] Figure 9 The first retrieval step is described, in which a large magnet positioned outside the nasal model is used to pull the bead into the nasal passage of the model.

[0028] Figure 10 The second retrieval step is described, in which a small magnet is used to retrieve the bead in the nasal passage of the model.

[0029] Figure 11Multiple sample wells are depicted, including turbid wells with 39 cP PEG+ water swabs (see white arrows).

[0030] Figure 12 A graph depicts the concentration of TAU measured in pure cerebrospinal fluid (CSF) and liquid swabs.

[0031] Figure 13 The images depict cerebrospinal fluid (CSF) floating on top of a 6 cP PEG swab (left) and a 39 cP PEG swab (right). The floating CSF is marked with arrows.

[0032] Figure 14 The measured TAU concentrations are plotted against the mixing method. The measurements correspond to simulated CSF (50 μL of PBS with 400 pg / mL TAU) on top of a 350 μL 6 cP glycerol liquid swab in a microcentrifuge tube.

[0033] Figure 15 A graph depicting the measured AB42 concentration is provided.

[0034] Figure 16 A graph depicting the TAU concentration corresponding to measurements of TAU protein eluted from beads containing multiple antibodies is presented.

[0035] Figure 17 A graph depicting the prepared TAU:AB42 ratio relative to the measured TAU:AB42 ratio was plotted.

[0036] Figure 18 A graph depicting the prepared ratio relative to the measured TAU concentration is presented.

[0037] Figure 19 A graph depicting simulated CSF versus simulated AB42 measurements in mucus was used.

[0038] Detailed Explanation This disclosure provides formulations, methods, and apparatus for intranasal biomarker sampling and therapeutic delivery using magnetic formulations. When combined with the application of an external magnetic field, the magnetic formulation moves within the nasal cavity. Intranasal magnetic formulations offer the benefits of capabilities including: targeting or guiding the placement of the formulation via a magnetic field, enhancing intranasal mixing of the formulation via a magnetic field, enhancing protein collection via antibody-coated magnetic beads, or enhancing sample retrieval via an inserter with a magnetic tip.

[0039] In some embodiments, a method for collecting biological material from a subject's intranasal region includes: delivering a magnetic formulation containing more than one magnetic particle to the subject's intranasal region, wherein the magnetic formulation is configured to capture biological material; and retrieving at least a portion of the magnetic formulation from the intranasal region, thereby collecting any biological material captured by that portion of the magnetic formulation. In some embodiments, the intranasal region is the subject's olfactory region. In some embodiments, the intranasal region is a target subregion of the nasal cavity.

[0040] In some embodiments, the magnetic particles are paramagnetic, diamagnetic, or ferromagnetic. In some embodiments, the biomaterial is captured from cerebrospinal fluid (CSF). In some embodiments, the magnetic particles are uncoated. In some embodiments, the magnetic particles are coated with or conjugated to one or more antibodies configured to bind to selected biomaterial. In some embodiments, each magnetic particle is coated with or conjugated to a single antibody. In some embodiments, each magnetic particle is coated with or conjugated to multiple antibodies.

[0041] In some embodiments, analysis of the captured biological material provides quantitative values ​​of the concentration of tau protein, the concentration of β-amyloid protein, or their ratio. In some embodiments, analysis of the captured biological material provides a quantitative value of the ratio of tau protein to β-amyloid protein 42, thereby enabling the diagnosis of Alzheimer's disease. In some embodiments, the method further includes eluting the captured biological material from magnetic particles and then analyzing the eluted biological material. In some embodiments, the biological material is analyzed via enzyme-linked immunosorbent assay (ELISA).

[0042] In some embodiments, the method further includes analyzing the captured biological material as a protein-bead complex. In some embodiments, the magnetic particles or protein-bead complex are washed and / or frozen prior to analysis of the captured biological material. In some embodiments, the biological material is analyzed via a self-administered test. In some embodiments, the biological material is analyzed via lateral flow assay, lateral flow immunoassay, lateral flow immunochromatography, or a magnetic lateral flow analyzer.

[0043] In some embodiments, a portion of the magnetic preparation is retrieved using a magnet. In some embodiments, the method further includes exposing the subject to a magnetic field before retrieving at least a portion of the magnetic preparation. In some embodiments, the magnetic field is an oscillating magnetic field. In some embodiments, the frequency of the oscillating magnetic field may be about 0.01-0.02, about 0.02-0.03, about 0.03-0.04, about 0.04-0.05, about 0.05-0.06, about 0.06-0.07, about 0.07-0.08, about 0.08-0.09, about 0.09-0.1, about 0.1-0.15, about 0.15-0.2, about 0.2-0.25, about 0.25-0.3, about 0.3-0.35, or about 0.35- 0.4, approximately 0.4-0.45, approximately 0.45-0.5, approximately 0.5-0.55, approximately 0.55-0.6, approximately 0.6-0.65, approximately 0.65-0.7, approximately 0.7-0.75, approximately 0.75-0.8, approximately 0.8-0.85, approximately 0.85-0.9, approximately 0.9-0.95, approximately 0.95-1, approximately 1-5, approximately 5-10, approximately 10-15, approximately 15-20, approximately 20-25, approximately 25-30, approximately 30-35, approximately 35-40 Approximately 40-45, Approximately 45-50, Approximately 50-55, Approximately 55-60, Approximately 60-65, Approximately 65-70, Approximately 70-75, Approximately 75-80, Approximately 80-85, Approximately 85-90, Approximately 90-95, Approximately 95-100, Approximately 100-110, Approximately 110-120, Approximately 120-130, Approximately 130-140, Approximately 140-150, Approximately 150-160, Approximately 160-170, Approximately 170-180, Approximately 180-190, Approximately 190-2 The Hz frequencies are approximately 0.00, 200-220, 220-240, 240-260, 260-280, 280-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, or 950-1000 Hz. In some embodiments, the method further includes positioning a magnet on or near the bridge of the subject's nose.

[0044] In some embodiments, the biomaterial includes cerebrospinal fluid, one or more microorganisms from the patient's microbiome, one or more components of the patient's metabolome, one or more pathogens, and / or one or more biomarkers of interest. In some embodiments, the biomaterial comprises amyloid-β, tau protein, or a combination thereof.

[0045] In some embodiments, the magnetic formulation is delivered via a delivery device comprising a cannula and / or microfluidic channels, wherein the cannula and / or microfluidic channels are configured for insertion into a subject's nasal cavity. In some embodiments, the formulation is delivered via a device comprising: a housing defining a first insertable portion and a second insertable portion, each insertable portion for insertion into a subject's nasal passage, wherein, upon insertion of the first insertable portion into the subject's nasal passage, at least one insertable portion engages tissue within the nasal passage to open or dilate the subject's internal nasal flap, thereby positioning at least one insertable portion for delivery of the formulation to the subject's olfactory region; and an actuator that delivers the formulation from any one or both of the insertable portions when the device is actuated.

[0046] In some embodiments, the method for diagnosing a subject includes (a) collecting biological material from the subject using the methods described herein; (b) analyzing the collected biological material; and (c) making a diagnosis based on the analysis of step b. In some embodiments, this method is used to diagnose Alzheimer's disease or another neurodegenerative disease. In some embodiments, analyzing the biological material includes identifying and / or quantifying biomarkers, pathogens, and / or microorganisms in the collected biological material. In some embodiments, the method further includes correlating the identified and / or quantified biomarkers, pathogens, and / or microorganisms with corresponding physiological characteristics and / or medical conditions. In some embodiments, analyzing the biological material includes using a point-of-care measurement system. In some embodiments, the point-of-care measurement system is configured to receive a sample of the collected biological material from a delivery device.

[0047] In some embodiments, a method of intranasal delivery of a therapeutic agent includes delivering a magnetic formulation to an intranasal region of a subject and exposing the subject to a magnetic field. In some embodiments, the magnetic formulation comprises more than one magnetic particle and a therapeutic agent. In some embodiments, the magnetic field causes movement of the magnetic particle, thereby enhancing the delivery of the therapeutic agent. In some embodiments, the magnetic formulation comprises a fluid suspension. More than one solid magnetic particle may be suspended in a fluid or fluid mixture to produce a formulation that behaves like a liquid and can be dispensed as a laminar jet.

[0048] In some embodiments, methods for enhancing intranasal contact of the formulation include: delivering a magnetic formulation containing more than one magnetic particle to an intranasal region of a subject; and enhancing intranasal contact of the magnetic formulation by exposing the subject to a magnetic field that induces oscillation or magnetophoretic movement of at least some of the magnetic particles. In some embodiments, the magnetic field is an oscillating magnetic field. In some embodiments, the magnetic field is provided via a permanent magnet. In some embodiments, the magnetic field reduces the necessary residence time of the formulation within the nasal cavity.

[0049] In some embodiments, a device for intranasally delivering and retrieving a magnetic preparation to and from the olfactory region of a subject includes: a housing including an insertable portion comprising a distal end, a proximal end, and a retrieval magnet; and a subject engagement portion engaging a subject's columellar region to position the distal end of the insertable portion within a jet region of the subject's nasal passage; wherein the device is configured to deliver the magnetic preparation to and retrieve at least a portion of the magnetic preparation from the subject's olfactory region. In some embodiments, the device includes a magnetic portion for retrieving a portion of the magnetic preparation. In some embodiments, the device dispenses the magnetic preparation as a laminar jet. In some embodiments, the device includes a compliant dispensing tip comprising a compliant and flexible soft tip. In some embodiments, the subject engagement portion applies pressure to the subject's columellar region such that and / or causes the preparation to be delivered from the insertable portion to the subject. In some embodiments, the insertable portion includes a dispensing element for delivering the magnetic preparation to the subject's olfactory region.

[0050] In some implementations, the device for intranasal retrieval of magnetic particles includes: a retrieval magnet; and a magnet support configured to position the retrieval magnet within a target area of ​​the subject's nasal cavity to facilitate the retrieval of more than one magnetic particle from the target area.

[0051] In some embodiments, a system for enhancing intranasal contact of a formulation includes: a device for intranasal delivery of a magnetic formulation comprising more than one magnetic particle; and a magnet for inducing intranasal movement of the magnetic formulation, thereby enhancing intranasal contact. In some embodiments, the system further includes a support configured to hold the magnet in a desired position on or near the bridge of the subject's nose. In some embodiments, the system further includes a retrieval magnet configured to be inserted into the subject's nasal cavity for retrieving a portion of the magnetic formulation. In some embodiments, the retrieval magnet has a diameter of approximately 2.5 mm. In some embodiments, the system further includes a retrieval magnet configured to be positioned externally to the subject's nasal cavity for retrieving a portion of the magnetic formulation.

[0052] In some embodiments, the magnetic formulation for intranasal biomarker sampling includes more than one magnetic particle configured to capture biological material upon delivery into the nasal cavity, wherein the delivered formulation is configured to be removed from the nasal cavity along with the biological material. In some embodiments, the magnetic particle is coated with tau antibody, β-amyloid antibody, or a combination thereof. In some embodiments, the magnetic particle comprises a first more than one magnetic particle coated with tau antibody and a second more than one magnetic particle coated with β-amyloid antibody. In some embodiments, the magnetic particle has an average diameter of about 50 micrometers.

