Hedgehog-shaped particles with multiscale chirality and mirror asymmetry for the analysis of nanostructured biological assemblies

Abstract

Materials and devices, such as microfluidic devices and isolation columns (e.g., spin-columns for centrifuges or syringe-fed columns), are provided for detecting, analyzing, and / or separating biomolecules. A plurality of hedgehog-shaped particles that exhibits a multiscale chirality can be used in an active or sensing region of a device. Each hedgehog-shaped particle comprises a core region and a plurality of projections connected thereto. Each hedgehog-shaped particle comprises a material selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and combinations thereof and an amino-acid derived chiral moiety. One or more target analytes associate with the plurality of hedgehog-shaped particles, for example, in the active region of a device, in the absence of any added targeting moiety to indicate the presence of, analyze, and / or separate the one or more target analytes. Methods of detecting or separating a target analyte in a biological fluid sample are also provided.

Description

HEDGEHOG-SHAPED PARTICLES WITH MULTISCALE CHIRALITY AND MIRROR ASYMMETRY FOR THE ANALYSIS OF NANOSTRUCTURED BIOLOGICAL ASSEMBLIESGOVERNMENT SUPPORT

[0001] This invention was made with government support under 2243104, 2317423, and 1566460 awarded by the National Science Foundation. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 668,567, filed on July 8, 2024. The entire disclosure of the above application is incorporated herein by reference.FIELD

[0003] The present disclosure relates to a material and / or a device, such as a microfluidic device, for detecting, analyzing, and / or separating one or more target analytes that includes an active region comprising a plurality of hedgehog-shaped particles that exhibits a multiscale chirality, as well as methods for detecting, analyzing, and / or separating a target analyte in a biological fluid sample.BACKGROUND

[0004] This section provides background information related to the present disclosure which is not necessarily prior art.

[0005] Improving an ability of devices to detect and / or analyze various biomolecules is important for a variety of fields, including in medicine, where treating, monitoring, and controlling biological systems is paramount. Chiral microstructures and chiral nanostructures have been used for detecting and analyzing biomolecules. Chirality is the foundational property of all biomolecules, which describes the fact the molecules and their mirror images cannot be superimposed, which manifests as different absorption coefficients for left-and right-circularly polarized light. Chiroptical activity of biomolecules is typically measured by circular dichroism (CD) spectrometry whose utilization, along with SERS, SPR and other methods, is highly desirable for biomolecule analysis because of the potentially large amount of information certain biomolecules, like membrane and cargo exosomes and other extracellular vesicles contained in polarization rotation spectra. Circular dichroism (CD) is differential absorption of left and rightcircularly polarized light. Circular dichroism may be expressed CD = extinction of left-handed circularly polarized light (LCP) - extinction of right-handed circularly polarized light (RCP), where cross-section extinction is a sum of cross-section absorption and cross-section scattering. However, extraction of this information and even their selective detection is challenging because chiroptical activity of proteins, lipids, and other biomolecules are typically confined to UV part of the spectrum, making detection of specific proteins in complex biological media very difficult using CD spectra. Chiral nanoparticles have been used to detect certain target biomolecules, such as mutations in cancer exosomes, by taking advantage of the chiral inorganic nanostructures that can both enhance and shift CD peaks associated with specific proteins. However, these chiral nanoparticles require the presence of a binding moiety / targeting ligand, such as an antigen, attached to the nanoparticle to bond with the target analytes (e.g., extracellular vesicles like exosomes).

[0006] Thus, detection and analysis of extracellular vesicles (EVs) and similar biomolecules can be important for various applications, in addition to detecting the presence of cancer. For example, extracellular vesicles (EVs) have emerged as being important for screening for conditions like sepsis, in that EVs derived from activated or apoptotic cells carry altered cargoes. For example, EVs originating from various cell types, including activated macrophages, monocytes, and neutrophils, have been found to have altered protein profiles. Moreover, EVs carry different types of nucleic acids, such as mRNA, miRNA, IncRNA, and circRNA, which change during sepsis progression.