[0053] In some embodiments, the magnetic particles have a molecular weight of about 0.01-0.015, about 0.015-0.02, about 0.02-0.025, about 0.025-0.03, about 0.03-0.035, about 0.035-0.04, about 0.04-0.045, about 0.045-0.05, about 0.05-0.055, about 0.055-0.06, about 0.06-0.065, about 0.065-0.07, about 0.07-0.075, about 0.075-0.08, about 0.08-0.085, about 0.085-0.09, about 0.09-0.095, or about 0.095-0. 1. Approximately 0.1-0.15, Approximately 0.15-0.2, Approximately 0.2-0.25, Approximately 0.25-0.3, Approximately 0.3-0.35, Approximately 0.35-0.4, Approximately 0.4-0.45, Approximately 0.45-0.5, Approximately 0.5-0.55, Approximately 0.55-0.6, Approximately 0.6-0.65, Approximately 0.65-0.7, Approximately 0.7-0.75, Approximately 0.75-0.8, Approximately 0.8-0.85, Approximately 0.85-0.9, Approximately 0.9-0.95, Approximately 0.95-1, Approximately 1-5, Approximately 5-10, Approximately 10-15, Approximately 15-20, Approximately 20-25, Approximately 25-30, Approximately 30-35, Approximately 3 5-40, approximately 40-45, approximately 45-50, approximately 50-55, approximately 55-60, approximately 60-65, approximately 65-70, approximately 70-75, approximately 75-80, approximately 80-85, approximately 85-90, approximately 90-95, approximately 95-100, approximately 100-105, approximately 105-110, approximately 110-115, approximately 115-120, approximately 120-125, approximately 125-130, approximately 130-135, approximately 135-140, approximately 140-145, approximately 145-150, approximately 150-155, approximately 155-160, approximately 160-165, approximately 165-170, approximately 170-175, approximately 17 Average diameter of 5-180, approximately 180-185, approximately 185-190, approximately 190-195, approximately 195-200, approximately 200-210, approximately 210-220, approximately 220-230, approximately 230-240, approximately 240-250, approximately 250-260, approximately 260-270, approximately 270-280, approximately 280-290, approximately 290-300, approximately 300-320, approximately 320-340, approximately 340-360, approximately 360-380, approximately 380-400, approximately 400-420, approximately 420-440, approximately 440-460, approximately 460-480, or approximately 480-500 micrometers.

[0054] In some embodiments, the formulation is delivered to the olfactory region of the nasal cavity. In some embodiments, the formulation is configured to capture biological material from a targeted subregion of the nasal cavity. In some embodiments, the delivered formulation is configured to retain the captured biological material upon retrieval. In some embodiments, the biological material includes cerebrospinal fluid (CSF), one or more microorganisms from the patient's microbiome, one or more components of the patient's metabolome, one or more pathogens, and / or one or more biomarkers of interest. In some embodiments, the formulation is configured to capture specific biological material.

[0055] In some embodiments, the formulation comprises a buffered saline solution, polyethylene glycol, glycerol, or a combination thereof. In some embodiments, the buffered saline solution is a 100 mM phosphate-buffered saline solution. In some embodiments, the formulation comprises one or more gelling agents and / or thickeners. In some embodiments, the formulation has a viscosity between about 20 cP and 40 cP. In some embodiments, the formulation has a viscosity between about 0-5, about 5-10, about 10-15, about 15-20, about 20-25, about 25-30, about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, about 90-95, or about 95-100 cP. In some embodiments, the formulation comprises a viscosity between about 100-110, about 110-125, about 125-150, about 150-200, about 200-250, about 250-300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-550, about 550-600, about 600-650, about 650-700, about 700-750, about 750-800, about 800-850, about 850-900, about 900-950, or about 950-1000 cP. In some embodiments, the formulation comprises a viscosity modifier to provide the desired viscosity of the formulation. In some embodiments, the viscosity modifier comprises at least one of glycerol, pectin, and polyethylene glycol. In some implementations, the viscosity modifier accounts for 25%-75% of the formulation by volume.

[0056] In some embodiments, the formulation is configured for removal from the nasal cavity using a magnet. In some embodiments, the formulation comprises one or more specific monoclonal or polyclonal antibodies to target specific biological materials. In some embodiments, the formulation comprises one or more specific aptamers to target specific biological materials. In some embodiments, the formulation is provided, delivered, and / or removed as a pellet of the formulation.

[0057] In some embodiments, the magnetic formulation for intranasal delivery of the therapeutic agent includes a magnetic formulation comprising more than one magnetic particle and the therapeutic agent, wherein the magnetic particle is configured to enhance intranasal contact of the therapeutic agent. In some embodiments, the formulation is delivered to the olfactory region of the nasal cavity.

[0058] Exemplary devices and methods As described in more detail below, Figures 2A-2F , Figures 3A-3D and Figures 4A-4F The figure illustrates an exemplary embodiment for collecting biomaterials from targeted areas of a patient's nasal cavity, such as the olfactory region of the nasal cavity. The exemplary embodiments and methods described herein are also applicable to collecting biomaterials from other areas of the nasal cavity, as disclosed herein. Figures 2A-2F The steps of an exemplary method using device 200 are shown. Device 200 includes a common sleeve 202 and separate containers 220 and 250 for delivery and recovery of the formulation, respectively. Figures 3A-3D The steps of an exemplary method using device 300 are shown. Device 300 includes a cannula 302 and an attached sphere 304 for both delivery and recovery of the formulation. Figures 4A-4F The steps of an exemplary method using apparatus 400 are illustrated. Apparatus 400 includes a container 420 with a sleeve 402 for formulation delivery and another container 450 with a sleeve 452 for formulation recovery. Details of these exemplary apparatuses and their operation are described below.

[0059] like Figure 2A As shown, device 200 includes a flexible, rigid, or compliant sheath 202. In some embodiments, the sheath at 202 is incorporated into a sheath mechanism to protect the olfactory region from contamination from the inferior nasal cavity and to protect the biomaterial from contamination during the dispensing phase. In some embodiments, device 200 includes a container 220 having a body for receiving formulation 226. Figure 2B In some embodiments, container 220 includes a deployment mechanism 224 for dispensing formulation 226 from container 220, such as... Figure 2B As shown. In some embodiments, container 220 is removably attached to sleeve 202. In some embodiments, container 220 includes carpule 222.

[0060] In some embodiments, the device 200 is positioned against the outer base of the nose. For example, in some embodiments, such as Figure 2A As shown, the device includes a clamp 212 mounted on a clamp base 206. The clamp 212 provides an anatomical reference point for accurately positioning the cannula 202 and maintaining the consistent placement of the cannula 202.

[0061] In some embodiments, the cannula 202 is a fixed length suitable for the general population. In some embodiments, the cannula 202 has a variable length set according to the specific measurements of the patient. For example, in some embodiments, the cannula 202 is slidably attached to the base 206 so that it can move relative to the clamp 212. In some embodiments, the cannula 202 includes graduations (e.g., markings on the cannula) to aid in placement. The graduations can be used to insert the cannula to a predetermined depth, for example, such that the tip of the cannula 202 reaches the olfactory region 210 without damaging the tissue. In some embodiments, the predetermined depth is determined, for example, by pre-insertion measurements on a single patient using a CT scan or otoscopy, or by using a maximum safe length determined, such as via a pooled anthropometric measurements database analyzing the nasal cavity.

[0062] In some implementations, the deployment agency includes any delivery agency as disclosed herein. For example, such as Figure 2B As shown, the deployment mechanism 224 is coupled to the container 220 and configured such that when the deployment mechanism 224 is activated, the formulation 226 is ejected from the container 220 through the sleeve 202 and deposited in the patient's olfactory region 210, as... Figure 2C As shown. In some embodiments, the deployment mechanism is activated by the user. In some embodiments, the cannula 202 includes an orifice positioned to deliver formulation 226 to a target subregion (not shown).

[0063] In some embodiments, the deployment mechanism 224 includes a button, a spring, and a plunger (not shown). When the button is pressed, this releases the spring, which moves the plunger to force the formulation 226 from the container 220 into the olfactory region 210. In other embodiments, the deployment mechanism 224 may take other forms.

[0064] As described above, in some embodiments, the formulation has a higher osmotic pressure than the mucus in olfactory region 210. In some embodiments, the formulation contains sugars to both increase osmotic pressure and generate a viscous fluid that can be completely extracted. In some embodiments, the increased osmotic pressure creates an osmotic pressure gradient that facilitates the absorption of biological materials, such as a specific biomarker target 240, into the formulation 226 deposited in olfactory region 210, such as... Figure 2D As shown. In some embodiments, formulation 226 is shear-thinned to facilitate its distribution into the narrow space within olfactory region 210.

[0065] In some implementations, the recycling of formulations and / or biological materials includes any recycling mechanism as disclosed herein. For example, such as Figure 2EAs shown, in some embodiments, container 220 is disconnected from sleeve 202 and replaced with recovery vessel 250. In some embodiments, recovery vessel 250 includes a body configured to contain the extracted formulation and / or biological material. In some embodiments, recovery vessel 250 includes a capillary 252, a deployment button 254, a spring (not shown), and / or a plunger (not shown). Figure 2F As shown, when deployment button 254 is pressed, a spring moves a plunger that draws formulation 226 and biomaterial 240 into recovery vessel 250 through sleeve 202. In some embodiments, the system geometry and plunger travel rate are controlled (e.g., by damping) to ensure that formulation 226 is not drawn in too quickly (e.g., to minimize the shear forces experienced by the captured biomaterial 240 or to prevent it from affecting analytical results by damaging the contents of the captured biomaterial, and to minimize air recovery or prevent air, rather than the full amount of formulation and biomaterial, from being recovered).

[0066] Formulation 226 is any formulation as disclosed herein. For example, in some embodiments, Figure 2F The formulation shown herein undergoes cross-linking upon exposure to air or other means (as described herein), thereby transforming the formulation into a semi-solid state. In some embodiments, the semi-solid formulation facilitates the preservation of captured biological material according to its target site, while simultaneously removing and storing the semi-solid formulation in container 252.

[0067] In some embodiments, the cannula 202 is fitted with a sheath to prevent or minimize cross-contamination of biological materials and / or contamination of the olfactory region via cannula access from the inferior nasal anatomy.

[0068] Figure 3A Another embodiment of the device 300, including a sleeve 302 and a flexible sphere 304, is shown. Figure 3B As shown, pressing the flexible sphere 304 propels the formulation 226 through the cannula 302 and into the olfactory region 210. In some embodiments, the cannula 302 includes an orifice positioned to deliver the formulation 226 to a target subregion (not shown). The formulation is any formulation disclosed herein.

[0069] Figure 3C Formulation 226 is shown to have a higher osmotic pressure than biomaterial 240, which generates an osmotic pressure gradient that facilitates the absorption of biomaterial 240 (including any biomarkers of interest contained therein) into formulation 226.

[0070] like Figure 3D As shown, the flexible sphere 302 is allowed to relax, thereby drawing the formulation 226 and the biomaterial 240 back into the sphere 304 through the sleeve 302.

[0071] In some embodiments, the cannula 302 is fitted with a sheath to prevent or minimize cross-contamination of biological materials and / or contamination of the olfactory region via cannula access from the inferior nasal anatomy.

[0072] Figures 4A-4D A device 400 is shown, comprising a container 420 containing formulation 226, having a deployment mechanism 424 configured to dispense the formulation through a sleeve 402, similar to the operation of the device 200 described above. In some embodiments, the container 420 includes a capillary 422. In some embodiments, the sleeve 402 includes an orifice positioned to deliver formulation 226 to a target subregion (not shown). In some embodiments, the deployment mechanism includes any delivery mechanism as disclosed herein. The formulation is any formulation disclosed herein.

[0073] like Figure 4E As shown, the retrieval device 450 can be inserted into a patient's nose. In some embodiments, the retrieval device includes a body for containing preparations and / or biological materials after removal. In some embodiments, the retrieval device includes a retrieval cannula, which is different from the cannula used for delivering preparations to the olfactory region. In some embodiments, the retrieval cannula is detachably coupled to a retrieval vessel. In some embodiments, the retrieval device includes any retrieval mechanism as disclosed herein. For example, in some embodiments, the retrieval device has a chamber 454 filled with wicking material and a retrieval cannula 452 filled with wicking material configured as a wicking fluid. In some embodiments, the wicking material includes woven, braided, or randomly oriented natural or synthetic fibers and / or capillaries of the wicking fluid. In some embodiments, such as Figure 4F As shown, the recovery device 450 and the wicking material are used to extract the preparation 226 and the biomaterial 240 from the patient's olfactory region 210.