[0007] Despite their immense diagnostic and therapeutic potential, current isolation methods, particularly one of the best techniques currently available — ultracentrifugation (UC) — remain inadequate due to high cost of equipment, relatively slow processing time, for example, greater than about 4 to 8 hours of processing, poor reproducibility, and further inflicting damage to the EVs. Alternative techniques like antibody capture, size-exclusion chromatography, and polymer precipitation each have significant drawbacks, as well. It would be desirable to provide chiral microparticles and nanoparticles that provide high specificity to such targets and thus capture biomolecules without any need for a targeting ligand, while leaving them intact without damage. Further, it would be advantageous to develop accurate, rapid, streamlined devices and methods of detecting and capturing target analytes of a biological origin, such as exosomes or other mutations in proteins, extracellular vesicles, derived from biological fluids in improved devices, such as microfluidic devices or isolation columns.SUMMARY

[0008] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0009] In certain aspects, the present disclosure relates to materials for detecting, analyzing, or separating one or more target analytes. The materials may be included in a device for detecting, analyzing, and / or separating one or more target analytes. The device includes an active region comprising a plurality of hedgehog-shaped particles that exhibit a multiscale chirality. Each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, as well as a shape directing organic chiral moiety. At least one of the core regions and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof. One or more target analytes associate with the plurality of hedgehog-shaped particles in the active region in the absence of any added targeting moiety.

[0010] In one aspect, the shape directing organic chiral moiety is an amino acid derived chiral moiety comprising a thiol group.

[0011] In one aspect, the shape directing organic chiral moiety is an amino acid derived chiral moiety selected from the group consisting of: cysteine, homocysteine, penicillamine, arginine, lysine, methionine, and / or combinations thereof.

[0012] In one aspect, the plurality of projections is substantially orthogonal to a surface of the core region and extend over an entire surface of the core region.

[0013] In one aspect, the plurality of projections has a rod-like needle shape or a sheetlike flat shape.

[0014] In one aspect, a branching pattern of the plurality of projections is stochastic.

[0015] In one aspect, the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0016] In one aspect, an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0017] In one aspect, at least one of the core regions and the plurality of projections comprises gold.

[0018] In one aspect, at least one of the core regions and the plurality of projections further comprises silver disposed over gold.

[0019] In one aspect, the material comprises copper (Cu) and is selected from the group consisting of: copper sulfide, copper iodide, and combinations thereof.

[0020] In one aspect, the core region and the plurality of projections are formed of the material that is the same composition.

[0021] In one aspect, the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm.

[0022] In one aspect, the multiscale chirality further comprises at least one third chirality selected from the group consisting of: a mesoscale chirality of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer); a microscale chirality of greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers); a sub-millimeter scale chirality of greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm); a millimeter scale chirality of greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters); and combinations thereof.

[0023] In one aspect, one or more target analytes indicates a presence of cancerous cells or mutated proteins.

[0024] In one aspect, the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

[0025] In certain further aspects, the present disclosure relates to a microfluidic device for detecting, analyzing, and / or separating one or more target analytes. The microfluidic device comprises a microfluidic channel having an inlet and an outlet that receives a fluid sample. The microfluidic channel comprises at least one active region having a surface comprising a plurality of hedgehog-shaped particles that exhibit a multiscale chirality and associates with one or more target analytes. Each respective hedgehog- shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core regions and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof. Each respective hedgehog-shaped particle also comprises a shape directing organic chiral moiety. The one or more target analytesassociate with the plurality of hedgehog shaped particles in the absence of any added targeting moiety.

[0026] In one aspect, the microfluidic channel is formed on a microchip.

[0027] In one aspect, the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

[0028] In one aspect, the shape directing organic chiral moiety is an amino acid derived chiral moiety is selected from the group consisting of: cysteine, homocysteine, penicillamine, arginine, lysine, methionine, and / or combinations thereof or of different amino acids.

[0029] In one aspect, the plurality of projections has a rod-like needle shape or a sheetlike flat shape and are substantially orthogonal to a surface of the core region and extend over an entire surface of the core region.

[0030] In one aspect, the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0031] In one aspect, an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0032] In one aspect, the material is selected from the group consisting of: gold, silver disposed over gold, copper sulfide, copper iodide, and combinations thereof.

[0033] In one aspect, the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm.

[0034] In one aspect, the multiscale chirality further comprises a third chirality on a mesoscale of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer).

[0035] In certain other aspects, the present disclosure relates to a method of separating one or more target analytes in a biological fluid sample obtained from a subject. The method comprises passing a biological fluid sample through a device comprising an active region having a plurality of hedgehog-shaped particles disposed thereon. Each respective hedgehog shaped particle comprises a core region and a plurality of projections connected to a surface of the core region. At least one of the core regions and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof. Eachrespective hedgehog-shaped particle also comprises a shape directing organic chiral moiety, such as an amino acid derived chiral moiety. The plurality of hedgehog-shaped particles is capable of associating with a target analyte optionally present in the biological fluid sample. The method further comprises separating the target analyte from the biological fluid sample by capturing the target analyte in a respective hedgehog-shaped particle in the active region.

[0036] In one aspect, the device comprises a microfluidic channel comprising the active region and the passing the biological fluid sample is through the microfluidic channel.

[0037] In one aspect, the device comprises a fluid receptacle comprising the active region and the passing the biological fluid sample is through the fluid receptacle.

[0038] In one aspect, the device is a spin-column for a centrifuge and the separating further comprises spinning the spin-column in the centrifuge.

[0039] In one aspect, the device is a column and the passing further comprises passing the biological fluid sample through the active region comprising a layer of the plurality of hedgehogshaped particles and a filter.

[0040] In one aspect, the method further comprises introducing the biological fluid sample into the device by pressurizing the biological fluid as it is introduced to the device.

[0041] In yet other aspects, the present disclosure relates to a method of detecting one or more target analytes in a biological fluid sample obtained from a subject. The method comprises passing a biological fluid sample through a microfluidic channel comprising an active region having a plurality of hedgehog-shaped particles disposed thereon. The method comprises directing circularly polarized light at the microfluidic channel while the biological fluid sample is disposed therein to measure a first level of at least one of magnitude of circular dichroism or peak wavelength. Each respective hedgehog shaped particle comprises a core region and a plurality of projections connected to a surface of the core region. At least one of the core regions and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, such as an amino acid derived chiral moiety. The plurality of hedgehog-shaped particles is capable of associating with a target analyte optionally present in the biological fluid sample. The method further comprises comparing the first level of at least one of magnitude of circular dichroism or peak wavelength to a baseline level of at least one of magnitude of circular dichroism or peak wavelength in the microfluidic channel in the absence of the biological fluid sample, wherein a difference between the first level and the baseline level indicates a presence of the target analyte in the biological fluid sample.

[0042] In one aspect, the method further comprises measuring the baseline level of at least one of magnitude of circular dichroism or peak wavelength by directing circularly polarized light at the microfluidic channel in the absence of the biological fluid sample.

[0043] In certain further aspects, the present disclosure relates to a device for detecting, analyzing, and / or separating one or more target analytes. The device may comprise a fluid receptacle configured to receive a fluid sample comprising one or more target analytes. The fluid receptacle comprises a layer of a plurality of hedgehog-shaped particles that exhibits a multiscale chirality. Each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region. At least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety. One or more target analytes associate with the plurality of hedgehog-shaped particles in the layer in the absence of any added targeting moiety.

[0044] In one aspect, the device is a spin-column and the fluid receptacle defines an open region configured to receive the fluid sample. The layer comprises the plurality of hedgehog- shaped particles is disposed adjacent to the open region, a filter having a first side disposed adjacent to the layer comprising the plurality of hedgehog-shaped particles and a second side disposed adjacent to an outlet, and a fluid containment region disposed adjacent to the fluid outlet.

[0045] In one aspect, the fluid receptacle, the layer comprising the plurality of hedgehog-shaped particles, the filter, and the outlet define an extraction component. The fluid containment region is a tube in which the extraction component seats and the device further comprises a cap configured to reversibly seal the device.

[0046] In one aspect, a centrifuge system comprises a centrifuge configured to receive the device, where the spin-column is configured to seat in the centrifuge and to separate the one or more target analytes associated with the plurality of hedgehog-shaped particles from the remainder of the fluid sample collected in the fluid containment region.

[0047] In one aspect, a kit comprises the device and further comprises a first container comprising a washing medium, a second container comprising an elution medium, and at least one additional tube.

[0048] In one aspect, the device is a syringe-fed column and the fluid receptacle defines an open region configured to receive the fluid sample from a syringe. The layer comprising the plurality of hedgehog-shaped particles is disposed adjacent to the open region. The syringe-fed column further comprises a filter and a fluid containment region that is a tube.

[0049] In one aspect, a kit comprises the device and further comprises a first container comprising a washing medium, a second container comprising an elution medium, at least one additional tube, and at least one syringe.

[0050] In one aspect, the fluid receptacle has a volume of greater than or equal to about 100 microliters.