[0074] In some embodiments, the cannula 402 is fitted with a sheath to prevent or minimize cross-contamination of biological materials and / or contamination of the olfactory region via cannula access from the inferior nasal anatomy.

[0075] The foregoing discussion provides many exemplary embodiments of the subject matter of this invention. Although each embodiment represents a single combination of inventive elements, the subject matter of this invention is considered to include all possible combinations of the disclosed elements. Thus, if an embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of this invention is considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0076] Exemplary Use Case Scenario A: In some implementations, the formulations, methods, and devices described herein provide the ability to test at home. As a non-limiting example, steps similar to the following can be used for at-home testing.

[0077] Step 1. The doctor prescribes a biomarker testing kit.

[0078] Step 2. Patients obtain test kits - Patients can pick up test kits at the pharmacy, receive them by mail, or obtain them at the pharmacy counter.

[0079] Step 3. Open the test kit – The patient opens the test kit, which contains two parts. Part 1: A precise olfactory delivery device containing a formulation including coated magnetic beads that will collect a specific biomarker of interest. Part 2: A nasal insert with a magnetic tip and a magnetic collection dish with a dilution fluid, connected to a lateral flow assay that has the ability to convey a positive or negative result, i.e., evidence of association with the biomarker of interest.

[0080] Step 4. Delivery of the formulation with selectively binding beads - The patient receives the fluid delivered to the olfactory area and waits for the predetermined number of minutes.

[0081] Step 5. Retrieve the fluid containing the bound biomarker – After a predetermined number of minutes have elapsed, the patient places the collection container under their nose and tilts their head downwards. The magnetic end of the collection container draws the preparation into the container.

[0082] Step 6. Analyze the sample by contact with the sideflow measurement.

[0083] Step 7. The results of the analysis are read by the patient.

[0084] Exemplary Use Case Scenario B: In some implementations, the formulations, methods, and devices described herein provide the capability for in-clinic testing. As a non-limiting example, steps similar to the following can be used for in-clinic testing.

[0085] Step 1. Doctors prescribe tests related to specific diseases or ailments based on clinical trials.

[0086] Step 2. The patient proceeds to the testing facility, possibly after some fluid intake requirements.

[0087] Step 3. The patient sits down, optionally secured in a suitable position, and may be upside down relative to gravity.

[0088] Step 4. Technicians retrieve a magnetic liquid swab containing multiple magnetic beads from a specialized storage device (such as a cooler that maintains a specific low temperature).

[0089] Step 4*. (Possible alternative to Step 4) The magnetic beads are coated in-situ to avoid potential degradation.

[0090] Step 5. The technician loads the device in a clean space (note that a pre-loaded device can be used as an alternative).

[0091] Step 6. Technicians perform a pre-application procedure on the patients and clip the external electromagnet onto their noses.

[0092] Step 7. The technician inserts the device into the patient's nose and sprays the sample. Step 8. The nose clip generates an oscillating magnetic field that targets the olfactory cleft (OC) to enhance mixing and guide the magnetic beads to the target site.

[0093] Step 9. Remove the spray device from the nose.

[0094] Step 10. After a predetermined time period, retrieve the bead by rinsing, by using the magnetic tip on the inserter, by blowing snot onto a special tissue, or by another suitable sample removal method or device.

[0095] Step 11. Take the sample to the cleaning station, rinse and package it.

[0096] Step 12. Label the cleaned samples and place them in the queue for analysis.

[0097] Step 13. Send the sample to the ELISA machine for analysis.

[0098] Step 14. Process the results and create a table to show which biomarkers were identified as present and the amount and ratio of the identified biomarkers.

[0099] Step 15. Share the results with your doctor for evaluation and possible diagnosis.

[0100] Example 1: A method for analyzing magnetic beads with attached proteins captured from olfactory cleavage. : The first exemplary method relates to the steps of eluting and measuring (measuring) separated proteins by a simple assay such as an ELISA kit, a lateral flow assay, or another immunoassay. The second exemplary method relates to the steps of measuring protein-bead complexes. Exemplary magnetic beads include superparamagnetic beads coated with MagSi-DNA 3.0 μm silica (Magtiviapart # MD0X022, available from Boca Scientific Inc, Dedham, MA); a Dynabeads Protein G Immunoprecipitation Kit (Invitrogen part # 10007D), optionally coated with AB42 antibody (anti-β-amyloid antibody, mouse monoclonal (Sigma part # A3981)); and superparamagnetic iron oxide nanoparticles (e.g., SPION, 80 nm). Lateral flow immunoassays (pregnancy strip tests) use beads as carriers for antibodies and proteins. The elution step of the first example can be eliminated, and the beads can be added to the lateral flow assay in some standard (buffer) solution. An exemplary sample pad may have two separate strips, one for β-amyloid 42 (“AB42”) and one for t-tau. Taking the AB42 strip as an example, the 'test' line would have an anti-AB42 antibody on it, which would bind to the AB42 protein picked up by the beads. The beads that actually pick up AB42 would be stuck on the test line. Downstream would be a 'control' line with AB42 protein (or another antibody binder) to bind to the remaining beads.

[0101] A similar mechanism on the t-tau strip will result in protein ratios. Measurements can be performed colorimetrically (i.e., with a camera), but this does not take into account the vast majority of beads located beneath the surface of the test strip. Here, considering the magnetic properties of the beads, an alternative option would be to use a magnetic lateral flow analyzer, which picks up signals from all beads through the thickness of the lateral flow membrane, thus significantly enhancing the signal and achieving higher analytical accuracy. Magnetic readers for lateral flow measurements allow for fully quantitative determinations, rather than semi-quantitative determinations, all at low cost and with simple operation.

[0102] Bead analysis can be performed via ELISA. After collecting proteins within antibody-coated beads, detection antibodies can be added. Detection antibodies can simply have tags (e.g., fluorescent or chemiluminescent tags) that can be detected directly or indirectly using imaging devices. For indirect detection, one option is to bind the detection antibody to horseradish peroxidase (HRP). This reacts with a liquid substrate, which in the case of HRP can be tetramethylbenzidine (TMB). HRP turns TMB blue. The more protein collected, the stronger the color intensity. Quantitative data can be provided by adding acid to quench the reaction and by measuring the absorbance at 450 nm (blue) light using an instrument.

[0103] Tau protein: Tau is a protein that helps stabilize the internal framework of nerve cells (neurons) in the brain. This internal framework has a tubular shape, through which nutrients and other essential substances travel to different parts of the neuron. In Alzheimer's disease (AD), abnormal forms of tau accumulate and cause the internal framework to break down. These abnormal forms of tau protein adhere to other tau proteins inside the neuron and form tau tangles. Tau tangles and amyloid-β plaques—large accumulations of microscopic brain protein fragments that slow down a person's thinking and memory abilities—are hallmarks of Alzheimer's disease. Total tau or t-tau is one option for measurement. Some portions of total tau can be phosphorylated tau.

[0104] Amyloid-β42 protein (AB42): Fragments of β-amyloid (or amyloid-β) protein, associated with Alzheimer's disease, aggregate between neurons in several different molecular forms. It is formed from the breakdown of a larger protein called amyloid precursor protein. One form, β-amyloid 42, is considered particularly toxic. In the brain of Alzheimer's patients, abnormal levels of β-amyloid 42 aggregate to form plaques that accumulate between neurons and disrupt cellular function.

[0105] The Tau:AB42 ratio as a diagnostic indicator for Alzheimer's disease: While individual concentrations of various CSF proteins have been used to aid in the diagnosis of AD, it has now been shown that ratios between multiple concentrations better distinguish AD patients from other patients. One promising ratio is the Tau:AB42 ratio.

[0106] The following represent possible thresholds for indicating physiological concentrations in healthy patients: - Tau <300 pg / mL 21-50 years old - Tau <450 pg / mL 51-70 years old - Tau <550 pg / mL 71-93 years old - AB42 >500 pg / mL for all ages Exemplary diagnostic biomarkers and their antibodies Tau protein exists naturally in six different forms, ranging in length from 352 to 441 amino acids. The longest form is called tau-441. This form of tau is known to be truncated by a protease (enzyme) exactly after the 421st amino acid. This truncated form of tau-441 is associated with Alzheimer's disease. The tau-441 (1-421) in this example is a truncated form of tau-441.

[0107] Example 2: Liquid swab preparation Magnetic beads in liquid swabs: Examples (including measurement of diagnostic biomarkers) used antibody-coated magnetic beads to capture biomarkers in CSF. To evaluate the effect of viscosity on the ease with which the beads can be moved using a magnetic field, the manipulation of the magnetic beads in the liquid swab formulation was tested.

[0108] Magnetic beads: In embodiments that include the measurement of diagnostic biomarkers, the diameter of the magnetic beads used is measured to be 2.8 μm. Beads with a diameter of 50 μm are used to manipulate beads in a liquid swab. This is the upper limit of typical bead size and maximizes the force generated by the magnetic field on the bead because: the force exerted by the magnetic field on the bead varies with the cube of the diameter. For example, the force exerted by the same magnetic field on a 50 μm bead is 5,694 times greater than the force exerted on a 2.8 μm bead.

[0109] Liquid swabs: The liquid swabs used consisted of 1 cP of fresh water and 39 cP of PEG hydrogel.

[0110] Nasal cavity model: Figure 5 A simplified nasal cavity constructed from three machined polycarbonate sheets is shown. These sheets are highly transparent, resulting in a nasal model that is significantly more transparent than a 3D-printed nasal model. The olfactory cleft inside the model measures 10 mm high × 30 mm wide × 3 mm thick.

[0111] Magnet: The magnet used (McMaster-Carr part number 5848K46) has a rated maximum tensile strength of 94 lb (based on direct contact with a rust-free, unpainted iron plate).

[0112] Bead movement with different liquid swab viscosities: Increasing the swab viscosity from 1 cP (water) to 39 cP (PEG) has a profound effect on the speed at which a 50 μm bead moves through the swab.

[0113] Example 2.1: By placing a magnet on top of the nose model, a bead is pulled from the bottom to the top of the olfactory cleft. The time required for the bead to reach the top of the olfactory cleft is reduced from 1 second in 1 cP water to 20+ seconds in 39 cP PEG.

[0114] Example 2.2: By removing the magnet from the top of the nose model, the bead was allowed to passively settle back to the bottom of the olfactory cleft. The time required for the bead to reach the bottom of the olfactory cleft was reduced from just a few seconds in water to 60+ seconds in PEG. See also Figure 6 and Figure 7 .

[0115] Bead retrieval using a small magnet in the nasal passage: The nasal model was used with a swab viscosity of 1 cP (water) to observe the retrieval of a 50 μm bead using a small magnet inserted into the nasal passage.

[0116] The small magnets used are McMaster-Carr part number 5862K426. These are cylindrical magnets with a rated maximum tensile force of 0.4 lbs. This is a rather weak tensile force, but the magnets were chosen with a diameter of 2.5 mm, which fits perfectly into the 3 mm thick nasal passages and olfactory cleft of the nasal model. A stack of 14 magnets was used to penetrate deep into the model.

[0117] The initial intention was to keep the magnet outside the sniffing crack area of ​​the model, but the magnet was too weak to retrieve the bead at any distance longer than ~5mm. Therefore, the magnet was inserted into the sniffing crack to retrieve any bead, such as... Figure 8 As shown. This is suboptimal because a hard object inserted into the olfactory fissure could damage the fragile cribriform plate.

[0118] Bead retrieval was performed first using a large magnet and then a small magnet: Bead retrieval using a specific sequence of magnets was observed using a nasal model with a swab viscosity of 1 cP (water): First, a large magnet outside the head was used to pull the bead from the olfactory cleft and into the nasal passage. Then, a small magnet was used to retrieve the bead from the nasal passage. This was successful, as shown in the image. Figure 9 and Figure 10 As shown.

[0119] Example 3: Household Management Biomarkers Household biomarkers are the only biomarkers that identify collected fluids as containing CSF. Household biomarkers need to be present in CSF and not in any other part of the nasal passages (such as mucous membranes, saliva, blood from incisions, etc.). Ideally, household biomarkers can also be measured using commonly available assays.