[0051] In one aspect, the fluid receptacle has a volume of greater than or equal to about 1 milliliter.

[0052] In one aspect, the fluid receptacle has a volume of greater than or equal to about 1 milliliter to less than or equal to about 100 milliliters.

[0053] In one aspect, the device further comprises a syringe in fluid communication with the fluid receptacle. The syringe is configured to transfer (e.g., inject) the fluid sample into the fluid receptacle.

[0054] In one aspect, the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0055] In one aspect, an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

[0056] In one aspect, at least one of the core regions and the plurality of projections comprises gold or silver disposed over gold.

[0057] In one aspect, the material comprises copper (Cu) and is selected from the group consisting of: copper sulfide, copper iodide, and combinations thereof.

[0058] In one aspect, the core region and the plurality of projections are formed of the material that is the same composition.

[0059] In one aspect, the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm, and at least one third chirality selected from the group consisting of: a mesoscale chirality of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer); a microscale chirality of greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers); a sub-millimeter scale chirality of greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm);a millimeter scale chirality of greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters); and combinations thereof.

[0060] In one aspect, the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins.

[0061] In one aspect, the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

[0062] In yet other aspects, the present disclosure relates to a method of separating one or more target analytes from a biological fluid sample obtained from a subject. The method comprises introducing a biological fluid sample into a column comprising a fluid receptacle and having a layer comprising a plurality of hedgehog- shaped particles. Each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region. At least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety. The plurality of hedgehogshaped particles is capable of associating with one or more target analytes optionally present in the biological fluid sample. The method comprises separating the one or more target analytes from the biological fluid sample by capturing the one or more target analytes in a respective hedgehogshaped particle in the active region by passing through the column, so that a remainder of the biological fluid sample passes through the layer for collection.

[0063] In one aspect, the column is a spin-column and the separating occurs by spinning the spin-column in a centrifuge.

[0064] In one aspect, the spinning in the centrifuge and conducted at a speed of less than or equal to about 10,000 rpm.

[0065] In one aspect, the spinning in the centrifuge is conducted at a speed of greater than or equal to about 50 rpm to less than or equal to about 3,000 rpm.

[0066] In one aspect, the column comprises the fluid receptacle defining an open region configured to receive the fluid sample, the layer comprising the plurality of hedgehog-shaped particles disposed adjacent to the open region, a filter, and a fluid containment region disposed downstream of the filter.

[0067] In one aspect, the column comprises the fluid receptacle defining an open region configured to receive the fluid sample, the layer comprising the plurality of hedgehog-shaped particles disposed adjacent to the open region, a filter having a first side disposed adjacent to thelayer comprising the plurality of hedgehog-shaped particles and a second side disposed adjacent to an outlet, and a fluid containment region disposed adjacent to the fluid outlet.

[0068] In one aspect, the fluid receptacle, the layer comprising the plurality of hedgehog-shaped particles, the filter, and the outlet define an extraction component and the fluid containment region is a tube in which the extraction component seats.

[0069] In one aspect, the separating occurs in less than or equal to about 30 minutes.

[0070] In one aspect, the separating occurs in less than or equal to about 10 minutes.

[0071] In one aspect, the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins.

[0072] In one aspect, the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

[0073] In one aspect, the one or more target analytes comprises extracellular vesicles (EVs) and the separating is marker-agnostic for separating heterogenous extracellular vesicle (EV) populations from the biological fluid sample.

[0074] In one aspect, greater than or equal to about 70% of an initial amount of the one or more target analytes present in the biological fluid sample are separated from the biological sample after the separating.

[0075] In one aspect, the method further comprises after the separating, introducing a washing liquid into the fluid receptacle so that it passes through the layer comprising a plurality of hedgehog-shaped particles.

[0076] In one aspect, the method further comprises after the separating, introducing an elution liquid into the fluid receptacle so that it passes through the layer comprising a plurality of hedgehog-shaped particles and elutes the one or more target analytes from the layer for collection.

[0077] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.DRAWINGS

[0078] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure.