[0120] β-2-Transferrin: β-2-Transferrin is a protein found only in CSF. It is used to diagnose a large amount of CSF leaking into the nasal cavity due to severe trauma.

[0121] Tau protein (TAU): Recent paper (Oudart et al.) Tau protein as a possible marker of cerebrospinal fluid leakage in cerebrospinal fluid rhinorrhoea: A pilot study Biochem Med (Zagreb). 2017 Oct 15;27(3):030703.) concluded that TAU ​​measurement can reliably detect the presence of CSF in nasal mucus and signal the presence of CSF leakage.

[0122] Uses of TAU as a Household Biomarker and Diagnostic Biomarker: TAU can be used as both a household biomarker and a diagnostic biomarker. When TAU is measured as a diagnostic biomarker, it may not be necessary to measure it separately as a household biomarker.

[0123] Example 4: Diagnostic Biomarkers Diagnostic biomarkers are those CSF biomarkers used to diagnose neurodegenerative diseases such as Alzheimer's disease (AD). Ideally, diagnostic biomarkers can be measured using commonly available assays.

[0124] Example 4.1: Selection of diagnostic biomarkers for testing Tau protein (TAU): Tau is a protein that helps stabilize the internal framework of nerve cells (neurons) in the brain. This internal framework has a tubular shape, through which nutrients and other essential substances travel to different parts of the neuron. In Alzheimer's disease, abnormal forms of tau accumulate and cause the internal framework to disintegrate. These abnormal forms of tau protein adhere to other tau proteins inside the neuron and form tau tangles. Tau tangles and amyloid-β plaques—large accumulations of microscopic brain protein fragments that slow down thinking and memory abilities—are hallmarks of Alzheimer's disease. This embodiment includes a measurement of total tau (also written as t-tau), designated as TAU. Some portions of total tau can be phosphorylated tau (also written as p-tau), and this form of tau is another marker of AD. However, p-tau is not used in this embodiment.

[0125] Amyloid-β42 protein (AB42): Fragments of β-amyloid protein (sometimes confusedly referred to as amyloid-β) associated with Alzheimer's disease aggregate between neurons in several different molecular forms. It is formed from the breakdown of a larger protein called amyloid precursor protein. One form, β-amyloid 42, is considered particularly toxic. In the brain of Alzheimer's patients, abnormal levels of β-amyloid 42 aggregate to form plaques that accumulate between neurons and disrupt cellular function.

[0126] The TAU:AB42 ratio as a diagnostic indicator for Alzheimer's disease: While individual concentrations of various CSF proteins have been used to aid in the diagnosis of AD, it has now been shown that ratios between multiple concentrations better distinguish AD patients from other patients. One promising ratio is the TAU:AB42 ratio. In fact, recent studies indicate that this specific ratio may have the best predictive value for the diagnosis of AD. Test for both TAU and AB42, as the TAU:AB42 ratio is very promising. Obtain the necessary supplies (TAU protein, AB42 protein, ELISA kits for both, and antibodies for both that can be attached to magnetic beads) and allow for the measurement of TAU and AB42 concentrations and the calculation of their ratio.

[0127] Example 4.2: Human CSF vs. Simulated CSF Sometimes, using simulated CSF is more convenient than using human CSF. The generation of simulated CSF provides known concentrations of biomarkers. Knowing the biomarker concentration before measuring with an ELISA kit allows us to interpret the measurement results.

[0128] The simulated CSF was prepared as follows: - Start with PBS (phosphate-buffered saline) as the liquid. -Then the biomarker is added at approximately physiological concentrations, which for healthy patients are: o TAU <300 pg / ml 21-50 years old o TAU <450 pg / ml 51-70 years old o TAU <550 pg / ml for ages 71-93 o AB42 >500 pg / ml for all ages The pooled human CSF was purchased from Innovative Research Inc. (product IRHUCSF 1ML). Detection of tau protein within the kit's detection limit (10 pg / mL) was expected but not achieved using the total tau ELISA kit (details below). It was determined that the processing of single-donor samples was much more careful than that of the pooled samples; they were rapidly frozen and typically did not undergo multiple freeze-thaw cycles, unlike the pooled samples.

[0129] Example 4.3: Liquid swab preparation Several different liquid swab formulations were tested, all with a viscosity <65 cP, as it was determined that the liquid swabs were optimally delivered to the middle of the olfactory cleft when the viscosity was maintained in the range of 1 cP–65 cP. Good results were obtained using glycerol + water as a formulation, and therefore this formulation was included along with the PEG + water formulation.

[0130] The following swab preparations are used throughout multiple diagnostic marker tests: - At 6 cP, PEG + water (1 part PEG: 3 parts water by weight or 0.25 PEG mass fraction) - At 39 cP, PEG + water (1 part PEG: 1 part water by weight or 0.50 PEG mass fraction) - At 2 cP, GLY + water (250 parts GLY: 1000 parts water by volume) - At 6 cP, GLY + water (800 parts GLY: 1000 parts water by volume) - At 40 cP, GLY + water (2500 parts GLY: 1000 parts water by volume) Example 4.4: ELISA Measurement - Simulated CSF in Liquid Swabs Objective: This ELISA measurement provides validation regarding whether the ELISA can: (1) measure biomarker concentrations in simulated CSF; (2) measure biomarker concentrations in PEG+ water swabs; and (3) measure biomarker concentrations in GLY+ water swabs. This experiment does not include the collection of CSF from liquid swabs. This experiment allows for the determination of whether liquid swabs interfere with the ELISA measurement and therefore includes the direct addition of TAU to the liquid swab.

[0131] Material: - TAU ELISA = TAU ELISA kit (Fisher / Invitrogen part # KHB4100) - TAU = tau-441 (1-421) protein (Sigma part # SRP0701) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) Method: First, the following formulation was prepared: - Prepared simulated CSF by adding TAU at 100 pg / ml to PBS - Prepared simulated CSF by adding TAU at 400 pg / ml to PBS - Prepare 6 cP PEG swabs and add TAU at 400 pg / ml - Prepare 39 cP PEG swabs and add TAU at 400 pg / ml - Prepare 2 cP GLY swabs and add TAU at 400 pg / ml - Prepare 40 cP GLY swabs and add TAU at 400 pg / ml The TAU concentration in these six sample fluids was then measured using an ELISA kit. During the ELISA kit procedure, wells containing 39 cP PEG swabs became cloudy. See [link to ELISA kit]. Figure 11 .

[0132] Discussion: Results in Figure 12As shown in the figure, the tau concentrations measured in 400 pg / ml and 100 pg / ml PBS samples correlated well with known concentrations. Tau was measured in all liquid swabs, indicating that these swabs do not preclude measurement using an ELISA kit. Additionally: - The measured values ​​for the two lower viscosity swabs (6 cP PEG and 2 cP GLY) were significantly higher than the 400 pg / ml at which they were produced.

[0133] - Higher viscosity 39 cP PEG swabs have a significant negative interference with the assay. - The higher viscosity 40 cP GLY swabs performed quite well, close to the values ​​measured in PBS.

[0134] Example 4.5: ELISA Measurement - Mixed CSF and Liquid Swabs Objective: This ELISA assay investigated how best to mix CSF into a liquid swab: CSF (ρ ~ 1 g / ml) would always float on any practical liquid swab (ρ > 1 g / ml). To illustrate this, red dye was added to some of our simulated CSF, and this CSF was then added to 6 cP PEG swabs and 39 cP PEG swabs. See [link to documentation]. Figure 13 .

[0135] Material: - TAU ELISA = TAU ELISA kit (Fisher / Invitrogen part # KHB4100) - TAU = tau-441 (1-421) protein (Sigma part # SRP0701) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Beads = MagSi-DNA 3.0μm silica-coated superparamagnetic beads (Magtivia part # MD0X022, purchased from Boca Scientific Inc, Dedham, MA. Boca part # MD01022) Method: First, generate all the liquids: - Prepared simulated CSF by adding TAU at 400 pg / ml to PBS - Preparation of 6 cP GLY swabs Then prepare 3 microcentrifuge tubes, as shown above. Figure 13 As shown: First, add 350 μl of GLY swab. Then add 50 μl of simulated CSF. Then, CSF was mixed into the liquid swab in three different ways: Tube 1. Do not mix at all, wait 5 min, then aspirate only 50 μl of the lower layer liquid (swab). This represents the worst possible mix: no mix at all. The only way TAU can enter a liquid swab is via diffusion, which is very slow. - Carefully retrieving the liquid swab means that any TAU measured must have already diffused into the liquid swab. 2. Vigorously mix the liquid in the dispensing tube by repeatedly aspirating and dispensing with a pipette. This represents the possible optimal mixture: vigorous mechanical mixing. This should result in the highest possible measurement of TAU concentration. Note that vigorous mechanical mixing may not be feasible in the olfactory slit due to the confined space and the fragility of the cribriform plate. 3. Add ordinary magnetic beads (3 μm diameter, uncoated with antibodies), and use a magnet to move the beads up and down to mix CSF into the liquid swab. This suggests that mixing methods are likely possible within the olfactory cleft. - In this embodiment, ordinary beads are used, but it is also possible to use beads coated with antibodies to directly capture biomarkers. - The mixing will not be as vigorous as in tube 2, and the measured TAU concentration is expected to be between the TAU concentrations measured in tube 1 and tube 2. Discussion: Results in Figure 14 As shown in the diagram. First, simulated CSF was measured as a control. The correct ELISA measurement was 400 pg / ml. Then, samples from 3 tubes were measured: Tube 1. Unmixed - As expected, this yielded the lowest measured TAU concentration. This reinforces the fact that isolated diffusion is a poor way to get biomarkers into swabs. Pipe 2. Mechanical mixing - As expected, this yielded the highest measured TAU concentration. - As with the first ELISA test, note that the TAU concentration measured with a GLY swab results in a measured TAU concentration that is much higher than the actual TAU concentration in the simulated CSF. Note that vigorous mechanical mixing may not be feasible in the olfactory slit due to the confined space and the fragility of the cribriform plate. Tube 3. Mixed with magnetic beads - As expected, the measured TAU concentration was between the TAU concentrations measured in tube 1 and tube 2. This indicates that magnetic mixing increases the efficiency of biomarker capture. Example 4.6: ELISA Measurement: Magnetic beads coated with AB42 antibody This embodiment relates to ELISA measurements to investigate the use of antibody-coated magnetic beads for capturing biomarkers.

[0136] Material - ELISA = Human Amyloid β42 ELISA Kit (Abcam Catalog No. AB289832) - AB42 = Human β-amyloid protein (1-42) PTD recombinant protein (Invitrogen part # 03111) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Beads = Dynabeads Protein G Immunoprecipitation Kit (Invitrogen part # 10007D) - AB42 antibody = anti-β-amyloid antibody, mouse monoclonal (Sigma part # A3981) method Step 1: Prepare simulated CSF by adding AB42 to PBS at 450 pg / ml.

[0137] Step 2: Coat the beads with AB42 antibody according to the Dynabeads kit instructions.

[0138] Step 3: Incubate the antibody-coated beads in 200 μl of simulated CSF for 1 hour.

[0139] Step 4: Collect the beads by placing a magnet outside the tube containing simulated CSF+ beads, causing the beads inside the tube to gather near the magnet. Discard the liquid supernatant and retain it for subsequent testing in the ELISA kit to observe how much AB42 protein was not collected by the beads.

[0140] Step 5: Remove the captured AB42 protein from the antibody on the beads using the low pH elution buffer provided in the Dynabeads kit, and add the eluted AB42 protein to 200 μl of PBS.

[0141] Step 6: Run the following sample liquid in the ELISA assay: A. A simulated CSF was used to verify the AB42 concentration in the simulated CSF.

[0142] B. AB42 protein eluted from the magnetic beads.

[0143] C. Supernatant containing all AB42 protein that was not captured by the beads.