[0079] FIGS. 1A-1D show an embodiment of a device for analyzing biomolecules according to certain aspects of the present disclosure in the form of a diagnostic microfluidic chip for a circularly polarized Raman Spectroscopy or chiral Raman Optical Activity extracellular vesicle detection chip (cROAev) for identifying sepsis phenotypes. FIG. 1A shows a diagnostic chip with an active sensing region (magnified) having a plurality of chiral hedgehog-shaped particles. FIG. IB shows circularly polarized light of a Raman spectroscopy device directed towards the active sensing region of diagnostic chip. FIG. 1C shows a schematic of the association of the biomolecules with a chiral hedgehog-shaped particle. FIG. ID reflects that Raman Optical Activity signal changes when biomolecules are present (associated with the hedgehog- shaped nanoparticles) in accordance with certain aspects of the present disclosure.

[0080] FIGS. 2A-2E show TEM tomography and circular dichroism and absorbance for hedgehog-shaped nanoparticles exhibiting multiscale chirality in accordance with certain aspects of the present disclosure. Left-handed chirality hedgehog- shaped nanoparticles with multiscale chirality (L-XND) are shown in FIGS. 2A-2B and right-handed chirality hedgehog-shaped nanoparticles having multiscale chirality (D-XND) are shown in FIGS. 2C-2D, a top view (FIGS. 2A, 2C) and side view (FIGS. 2B, 2D). FIG. 2C shows UV-Vis (dotted lines) and CD (solid lines) spectra of L-XND (red) and D-XND (blue), where cysteine concentration [Cys] is 3.6 pM when the hedgehog-shaped nanoparticles are formed. FIG. 2E shows circular dichroism (CD) and absorbance versus wavelength for the L-XND and D-XND particles.

[0081] FIGS. 3A-3I show SEM images and optical spectra of left-handed chirality hedgehog-shaped nanoparticles with multi scale chirality (L-XND) at different time points of their synthesis and particle growth. FIGS. 3A-3F show SEM images of particles synthesized at 10, 20, 30, 45, 60 and 90 minutes. FIG. 3G shows UV-Vis absorbance, FIG. 3H shows circular dichroism (CD) and FIG. 31 shows g-factor spectra for transversal plasmon peaks of L-XND synthesized during 10 to 90 minutes synthesis time with a cysteine concentration [Cys] = 3.6 pM. The nanoparticle dispersions become chemically unstable after removal of the growth solutions for time (t) of less than 10 minutes.

[0082] FIGS. 4A-4F show a microscopy study of the nanohedgehog-shaped particle growth. FIG. 4A shows typical TEM image of the dispersion at t = 5 minutes. Small spheroidal NPs formed are abundantly along with the growing seed NPs (See inserts, scale up 3 times). TEM images of the growing L-XNDs: after (FIG. 4B) 1 minute, (FIG. 4C) 3 minutes, (FIG. 4D) 5 minutes, and (FIG. 4E) 10 minutes of synthesis. FIG. 4D shows 3D image reconstruction of L- XND particle with t = 90 minutes by TEM tomography.

[0083] FIG. 5 shows STEM images and graphic representations of nanohedgehog-shaped particles for different cysteine concentration levels, more specifically from 0.125 pM < cysteine concentration [Cys] < 36 pM. Scale bar in all images is 100 nm.

[0084] FIGS. 6A-6F show STEM images and optical activities for particles after additional growth with left-handed chirality hedgehog-shaped nanoparticles having multiscale chirality (L-XND). FIGS. 6A-6B show a seed concentration of 40 pM and FIGS. 6C-6D show a seed concentration of 0.04 pM, respectively. Original XND and HH are hedgehog- shaped particles with left-handed chirality without any excess seed concentration. Optical activities or g- factor (FIG. 6E) and extinction spectra (FIG. 6F) of the particles before and after additional growth are shown.

[0085] FIG. 7 shows various gold hedgehog-shaped nanoparticles having multiscale chirality prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles. Images for cumulative, gold, sulfur, nitrogen, phosphorus, oxygen, sodium, and calcium are shown. Scale bars are 20 nm.

[0086] FIGS. 8A-8B show gold hedgehog-shaped nanoparticles at different magnification levels having multiscale chirality prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles. FIG. 8A has a scale bar of 1 micrometer and FIG. 8B is a magnified view showing a single extracellular vesicle.

[0087] FIG. 9 shows TEM images with a scale bar of 1 micrometer of gold hedgehogshaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles, including mapping of cumulative, potassium, sodium, and phosphorus, and an intensity (kCounts) versus energy (keV).

[0088] FIG. 10 shows STEM images with scale bars of 100 nm of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles, including elemental mapping of cumulative, gold, potassium, phosphorus, and sodium.

[0089] FIG. 11 shows STEM images with scale bars of 200 nm of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles, including elemental mapping of gold and phosphorus.

[0090] FIG. 12 shows STEM images with scale bars of 100 nm of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing liposomes, including elemental mapping of gold and phosphorus.

[0091] FIG. 13 shows STEM images with scale bars of 200 nm of silver-coated gold hedgehog- shaped nanoparticles prepared in accordance with certain aspects of the presentdisclosure capturing extracellular vesicles, including elemental mapping of gold and combined gold, silver, and phosphorus.

[0092] FIG. 14 shows STEM images with scale bars of 200 nm of silver-coated gold hedgehog- shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing liposomes, including elemental mapping of gold and silver combined and gold, silver, and phosphorus.

[0093] FIGS. 15A-15C show gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure. FIG. 15A shows circular comparative circular dichroism (CD) and FIG. 15B shows g-factor spectra for peaks of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure having no extracellular vesicles or having extracellular vesicles. FIG. 15C shows a table of gold hedgehogshaped nanoparticles with and without extracellular vesicles, including zeta potential, mobility, and conductivity values for comparison.

[0094] FIGS. 16A-16B show gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure having different amino acid-derived shaped directing moi eties and show the comparative circular dichroism (CD) (FIG. 16A) and g-factor spectra for peaks (FIG. 16B). More specifically, a gold hedgehog-shaped nanoparticle having 100% cysteine as an amino-acid derived chiral moiety is compared to a gold hedgehog-shaped nanoparticle having 50% cysteine to 50% methionine as an amino-acid derived chiral moiety.

[0095] FIGS. 17A-17B show STEM images of gold hedgehog- shaped nanoparticles prepared in accordance with certain aspects of the present disclosure having different amino acid- derived shaped directing moi eties. FIG. 17A has a scale bar of 100 nm and shows a gold hedgehogshaped nanoparticle having 100% cysteine as an amino-acid derived chiral moiety. FIG. 17B has a scale bar of 50 nm and shows a gold hedgehog-shaped nanoparticle with 50% cysteine to 50% methionine as an amino-acid derived chiral moiety.

[0096] FIGS. 18A-18H show pH-driven self-assembly of copper-based complex supraparticles (SPs). FIG. 18A shows a schematic representation where charged nanoclusters reversibly self-assemble into SPs of atomically thin nanosheets (NSs), depending on their electrostatic attraction-repulsion. FIG. 18B shows on the left: simplified structures of pH- dependent CuS-Pen (penicillamine) building blocks used in the SPs self-assembly. FIG. 18B shows on the right: prismatic fragment of the nanosheet network comprising CU3S3 hexagonal units. FIG. 18C shows various SEM images of SPs prepared at different 5 values varying from - 0.50 to +0.40 formed with either right-handed penicillamine (D-SPs), left-handed penicillamine (L-SPs), or racemic penicillamine. FIG. 18D shows XRD patterns of D-, L- and rac-SPssynthesized at 5 = 0. The sharpest peak indicates the nanosheet interlayer spacing of 1.28 nm. FIG. 18E shows organization of ligands within a single NSs obtained from MD. FIGS. 18F-18G show MD-simulated NSs self-assembly. FIG. 18F shows side-views on the lamellar L-SPs fragment after 500 ns simulation time at different perspectives. More specifically, the material comprises periodic layers of copper sulfide and chiral penicillamine ligands without a metal-based core region. FIG. 18G shows a top view on the D- (left) and L-NSs (right) to reveal their left- and right- handedness, respectively. FIG. 18H shows magnified SEM images of D-, rac- and L-SPs display the same handedness as those obtained from the MD simulations; rac-SPs comprise achiral needles.

[0097] FIGS. 19A-19B show various chiral hedgehog-shaped particles comprising copper iodide having multiscale chirality prepared in accordance with certain aspects of the present disclosure. FIG. 19A shows the SEM images of the D-copper iodi de-penicillamine chiral particles at different scales and FIG. 19B shows analogous copper iodide-particles comprising a chiral L- penicillamine ligand instead of D-penicillamine.

[0098] FIGS. 20A-20C show TEM images of various chiral hedgehog-shaped supraparticles (SPs) comprising copper sulfide having multiscale chirality prepared with lefthanded penicillamine (L-SPs) (Z-copper sulfide-penicillamine (L-CuS-Pen)) where the supraparticles comprising copper formed have left-handed chirality. FIG. 20A shows images captured using BF-STEM, FIG. 20B shows HAADF-STEM, and FIG. 20C shows TEM modes.