[0144] result The AB42 concentration, measured in simulated CSF, was ~450 pg / ml. Protein eluted from the antibody-coated beads was ~80 pg / ml. The supernatant concentration was ~150 pg / ml. See also... Figure 15 .

[0145] discuss These results allow for comparisons of the total amount of protein in the simulated CSF, the amount of that protein captured by the antibody-coated beads, and the amount remaining in the simulated CSF. Placing the antibody-coated beads in 200 μl of simulated CSF containing 450 pg / ml meant a total of 90 pg of AB42 protein.

[0146] The supernatant is a simulated CSF after the antibody-coated beads pick up their AB42. The supernatant was measured at ~150 pg / ml, which is 1 / 3 of the original 450 pg / ml. This means that the antibody-coated beads picked up 2 / 3 of the usable 90 pg of Ab42, i.e., 60 pg of Ab42.

[0147] If the elution step has removed 100% of the captured AB42 from the antibody, then all 60 pg of AB42 will be added to 200 μl of PBS, which should be measured as: 60 pg into 200 μl of PBS = 300 pg / ml.

[0148] Only 80 pg / ml was measured in the eluted AB42 sample. This means that the elution step removed only 80 / 300 = 27% of the antibody-bound AB42. This can be optimized to improve protein recovery.

[0149] Example 4.7: ELISA Measurement - TAU Capture with Magnetic Beads Objective: This ELISA assay was used to investigate the application of capturing TAU protein with antibody-coated magnetic beads.

[0150] Material - TAU ELISA = TAU ELISA kit (Fisher / Invitrogen part # KHB0041) - TAU = tau-441 (1-421) protein (Sigma-Aldrich part # SRP0701) - TAU antibody 1 = TAU monoclonal antibody (EPR22524-95) (Abcam part # ab254256) - TAU antibody 2 = TAU monoclonal antibody (EPR25205-233) (Abcam part # ab308439) - TAU antibody 3 = Tau monoclonal antibody (Tau-5) (Fisher / Invitrogen part # AHB0042) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Beads = Dynabeads Protein G Immunoprecipitation Kit (Fisher / Invitrogen part # 10007D) - TRIS-HCl = 1M Tris-HCl buffer, pH 7.5 (Fisher / Invitrogen part #15567027) method First, all liquids were generated: a simulated CSF was prepared by adding TAU at 400 pg / ml to PBS. Then, according to the Dynabeads kit instructions, the first group of beads was coated with TAU antibody 1, the second group of beads was coated with TAU antibody 2, and the third group of beads was coated with TAU antibody 3.

[0151] Then, the three groups of individually antibody-coated beads were incubated in three separate simulated CSF aliquots: - Add TAU antibody-coated beads to 400 μL of simulated CSF. - Add TAU antibody 2-coated beads to 400 pL of simulated CSF. - Add TAU antibody 3-coated beads to 400 pL of simulated CSF. - Let stand for 1 hour After incubation, collect the beads: - Place the magnet on the outside of a tube containing simulated CSF+ beads. - Gather the beads inside the tube near the magnet - Remove the liquid, now known as the supernatant. - Retain this supernatant for subsequent tests in the ELISA kit to observe how much TAU protein was not collected by the beads. The captured TAU protein is then removed from the antibodies on the beads: - Use the low pH elution buffer provided in the Dynabeads kit to elute (remove) the captured TAU protein from the antibody on the beads. - Resuspend the eluted TAU protein in 120 μL of TRIS-HCl (total volume). Then run the following sample liquid in the ELISA assay: - Simulated CSF to verify the TAU concentration in the simulated CSF. - TAU protein eluted from antibodies on beads - Supernatant containing all TAU proteins not captured by beads result The TAU concentration measured in the simulated CSF was ~385 pg / ml.

[0152] The protein eluted from the antibody-coated beads was measured to be ~80 pg / ml.

[0153] The supernatant was measured to be ~15 pg / ml.

[0154] The protein eluted from the antibody-coated beads was measured to be ~305 pg / ml.

[0155] The supernatant was measured to be ~60 pg / ml.

[0156] The protein eluted from the antibody-coated beads was measured to be ~140 pg / ml.

[0157] The supernatant concentration was ~90 pg / ml. See [link / reference] Figure 16 discuss The TAU concentration measured in the simulated CSF was very close to its original concentration of 400 pg / ml. This indicates that the CSF was prepared correctly and that the ELISA kit works as it should.

[0158] All three antibody-coated beads removed at least 75% of TAU protein, as all three supernatants measured <100 pg / ml, indicating that >300 pg / ml had been removed from the simulated CSF by the beads.

[0159] Although the supernatant measurements indicated that at least 300 pg / ml of antibody-coated beads were removed from the simulated beads, the measurements for the three elution buffers were significantly less than 300 pg / ml. This is partly due to the fact that not all proteins are recovered from the antibody and partly due to the determination of how many volumes of liquid (TRIS-HCl) the eluted proteins should be resuspended (placed) in.

[0160] These results can be interpreted by considering the total amount of protein in the simulated CSF and then determining how much of that protein is captured by the antibody-coated beads and how much of that protein remains in the simulated CSF.

[0161] Considering the best-performing antibody, TAU antibody 2 (Abcam part # ab308439): the antibody-coated beads were placed in 400 plc of simulated CSF containing 400 pg / ml, which means there were a total of 160 pg of TAU in the sample.

[0162] The supernatant is a simulated CSF after the antibody-coated beads pick up their TAU. The supernatant was measured at ~60 pg / ml in 400 plc, which means that a total of 24 pg of TAU remained in the supernatant.

[0163] Calculate how much TAU is picked up by the antibody-coated beads: the antibody-coated beads pick up 160 pg - 24 pg = 136 pg of TAU. Then elute the TAU from the antibody and resuspend the eluted TAU in 120 pL of PBS.

[0164] If the elution step has removed 100% of the 136 pg captured TAU from the antibody, then all 136 pg will be added to 120 plc of PBS, and it should be measured as: 136 pg into 120 plc of PBS = 1133 pg / ml.

[0165] However, only 305 pg / ml was measured in the eluted TAU sample. This means that the elution step only removed 305 / 1133 = 27% of the antibody-bound TAU.

[0166] This is the same elution efficiency obtained in the AB42 ELISA test (see Example 4.6).

[0167] Example 4.8: ELISA Measurement - TAU:AB42 Ratio Using Magnetic Bead 1 Objective: This ELISA study investigated the use of antibody-coated magnetic beads to capture both TAU and AB42 proteins, and thus measured the TAU:AB42 ratio.

[0168] Material - TAU ELISA = TAU ELISA kit (Fisher / Invitrogen part # KHB0041) - TAU = tau-441 (1-421) protein (Sigma-Aldrich part # SRP0701) - TAU antibody = TAU monoclonal antibody (EPR25205-233) (Abcam part # ab308439) - AB42 ELISA = Amyloid β 42 ELISA Kit (Abcam catalog number AB289832) - AB42 = Amyloid β protein (AB42 ELISA kit protein standard) - AB42 antibody = Amyloid β monoclonal antibody (Sigma part # A3981) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Beads = Dynabeads Protein G Immunoprecipitation Kit (Fisher / Invitrogen part # 10007D) - TRIS-HCl = 1M Tris-HCl buffer, pH 7.5 (Fisher / Invitrogen part #15567027) method Simulated CSF with various TAU:AB42 ratios was generated by adding proteins to PBS: - CSF 1 = 500 pg / ml TAU and 333 pg / ml AB42. The TAU:AB42 ratio is 1.5. - The TAU:AB42 ratio of 1000 pg / ml CSF2 and 333 pg / ml AB42 is 3.0. - The ratio of TAU:AB42 to TSF3 (1500 pg / ml) and AB42 (333 pg / ml) is 4.5. According to the Dynabeads kit instructions, the first group of beads was coated with TAU antibody and the second group of beads was coated with AB42 antibody. After coating, the two groups of beads were mixed together.

[0169] Add the bead mixture to the simulated CSF and incubate: Add 1 / 3 of the bead mixture to CSF ​​1. Add 1 / 3 of the bead mixture to CSF ​​2. Add 1 / 3 of the bead mixture to CSF ​​3. - Incubation lasts for 1 hour After incubation, collect the beads: - Place the magnet on the outside of a tube containing simulated CSF+ beads. - Gather the beads inside the tube near the magnet - Remove the liquid, now known as the supernatant. - Retain this supernatant for subsequent tests in the ELISA kit to observe how much TAU and AB42 protein was not collected by the beads. After the incubation period, the captured TAU and AB42 proteins were removed from the beads using the low-pH elution buffer provided in the Dynabeads kit: - TAU and AB42 proteins captured by elution from CSF 1 - Resuspend the eluted TAU and AB42 proteins in 190 μl of TRIS-HCl. - This is called resuspension 1 - TAU and AB42 proteins captured by CSF 2 elution - Resuspend the eluted TAU and AB42 proteins in 190 μl of TRIS-HCl. - This is called resuspension 2 - TAU and AB42 proteins captured by CSF 3 elution - Resuspend the eluted TAU and AB42 proteins in 190 μl of TRIS-HCl. - This is called resuspension 3 Then run the resuspension in the ELISA assay: - Measure TAU protein eluted from CSF 1 by running resuspension 1 in the TAU ELISA kit. - Measure TAU protein eluted from CSF 2 by running resuspension 2 in the TAU ELISA kit. - Measure TAU protein eluted from CSF 3 by running resuspension 3 in the TAU ELISA kit. - Measure AB42 protein eluted from CSF 1 by running resuspension 1 in the AB42 ELISA kit. - Measure AB42 protein eluted from CSF 2 by running resuspension 2 in the AB42 ELISA kit. - Measure AB42 protein eluted from CSF 3 by running resuspension 3 in the AB42 ELISA kit. Then, the TAU and AB42 measurements are used to calculate the TAU:AB42 ratio.

[0170] result For CSF 1, no meaningful ratio was obtained. For CSF 2, TAU was measured at 106 pg / ml and AB42 at 369 pg / ml, with a TAU:AB42 ratio of 0.287. For CSF 3, TAU was measured at 179 pg / ml and AB42 at 416 pg / ml, with a TAU:AB42 ratio of 0.430. See also Figure 17 discuss Although only two of the three preparation ratios were successfully measured, additional data points can be assumed because reducing TAU to 0 pg / ml would result in a TAU:AB42 ratio of 0, and indeed, the linear fit of the two data points does pass through the origin.

[0171] This experiment mixed TAU protein, AB42 protein, magnetic beads, TAU antibody, and AB42 antibody together in a simulated CSF. The successful collection, elution, and measurement of both TAU and AB42 proteins were very positive. The proteins, beads, and antibodies did not interfere with each other. The presence of TAU protein does not interfere with the binding of AB42 protein to its antibody, and vice versa.

[0172] The presence of TAU protein does not interfere with the elution of AB42 protein from its antibody, and vice versa.

[0173] The measured TAU:AB42 ratio differed from the prepared ratio (approximately 10 times lower). This could be because: Different antibodies have different protein-binding affinities. During the incubation step, the TAU antibody will capture a certain percentage of available TAU protein, while the AB42 antibody will capture a different percentage of available AB42 protein. Therefore, the ratios are different after the protein capture step.

[0174] Different antibodies will have different elution characteristics. During the protein elution step, the TAU antibody will release a certain percentage of the TAU protein it captures, while the AB42 antibody will release a different percentage of the AB42 protein it captures. Therefore, the ratio changes again.