[0099] FIGS. 21A-21B show various chiral hedgehog-shaped suprap articles (SPs) comprising copper sulfide and penicillamine having multiscale chirality. FIG. 21A shows the particles prepared with right-handed penicillamine (D-SPs) where the supraparticles comprising copper sulfide formed have right-handed chirality. FIG. 2 IB shows the particles prepared with left-handed penicillamine (L-SPs) where the supraparticles comprising copper sulfide formed have left-handed chirality. All scale bars are 5 micrometers.

[0100] FIGS. 22A-22C show tunable chiroptical properties of copper sulfide-penicillamine (CuS-Pen) particles formed in accordance with certain aspects of the present disclosure. More particularly, FIGS. 22A-22C show optical properties of different chiral particles prepared at constant 5 value of 0 at varying temperatures. FIG. 22A shows the CD spectra (in a form of anisotropy g-factors) of 5 value of 0 of D- and L- copper sulfide-penicillamine particles. FIG. 22B shows the vibrational circular dichroism (VCD) spectra of 5 value of 0 of copper sulfide- penicillamine hedgehog-shaped supraparticles formed with right-handed penicillamine (D-SPs) and left-handed penicillamine (L-SPs). FIG. 22C shows circular polarized light emission (CPLE) spectra of D- and L-SPs prepared at different temperatures at a 5 value of 0.

[0101] FIG. 23 shows copper sulfide-penicillamine hedgehog-shaped supraparticles formed with right-handed penicillamine (D-SPs), left-handed penicillamine (L-SPs), or racemic penicillamine at scales indicated in each SEM image. Images have scale bars of 3 micrometers, 5 micrometers, 10 micrometers, 15 micrometers, 30 micrometers, or 50 micrometers, as designated.

[0102] FIG. 24 shows SEM images of copper sulfide-penicillamine hedgehog-shaped supraparticles (SPs) synthesized at different enantiomeric excess (X), at different temperatures and shown at different magnifications. The X value denotes the chirality of used Pen; X = +100 for pure D- enantiomer, X = -100 for pure L- enantiomer, and X = 0 for racemate (DL-Pen). Scale bars are 100 micrometers.

[0103] FIGS. 25A-25B show confocal microscopy images of copper sulfide- penicillamine hedgehog-shaped supraparticles formed with right-handed penicillamine (D-SPs) prepared in accordance with certain aspects of the present disclosure capturing biomolecules / bioassemblies in the form of exosomes. FIG. 25 A shows a 3D reconstructed, z-stack overlap with a scale bar of 20 micrometers, while FIG. 25B shows a confocal image of the D-SPs associated with free exosomes.

[0104] FIGS. 26A-26B show two different confocal microscopy images of copper sulfide- penicillamine hedgehog-shaped supraparticles, prepared in accordance with certain aspects of the present disclosure, capturing biostructures in the form of exosomes. The exosomes are visualized in a form of green-luminescent aggregates due to the labeling with green fluorescent protein (GFP).

[0105] FIG. 27 shows SEM images (at different magnification levels) of copper sulfide- penicillamine hedgehog-shaped supraparticles formed with left-handed penicillamine (L-SPs) prepared in accordance with certain aspects of the present disclosure capturing biomolecules in the form of bovine serum albumin (BSA). Scale bars are at 1 micrometer, 3 micrometers, or 10 micrometers, as designated.

[0106] FIG. 28 shows SEM images (at different magnification levels) of copper sulfide-penicillamine hedgehog-shaped supraparticles formed with left-handed penicillamine (L- SPs) prepared in accordance with certain aspects of the present disclosure capturing biomolecules in the form of specific proteins from fetal bovine serum (FBS). Scale bars are at 1 micrometer or 4 micrometers, as designated.

[0107] FIGS. 29A-29C show the major proteins selectively captured and isolated from fetal bovine serum by using copper sulfide-penicillamine hedgehog-shaped supraparticles of different chirality according to certain aspects of the present disclosure. FIG. 29A shows the composition of the isolated protein concentrate obtained by the separation using chiral D-coppersulfide-penicillamine particles prepared with right-handed D-Pen (D-SPs). FIG. 29B shows the composition of the isolated protein concentrate obtained by the separation using chiral L-copper sulfide-penicillamine particles prepared with left-handed L-Pen (L-SPs). FIG. 29C shows the composition of the isolated protein concentrate obtained by separation using achiral D / L-copper sulfide-penicillamine particles prepared with racemic D / L-Pen (rac-SPs).