[0175] The difference between the measured ratio and the actual ratio can be addressed by calibrating the ratio measurement: - Preparation of sample fluids with a known TAU:AB42 ratio - Measure the TAU:AB42 ratio in the prepared sample fluid. - Calculate the calibration factor to convert the measured ratio to the actual ratio: For example, in this case: - For CSF 2, the preparation ratio = 3.0 and the measurement ratio = 0.287, which is 10.45 times smaller. - For CSF 3, the preparation ratio = 4.5 and the measurement ratio = 0.430, which is 10.47 times smaller. Since the linear fitting line passes through the origin, no offset term is needed, and the calibration equation simplifies to: Actual TAU:AB42 ratio = 10.46 × Measured TAU:AB42 ratio Example 4.9: ELISA Measurement - TAU:AB42 Ratio Using Magnetic Beads Objective: This is the second ELISA measurement of the TAU:AB42 ratio. The first ELISA measurement of the TAU:AB42 ratio (see Example 4.8) used the AB42 protein included with the AB42 ELISA kit because this lyophilized AB42 protein is reliably reconstructed and shows an accurate signal on the kit. Subsequent measurements used Abcam product number AB120301.

[0176] Material - TAU ELISA = TAU ELISA kit (Fisher / Invitrogen part # KHB0041) - TAU = tau-441 (1-421) protein (Sigma-Aldrich part # SRP0701) - TAU antibody = TAU monoclonal antibody (EPR25205-233) (Abcam part # ab308439) - AB42 ELISA = Amyloid β 42 ELISA Kit (Abcam catalog number AB289832) - AB42 = Amyloid β (1-42) protein (Abcam product number AB120301) - AB42 antibody = Amyloid β monoclonal antibody (Sigma part # A3981) - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Beads = Dynabeads Protein G Immunoprecipitation Kit (Fisher / Invitrogen part # 10007D) - TRIS-HCl = 1M Tris-HCl buffer, pH 7.5 (Fisher / Invitrogen part #15567027) method Simulated CSFs with various TAU:AB42 ratios were prepared by adding proteins to PBS: - CSF 0 = 0 pg / ml TAU and 1000 pg / ml AB42. The TAU:AB42 ratio is 0. - The ratio of TAU:AB42 to CSF ​​1 (250 pg / ml) and AB42 (1000 pg / ml) is 0.25. - TAU (1000 pg / ml) and AB42 (1000 pg / ml) were combined, with a TAU:AB42 ratio of 1.00. - The ratio of TAU:AB42 to CSF3 (2000 pg / ml) and AB42 (1000 pg / ml) is 2.00. According to the Dynabeads kit instructions, the first group of beads was coated with TAU antibody and the second group of beads was coated with AB42 antibody. After coating, the two groups of beads were mixed together.

[0177] Add the bead mixture to the simulated CSF and incubate: Add 1 / 3 of the bead mixture to CSF ​​1. Add 1 / 3 of the bead mixture to CSF ​​2. Add 1 / 3 of the bead mixture to CSF ​​3. - Incubation lasts for 1 hour After incubation, collect the beads: - Place the magnet on the outside of a tube containing simulated CSF+ beads. - Gather the beads inside the tube near the magnet - Remove the liquid, now known as the supernatant. - Retain this supernatant for subsequent tests in the ELISA kit to observe how much TAU and AB42 protein was not collected by the beads. After the incubation period, the captured TAU and AB42 proteins were removed from the beads using the low-pH elution buffer provided in the Dynabeads kit: - TAU and AB42 proteins captured by elution from CSF 1 - Resuspend the eluted TAU and AB42 proteins in 220 μl of TRIS-HCl. - This is called resuspension 1 - TAU and AB42 proteins captured by CSF 2 elution - Resuspend the eluted TAU and AB42 proteins in 220 μl of TRIS-HCl. - This is called resuspension 2 - TAU and AB42 proteins captured by CSF 3 elution - Resuspend the eluted TAU and AB42 proteins in 220 μl of TRIS-HCl. - This is called resuspension 3 Then run the resuspension in the ELISA assay: - Measure TAU protein eluted from CSF 1 by running resuspension 1 in the TAU ELISA kit. - Measure TAU protein eluted from CSF 2 by running resuspension 2 in the TAU ELISA kit. - Measure TAU protein eluted from CSF 3 by running resuspension 3 in the TAU ELISA kit. - Measure AB42 protein eluted from CSF 1 by running resuspension 1 in the AB42 ELISA kit. - Measure AB42 protein eluted from CSF 2 by running resuspension 2 in the AB42 ELISA kit. - Measure AB42 protein eluted from CSF 3 by running resuspension 3 in the AB42 ELISA kit. Then, the TAU and AB42 measurements are used to calculate the TAU:AB42 ratio.

[0178] result The TAU protein yielded good results, but the AB42 protein provided a poor signal. For the TAU protein, the results are as follows: - For CSF 0, TAU was measured at 9 pg / ml - For CSF 1, TAU was measured at 74 pg / ml - For CSF 2, TAU was measured at 299 pg / ml - For CSF 3, TAU was measured at 689 pg / ml. Although the AB42 protein does not provide a perceptible signal, the known TAU:AB42 ratio during CSF preparation can be plotted against the measured TAU protein concentration (see [link to CSF ​​preparation]). Figure 18): - For CSF 0, the preparation ratio was 0.00, and TAU was measured at 9 pg / ml. - For CSF 1, the preparation ratio was 0.25, and TAU was measured at 74 pg / ml. - For CSF 2, the preparation ratio was 1.00, and the TAU was measured at 299 pg / ml. - For CSF 3, the preparation ratio was 2.00, and the TAU was measured at 689 pg / ml. Discussion: This example demonstrates a strong correlation between the TAU protein eluted from the magnetic beads and the TAU protein concentration in the original sample (in which the beads were incubated), and across a wide concentration range (0 pg / ml–2000 pg / ml) covering the entire physiological range (and beyond) of TAU protein concentration in the CSF. When two data points from previous experiments are also considered (see Example 4.8), the correlation spans six steps across this 0 pg / ml–2000 pg / ml range. This is striking because such a strong correlation—or, for that matter, any correlation at all—is unexpected. For example, the beads can be saturated with a certain concentration of TAU protein, after which the eluted protein concentration will stabilize. Considering the two experiments, in the presence of two substantially different concentrations of AB42 (333 pg / ml and 1,000 pg / ml), the aforementioned correlation between the eluted TAU and the TAU concentration in the original sample exists. If the AB42 signal is not disrupted, it is most likely constant, demonstrating the preservation of the ratio. The next step is to change the concentrations of AB42 and TAU in the simulated CSF, not just the TAU concentration.

[0179] Example 4.10: ELISA Measurement of AB42 in Mucus Objective: This is the first ELISA measurement of proteins in the presence of mucus. This experiment allows determination of the effect of large amounts of mucus on the ability to detect proteins. Magnetic beads are not used.

[0180] Material - AB42 ELISA = Amyloid β 42 ELISA Kit (Abcam catalog number AB289832) - AB42 = amyloid β (1-42) protein from the above ELISA kit - PBS = Phosphate-buffered saline, pH 7.4 (Sigma part # P3813-10PAK) - Mucus = Artificial nasal mucus, 3000-5000 cP (Biochemazone, catalog number BZ253) method First, a sample containing only PBS as the liquid component is prepared. This is the typical simulated CSF: Simulated CSF = PBS with 700 pg / ml AB42. Then, a sample containing only mucus as a liquid component was prepared. This extreme case determined the effect of 100% mucus on protein measurements. Simulated mucus = mucus with 700 pg / ml AB42. Now these samples will be incubated for different durations: - Simulated CSF incubation lasts for 1 hour before measurement by ELISA. - Simulated mucus incubation lasts for 10 minutes before measurement by ELISA. - Simulated mucus incubation lasts for 1 hour before measurement by ELISA. The concentration of AB42 was measured using the AB42 ELISA kit.

[0181] result The simulated CSF measurement was 775 pg / ml, close to the target AB42 concentration of 700 pg / ml. See also Figure 19 The simulated mucus samples were all measured to be just over 400 pg / ml, or roughly half the prepared concentration.

[0182] Discussion: Mucus has the effect of reducing apparent AB42 concentration. The fact that the two mucus samples measured the same concentration likely means that the measurements were reproducible and unaffected by the residence time of AB42 in the mucus. If AB42 were actively digested by proteases in the mucus, different concentrations would be expected in the two measurements. This further suggests that the signal reduction relative to the signal measured in simulated CSF may be a matrix effect (i.e., the viscous mucus itself causes a reproducible reduction in signal from the ELISA kit). This is not a problem, and such an effect has been observed in previous experiments (see Example 4.4) where apparent TAU ​​concentrations were reduced using a high-viscosity PEG and glycerol liquid swab formulation.

[0183] Interestingly, the signal inhibition effect of viscosity is much lower in mucus than in PEG or glycerol. According to the manufacturer, PEG and glycerol were only tested up to 40 cP, while synthetic mucus was around 4,000 cP (100 times larger).

[0184] Example 5: Magnetic Bead Toxicity This embodiment relates to the safety and toxicity of magnetic beads, particularly when it involves magnetic beads held in a liquid swab deposited in the olfactory crack.

[0185] Magnetic beads: The beads used in the above embodiments are superparamagnetic beads. Such beads exhibit magnetic behavior only in the presence of a magnetic field. The magnetic behavior disappears when the magnetic field is removed.

[0186] The fact that superparamagnetic beads only exhibit magnetism in the presence of an external magnetic field is an attractive feature in life sciences, as they can be brought together or displaced when needed by introducing a magnetic field, otherwise remaining free to move around. Furthermore, permanent (or even temporary) retention of magnetization could increase additional health risks within the body (e.g., clamping a membrane between two magnetic beads that attract each other could damage the membrane and—if the magnetization is permanent—potentially eliminate the possibility of them being expelled from the body). The absence of such conditions, their generally high biocompatibility, and many other factors make superparamagnetic beads a very useful tool in medical devices.

[0187] The shorter term 'magnetic bead' or simply 'bead' is used to refer to both paramagnetic beads and superparamagnetic beads. The term 'superparamagnetic' refers to a class of paramagnetic beads containing materials that have a particularly high magnetic response to an external magnetic field (technically, superparamagnetic beads have a higher magnetic susceptibility than other paramagnetic beads).

[0188] The beads used to collect TAU ​​and AB42 proteins (see Example 4) are 2.8 μm diameter Dynabeads, which are superparamagnetic iron oxide beads coated with an inert polymer shell. These specific Dynabeads have a polymer shell coated with G proteins. G proteins can be used to attach any antibody conjugated to a G protein. The examples above include TAU and AB42 antibodies that are well conjugated to G proteins.

[0189] The beads used to study the movement of beads in a liquid swab (see Example 2.2) are very large (50 μm diameter) superparamagnetic iron oxide beads covered with an inert silica shell.

[0190] Bead coatings are frequently used to reduce in vivo exposure to internal magnetic materials (typically iron oxide) and to impart desired functions (including antibody binding, soluble drug layers, and improved contrast for a variety of imaging techniques).

[0191] Iron oxide particles such as magnetite (Fe3O4) or its oxidized and more stable form, maghemite (γ-Fe2O3), are superior to other metal oxide particles in terms of biocompatibility and stability, and are by far the most commonly used magnetic particles in biomedical applications. Iron oxide eventually decomposes to form hemoglobin.

[0192] Another embodiment: Magnetic beads in liquid swabs: Adding magnetic beads to liquid swabs can aid in biomarker collection. Magnetic beads in liquid swabs can be used to mix CSF into the liquid swab and / or capture biomarkers directly from the CSF. It is expected that the CSF will float on top of the denser liquid swab. Magnetic beads in liquid swabs can help overcome this stratification and aid in the retrieval of CSF biomarkers.

[0193] Mixing without directly capturing biomarkers: Ordinary magnetic beads without antibody coating can be used. A moving magnetic field can cause the beads to move around to actively mix CSF into the liquid swab. Such mixing will increase the volume of CSF collected with the liquid swab when it is retrieved. Ordinary beads do not require any special coating layer required for antibody binding; they are freely coated with various inert coating layers, for example, to reduce toxicity.

[0194] Direct capture of biomarkers without mixing: Antibody-coated magnetic beads can be used to directly capture biomarkers. A static magnetic field can pull the beads to the top of the olfactory cleft, where they can come into direct contact with CSF floating on a liquid swab. Biomarkers in the CSF can then be captured by the antibody-coated beads.

[0195] Direct biomarker capture + mixing: Antibody-coated magnetic beads can directly capture biomarkers. A moving magnetic field causes the beads to move around, actively mixing CSF into the liquid swab. This mixing increases the contact between the biomarker and the antibody-coated beads, maximizing biomarker capture efficiency.

[0196] Biomarker concentration and washing: After the liquid swabs are retrieved, antibody-coated magnetic beads can be used to concentrate the biomarkers (the liquid swabs are collected in a container, the beads are pulled into a single spot using a magnet, and then the liquid swabs are discarded). Similarly, the magnetic beads can be washed (to remove enzymes that may damage the biomarkers) and the wash fluid can be discarded.

[0197] Biomarker elution and analysis: After washing, biomarkers can be eluted (removed) from the antibody and analyzed immediately or frozen for subsequent analysis.

[0198] Biomarkers are held on beads: Biomarkers that bind to antibodies can be held on magnetic beads. Lateral flow immunoassays use antibodies attached to beads (including magnetic beads). The complete bead-antibody-biomarker complex can be fed directly into the lateral flow strip for very inexpensive analysis. While many lateral flow assays are qualitative, there are ways to make them more quantitative, including using instruments that more accurately measure the amount of beads captured at the test and control lines.

[0199] List of implementation plans: Listed Implementation Scheme 1. A method for collecting biomaterials from a target region of a subject, the method comprising: a. Delivering a magnetic formulation containing more than one magnetic particle to a target region of a subject, wherein the magnetic formulation is configured to capture biomaterial; and b. Retrieve at least a portion of the magnetic formulation from the target region, thereby collecting any biological material captured by that portion of the magnetic formulation.

[0200] Example 2. The method described in Example 1, wherein the target region is the subject's olfactory region or a subregion of the nasal cavity.

[0201] Implementation scheme 3. The method according to any one of the foregoing implementation schemes, wherein the sub-region is a sub-region of the nasal turbinate, nasopharynx, or nasal lymphatic system.

[0202] 4. The method according to any one of the foregoing embodiments, wherein the magnetic particles are paramagnetic, diamagnetic, or ferromagnetic.

[0203] Example 5. The method according to any one of the foregoing embodiments, wherein the formulation comprises a liquid suspension.

[0204] 6. The method according to any one of the foregoing embodiments, wherein the biological material is captured from cerebrospinal fluid (CSF).

[0205] 7. The method according to any one of the foregoing embodiments, wherein the magnetic particles are uncoated.

[0206] 8. The method according to any one of the foregoing embodiments, wherein the magnetic particles are coated with or coupled to one or more antibodies configured to bind to selected biological material.

[0207] Example 9. The method according to any one of the foregoing embodiments, wherein each magnetic particle is coated with or conjugated to a single antibody.

[0208] Example 10. The method according to any one of the foregoing embodiments, wherein each magnetic particle is coated with or coupled to multiple antibodies.

[0209] Example 11. The method according to any one of the foregoing embodiments, wherein analysis of the captured biological material provides quantitative values ​​of the concentration of tau protein, the concentration of β-amyloid protein, or their ratio.

[0210] Example 12. The method according to any one of the foregoing embodiments, wherein analysis of the captured biological material provides a quantitative value of the ratio of tau protein to β-amyloid protein 42, thereby enabling the diagnosis of Alzheimer's disease.

[0211] Example 13. The method according to any one of the foregoing embodiments further includes eluting the captured biological material from the magnetic particles and then analyzing the eluted biological material.

[0212] Example 14. The method according to any one of the foregoing embodiments, wherein the biological material is analyzed by enzyme-linked immunosorbent assay (ELISA).

[0213] Example 15. The method according to any one of the foregoing embodiments further includes analyzing the captured biological material as a protein-bead complex.

[0214] Example 16. The method according to any one of the foregoing embodiments, wherein the magnetic particles or protein-bead complexes are washed and / or frozen prior to analysis of the captured biological material.

[0215] Example 17. The method according to any one of the foregoing embodiments, wherein the biological material is analyzed via a self-administered test.

[0216] Example 18. The method according to any one of the foregoing embodiments, wherein the biological material is analyzed by lateral flow assay, lateral flow immunoassay, lateral flow immunochromatography or magnetic lateral flow analyzer.

[0217] Example 19. The method according to any one of the foregoing embodiments, wherein a portion of the magnetic agent is retrieved using a magnet.

[0218] Example 20. The method according to any one of the foregoing embodiments further includes exposing the subject to a magnetic field before retrieving at least a portion of the magnetic preparation.

[0219] 21. The method according to any one of the foregoing embodiments, wherein the magnetic field is an oscillating magnetic field.

[0220] 22. The method according to any one of the foregoing embodiments further includes positioning a magnet on or near the bridge of the subject's nose.

[0221] Example 23. The method according to any one of the foregoing embodiments, wherein the biological material includes cerebrospinal fluid, neural stem cells, proteins, enzymes, oligonucleotides, one or more microorganisms from the patient's microbiome, one or more components of the patient's metabolome, one or more pathogens, and / or one or more biomarkers of interest.

[0222] Example 24. The method according to any one of the foregoing embodiments, wherein the biological material comprises amyloid-β, tau protein, or a combination thereof.

[0223] Example 25. The method according to any one of the foregoing embodiments, wherein the magnetic preparation is delivered via a delivery device comprising a cannula and / or a microfluidic channel, wherein the cannula and / or the microfluidic channel is configured for insertion into the nasal cavity of the subject.

[0224] Example 26. The method according to any one of the foregoing embodiments, wherein the formulation is delivered via an apparatus comprising: a. A housing defining a first insertable portion and a second insertable portion, each insertable portion for insertion into a subject's nasal passage, wherein, when the first insertable portion is inserted into the subject's nasal passage, at least one insertable portion engages tissue within the nasal passage to open or dilate the subject's internal nasal flap, thereby positioning at least one of the insertable portions for delivery of a formulation to the subject's olfactory region or a subregion of the subject's nasal cavity; and b. An actuator that delivers a formulation from any one or both of the insertable portions when the device is actuated.

[0225] Example 27. A method for diagnosing a subject, comprising: a. To collect biological materials from a subject by performing the method according to any one of the foregoing embodiments; b. Analyze the collected biological materials; and c. Based on the analysis in step b, make a diagnosis.

[0226] 28. The method according to any one of the foregoing embodiments, wherein the method is used to diagnose Alzheimer's disease or another neurodegenerative disease.

[0227] Example 29. The method according to any one of the foregoing embodiments, wherein the analysis of biological materials includes identifying and / or quantifying biomarkers, pathogens and / or microorganisms in the collected biological materials.

[0228] The listed implementation scheme 30. The method according to any one of the foregoing implementation schemes further includes associating identified and / or quantified biomarkers, pathogens and / or microorganisms with corresponding physiological characteristics and / or medical conditions.

[0229] 31. The method according to any one of the foregoing embodiments, wherein the analysis of biological materials includes the use of a point-of-care assay system.

[0230] Example 32. The method according to any one of the foregoing embodiments, wherein the point-of-care measurement system is configured to receive a sample of collected biological material from a delivery device.

[0231] Listed implementation scheme 33. A method for delivering a therapeutic agent into the nasal cavity of a subject, the method comprising: a. Delivering a magnetic formulation to a targeted area in the nasal cavity, the magnetic formulation comprising: i. More than one magnetic particle; and ii. therapeutic agents; and b. Expose the subject to a magnetic field; The magnetic field causes the magnetic particles to move, thereby enhancing the delivery of the therapeutic agent.

[0232] Listed implementation scheme 34. A method for enhancing contact between a formulation and a target region within the nasal cavity of a subject, the method comprising: a. Delivering a magnetic formulation containing more than one magnetic particle to a targeted area within the nasal cavity; and b. By exposing the subject to a magnetic field that causes at least some magnetic particles to oscillate or magnetophoretically move, the contact between the magnetic agent and the target area is enhanced.

[0233] 35. The method according to any one of the foregoing embodiments, wherein the magnetic field is an oscillating magnetic field.

[0234] 36. The method according to any one of the foregoing embodiments, wherein the frequency of the oscillating magnetic field is about 0.01-0.02, about 0.02-0.03, about 0.03-0.04, about 0.04-0.05, about 0.05-0.06, about 0.06-0.07, about 0.07-0.08, about 0.08-0.09, about 0.09-0.1, about 0.1-0.15, about 0.15-0.2, about 0.2-0.25, about 0.25-0.3, or about 0.3-0.35. Approximately 0.35-0.4, approximately 0.4-0.45, approximately 0.45-0.5, approximately 0.5-0.55, approximately 0.55-0.6, approximately 0.6-0.65, approximately 0.65-0.7, approximately 0.7-0.75, approximately 0.75-0.8, approximately 0.8-0.85, approximately 0.85-0.9, approximately 0.9-0.95, approximately 0.95-1, approximately 1-5, approximately 5-10, approximately 10-15, approximately 15-20, approximately 20-25, approximately 25-30, approximately 30-35, approximately 35-40, approximately 40-45, approximately 45-50, approximately 50-55, approximately 55-60, approximately 60-65, approximately 65-70, approximately 70-75, approximately 75-80, approximately 80-85, approximately 85-90, approximately 90-95, approximately 95-100, approximately 100-110, approximately 110-120, approximately 120-130, approximately 130-140, approximately 140-150, approximately 150-160, approximately 160-170, approximately 170-180, approximately 180-190, approximately 190 -200, approximately 200-220, approximately 220-240, approximately 240-260, approximately 260-280, approximately 280-300, approximately 300-350, approximately 350-400, approximately 400-450, approximately 450-500, approximately 500-550, approximately 550-600, approximately 600-650, approximately 650-700, approximately 700-750, approximately 750-800, approximately 800-850, approximately 850-900, approximately 900-950 or approximately 950-1000 Hz.

[0235] 37. The method according to any one of the foregoing embodiments, wherein the magnetic field is provided via a permanent magnet.

[0236] Example 38. The method according to any one of the foregoing embodiments, wherein the magnetic field reduces the necessary residence time of the preparation in the nasal cavity.

[0237] Listed embodiment 39. A device for delivering and retrieving a magnetic preparation to a target area of ​​a subject's nasal cavity, the device comprising: a. A housing including an insertable portion comprising a distal end, a proximal end, and a retrieval magnet; and b. Subject engagement portion, which engages with the columellar region of the subject to place the distal end of the insertable portion within the jet area of ​​the subject's nasal passage; The device is configured to deliver a magnetic agent to a target region and retrieve at least a portion of the magnetic agent from the target region.

[0238] 40. The device according to any one of the foregoing embodiments, wherein the target region is an olfactory region.

[0239] Example 41. The apparatus according to any one of the foregoing embodiments, wherein the apparatus includes a magnetic portion for retrieving a portion of the magnetic preparation.

[0240] Example 42. The apparatus according to any one of the foregoing embodiments, wherein the apparatus dispenses the magnetic agent as a laminar jet.

[0241] Example 43. The apparatus according to any one of the foregoing embodiments, wherein the apparatus includes a compliant dispensing tip, the compliant dispensing tip including a compliant and flexible soft tip.

[0242] Example 44. The device according to any one of the foregoing embodiments, wherein the subject engagement portion applies pressure to the columellar region of the subject such that and / or causes the preparation to be delivered from the insertable portion to the subject.

[0243] Example 45. The apparatus according to any one of the foregoing embodiments, wherein the insertable portion includes a dispensing element for delivering a magnetic agent to a target subregion of the subject.

[0244] Listed Implementation Scheme 46. An apparatus for retrieving magnetic particles from the nasal cavity of a subject, the apparatus comprising: a. Retrieve the magnet; and b. A magnet support configured to position a retrieved magnet within a target area of ​​the nasal cavity to facilitate the retrieval of more than one magnetic particle from the target area.

[0245] Listed Embodiment 47. A system for enhancing contact between a formulation and a target region within the nasal cavity of a subject, the system comprising: a. A device for delivering a magnetic formulation containing more than one magnetic particle to a targeted area of ​​the nasal cavity; and b. A magnet, which is used to induce movement of the magnetic preparation within the nasal cavity, thereby enhancing contact with the target area.

[0246] Example 48. The system according to any one of the foregoing embodiments further includes a support configured to hold the magnet in a desired position on or near the bridge of the subject's nose.

[0247] Example 49. The system according to any one of the foregoing embodiments further includes a retrieval magnet configured to be inserted into the subject's nasal cavity for retrieving a portion of the magnetic preparation.

[0248] Example 50. The system according to any one of the foregoing embodiments, wherein the retrieved magnet has a diameter of about 2.5 mm.

[0249] 51. The system according to any one of the foregoing embodiments further includes a retrieval magnet configured to be positioned outside the subject's nasal cavity for retrieving a portion of the magnetic preparation.

[0250] Example 52. A magnetic formulation for sampling biomarkers from the nasal cavity of a subject, the formulation comprising more than one magnetic particle and configured to capture biological material upon delivery into the nasal cavity, wherein the delivered formulation is configured to be removed from the nasal cavity together with the biological material.

[0251] Example 53. The formulation according to any one of the foregoing embodiments, wherein the magnetic particles are coated with tau antibody, β-amyloid antibody or a combination thereof.

[0252] Example 54. The formulation according to any one of the foregoing embodiments, wherein the magnetic particles comprise more than one first magnetic particle coated with tau antibody and more than one second magnetic particle coated with β-amyloid antibody.

[0253] 55. The formulation according to any one of the foregoing embodiments, wherein the magnetic particles have an average diameter of about 50 micrometers.

[0254] Example 56. The formulation according to any one of the foregoing embodiments, wherein the magnetic particles have a particle size of about 0.01-0.015, about 0.015-0.02, about 0.02-0.025, about 0.025-0.03, about 0.03-0.035, about 0.035-0.04, about 0.04-0.045, about 0.045-0.05, about 0.05-0.055, about 0.055-0.06, about 0.06-0.065, about 0.065-0.07, about 0.07-0.075, about 0.075-0.08, about 0.08-0.085, about 0.085-0.09, about 0.09-0.095, about 0.095-0.1, approximately 0.1-0.15, approximately 0.15-0.2, approximately 0.2-0.25, approximately 0.25-0.3, approximately 0.3-0.35, approximately 0.35-0.4, approximately 0.4-0.45, approximately 0.45-0.5, approximately 0.5-0.55, approximately 0.55-0.6, approximately 0.6-0.65, approximately 0.65-0.7, approximately 0.7-0.75, approximately 0.75-0.8, approximately 0.8-0.85, approximately 0.85-0.9, approximately 0.9-0.95, approximately 0.95-1, approximately 1-5, approximately 5-10, approximately 10-15, approximately 15-20, approximately 20-25, approximately 25-30, approximately 30- 35, approximately 35-40, approximately 40-45, approximately 45-50, approximately 50-55, approximately 55-60, approximately 60-65, approximately 65-70, approximately 70-75, approximately 75-80, approximately 80-85, approximately 85-90, approximately 90-95, approximately 95-100, approximately 100-105, approximately 105-110, approximately 110-115, approximately 115-120, approximately 120-125, approximately 125-130, approximately 130-135, approximately 135-140, approximately 140-145, approximately 145-150, approximately 150-155, approximately 155-160, approximately 160-165, approximately 165-170, approximately 170-175, approximately Average diameter of 175-180, approximately 180-185, approximately 185-190, approximately 190-195, approximately 195-200, approximately 200-210, approximately 210-220, approximately 220-230, approximately 230-240, approximately 240-250, approximately 250-260, approximately 260-270, approximately 270-280, approximately 280-290, approximately 290-300, approximately 300-320, approximately 320-340, approximately 340-360, approximately 360-380, approximately 380-400, approximately 400-420, approximately 420-440, approximately 440-460, approximately 460-480, or approximately 480-500 micrometers.

[0255] Example 57. The formulation according to any one of the preceding embodiments, wherein the formulation is delivered to the olfactory region of the nasal cavity.

[0256] Example 58. A formulation according to any one of the foregoing embodiments, wherein the formulation is configured to capture biomaterial from a target subregion of the nasal cavity.

[0257] Example 59. The formulation according to any one of the foregoing embodiments, wherein the delivered formulation is configured to preserve the captured biological material upon retrieval.

[0258] Example 60. An formulation according to any one of the foregoing embodiments, wherein the biomaterial comprises cerebrospinal fluid (CSF), one or more microorganisms in the patient's microbiome, one or more components in the patient's metabolome, one or more pathogens and / or one or more biomarkers of interest.

[0259] Listed embodiments 61. A formulation according to any one of the foregoing embodiments, wherein the formulation is configured to capture specific biological material.

[0260] Example 62. The formulation according to any one of the foregoing embodiments, wherein the formulation comprises a buffered saline solution, polyethylene glycol, glycerin, or a combination thereof.

[0261] Example 63. The formulation according to any one of the foregoing embodiments, wherein the buffer saline solution is a 100 mM phosphate buffered saline solution.

[0262] Example 64. The formulation according to any one of the foregoing embodiments, wherein the formulation comprises one or more gelling agents and / or thickeners.

[0263] Example 65. The formulation according to any one of the foregoing embodiments, wherein the formulation contains a viscosity between about 20 cP and 40 cP.

[0264] Example 66. The formulation according to any one of the preceding embodiments, wherein the formulation comprises a viscosity between about 0-5, about 5-10, about 10-15, about 15-20, about 20-25, about 25-30, about 30-35, about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about 60-65, about 65-70, about 70-75, about 75-80, about 80-85, about 85-90, about 90-95, or about 95-100 cP.

[0265] Example 67. The formulation according to any one of the preceding embodiments, wherein the formulation comprises a viscosity between about 100-110, about 110-125, about 125-150, about 150-200, about 200-250, about 250-300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-550, about 550-600, about 600-650, about 650-700, about 700-750, about 750-800, about 800-850, about 850-900, about 900-950, or about 950-1000 cP.

[0266] Example 68. A formulation according to any one of the foregoing embodiments, wherein the formulation comprises a viscosity modifier to provide a desired viscosity for the formulation.

[0267] Example 69. The formulation according to any one of the foregoing embodiments, wherein the viscosity modifier comprises at least one of glycerol, pectin and polyethylene glycol.

[0268] Example 70. The formulation according to any one of the foregoing embodiments, wherein the viscosity modifier accounts for 25%-75% of the formulation by volume.

[0269] 71. An formulation according to any one of the foregoing embodiments, wherein the formulation is configured for removal from the nasal cavity using a magnet.

[0270] Example 72. A formulation according to any one of the foregoing embodiments, wherein the formulation comprises one or more specific monoclonal or polyclonal antibodies for targeting specific biological materials.

[0271] 73. An embodiment of any of the foregoing embodiments, wherein the embodiment comprises one or more specific aptamers for targeting specific biological materials.

[0272] Example 74. A formulation according to any one of the foregoing embodiments, wherein the formulation is provided, delivered and / or removed as a pellet of the formulation.

[0273] Example 75. A magnetic formulation for delivering a therapeutic agent to a target region of a subject's nasal cavity, the formulation comprising a magnetic formulation containing more than one magnetic particle and a therapeutic agent, wherein the magnetic particle is configured to enhance contact between the therapeutic agent and the target region.

[0274] Example 76. The formulation according to any one of the foregoing embodiments, wherein the formulation is delivered to the olfactory region of the nasal cavity.

[0275] Listed Implementation Scheme 77. A method for collecting biomaterials from a target region of a subject, the method comprising: a. Delivering a magnetic formulation containing more than one magnetic particle to a target region of a subject, wherein the magnetic formulation is configured to capture biomaterial; and b. Retrieve at least a portion of the magnetic formulation from the target region, thereby collecting any biological material captured by that portion of the magnetic formulation.

[0276] 78. The method according to any one of the foregoing embodiments, wherein the target area is the rectal region, vaginal region, ear sub-region, navel, skin sub-region, or another body surface or orifice.

Claims

1. A method for collecting biomaterials from a target region of a subject, the method comprising: a. Delivering a magnetic formulation containing more than one magnetic particle to the target region of a subject, wherein the magnetic formulation is configured to capture the biomaterial; as well as b. Retrieve at least a portion of the magnetic formulation from the target region, thereby collecting any biological material captured by the portion of the magnetic formulation.

2. The method according to claim 1, wherein the target region is the olfactory region or a subregion of the nasal cavity of the subject.

3. The method according to any one of the preceding claims, wherein the sub-region is a sub-region of the nasal turbinate, nasopharynx, or nasal lymphatic system.

4. The method according to any one of the preceding claims, wherein the magnetic particles are paramagnetic, diamagnetic, or ferromagnetic.

5. The method according to any one of the preceding claims, wherein the formulation comprises a liquid suspension.

6. The method according to any one of the preceding claims, wherein the biomaterial is captured from cerebrospinal fluid (CSF).

7. The method according to any one of the preceding claims, wherein the magnetic particles are coated with or coupled to one or more antibodies, the one or more antibodies being configured to bind to selected biological material.

8. The method according to any one of the preceding claims, wherein analysis of the captured biological material provides quantitative values ​​of the concentration of tau protein, the concentration of β-amyloid protein, or their ratio.

9. The method according to any one of the preceding claims further includes eluting the captured biological material from the magnetic particles and then analyzing the eluted biological material.

10. The method according to any one of the preceding claims further includes analyzing the captured biomaterial as a protein-bead complex.

11. The method according to any one of the preceding claims, wherein the magnetic particles or protein-bead complex are washed and / or frozen prior to analysis of the captured biological material.

12. The method according to any one of the preceding claims, wherein the portion of the magnetic agent is retrieved using a magnet.

13. The method according to any one of the preceding claims, further comprising exposing the subject to a magnetic field before retrieving at least a portion of the magnetic preparation.

14. The method according to any one of the preceding claims, further comprising positioning a magnet on or near the bridge of the subject's nose.

15. The method according to any one of the preceding claims, wherein the biological material comprises cerebrospinal fluid, neural stem cells, proteins, enzymes, oligonucleotides, one or more microorganisms of the patient's microbiome, one or more components of the patient's metabolome, one or more pathogens and / or one or more biomarkers of interest.

16. The method according to any one of the preceding claims, wherein the formulation is delivered via a device comprising: a. A housing defining a first insertable portion and a second insertable portion, each insertable portion for insertion into the nasal passage of the subject, wherein when the first insertable portion is inserted into the nasal passage of the subject, at least one insertable portion engages tissue within the nasal passage to open or dilate the subject's internal nasal flap, thereby positioning at least one of the insertable portions for delivery of the formulation to the subject's olfactory region or a subregion of the subject's nasal cavity; as well as b. An actuator that delivers the formulation from any one or both of the insertable portions when the device is actuated.

17. A method of delivering a therapeutic agent into the nasal cavity of a subject, the method comprising: a. Delivering a magnetic formulation to a targeted area of ​​the nasal cavity, the magnetic formulation comprising: i. More than one magnetic particle; and ii. therapeutic agents; as well as b. Expose the subject to a magnetic field; The magnetic field causes the magnetic particles to move, thereby enhancing the delivery of the therapeutic agent.

18. An apparatus for delivering and retrieving a magnetic formulation to a target area in the nasal cavity of a subject, the apparatus comprising: a. A housing, the housing including an insertable portion, the insertable portion including a distal end, a proximal end, and a retrieval magnet; as well as b. Subject engagement portion, wherein the subject engagement portion engages the columellar region of the subject to place the distal end of the insertable portion within the jet area of ​​the subject's nasal passage; The device is configured to deliver a magnetic agent to the target region and retrieve at least a portion of the magnetic agent from the target region.

19. The device of claim 18, wherein the targeting region is an olfactory region.

20. The apparatus of claim 18, wherein the apparatus includes a magnetic portion for retrieving a portion of the magnetic preparation.

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