[0108] FIG. 30 show SEM images of copper sulfide-penicillamine Z>-SPs prepared with different cationic and anionic surfactants rather than cetyltrimethylammonium chloride (CTAC); in pure water; cetyltrimethylammonium bromide (CTAB); poly(diallyldimethylammonium chloride) (PDADMAC) of varying molecular weights; choline chloride; (3-chloro-2- hydroxypropyl)trimethylammonium chloride (3-CHTMAC); sodium dodecyl sulfate (SDS); poly(sodium 4-styrenesulfonate) (PSS), and l-butyl-3-methylimidazolium hydrogen sulfate ([BMIM][HS04]). Scale bars are at 500 nanometers, 5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or 100 micrometers, as designated.

[0109] FIGS. 31A-31C show a device in a form of an isolation column, or more specifically a spin-column for use in a centrifuge, for detecting, analyzing, and / or separating one or more target analytes in accordance with certain variations of the present disclosure. FIG. 31A shows an extraction component for the spin-column device having an active region comprising a layer with a plurality of hedgehog-shaped particles according to certain aspects of the present disclosure. FIG. 3 IB shows the device of FIG. 31A after conducting a separation process of a fluid sample under UV light demonstrating capture of target analytes in the active region layer comprising the hedgehog-shaped particles. FIG. 31C shows an overall schematic of an isolation / spin-column incorporating the extraction component in FIGS. 31A and 3 IB along with a containment vessel in the form of a collection tube, wherein the device further includes a reversibly sealable cap for forming a fluid tight seal for spinning in a centrifuge for separation.

[0110] FIG. 32 shows a kit with various components, including a separation device in the form of a column comprising an active region having a plurality of hedgehog-shaped particles for associating with and capturing extracellular vesicles (EVs). FIG. 32 also shows processes for preparing and conducting separation of extracellular vesicles (EV) from a biological fluid sample for certain therapeutic modes according to certain variations of the present disclosure, which may be used for diagnostic and / or therapeutic applications.[oni] FIGS. 33A-33E show another variation of a device in the form of a syringe-fed isolation column for detecting, analyzing, and / or separating one or more target analytes from a fluid sample in accordance with certain variations of the present disclosure. FIG. 33 A shows threecomponents prior to and FIG. 33B shows the components after assembly to form a device that is a part of a syringe-fed isolation column, having an active region comprising a layer with a plurality of hedgehog-shaped particles according to certain aspects of the present disclosure. FIG. 33C shows a sectional side view of a sleeve component that receives an extraction / separation component and defines an interlocking region, as well as a top view of the extraction component seated in the sleeve. FIG. 33D shows a syringe secured by screwing the syringe tip into an interlocking region of the sleeve prior to introduction into the collection tube. FIG. 33E shows the syringe and sleeve secured together and disposed within the collection tube so that the fluid sample may be transferred from the syringe into the extraction component.

[0112] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.DETAILED DESCRIPTION

[0113] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0114] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and / or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recitedcompositions, materials, components, elements, features, integers, operations, and / or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and / or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0115] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0116] When a component, element, or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0117] Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and / or sections, these steps, elements, components, regions, layers and / or sections should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer, or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer, or section discussed below could be termed a second step, element, component, region, layer, or section without departing from the teachings of the example embodiments.

[0118] Spatially or temporally relative terms, such as “before,” “after,” “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially or temporally relative terms may be intended to encompassdifferent orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

[0119] Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.

[0120] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges.

[0121] As used herein, the terms “composition” and “material” are used interchangeably to refer broadly to a substance containing at least the preferred chemical constituents, elements, or compounds, but which may also comprise additional elements, compounds, or substances, including trace amounts of impurities, unless otherwise indicated.

[0122] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0123] The present disclosure relates to the use of hedgehog-shaped particles that exhibit a multiscale chirality. The hedgehog- shaped particles may be an active material that can be disposed in a device, such as a sensor, detector, or analyzer. In certain variations, the hedgehogshaped particles may be included in a microfluidic device. Hedgehog-shaped particles may be microparticles or nanoparticles that generally emulate the shape of a hedgehog animal in that the particles have projections or spikes protruding from a central core region. Chiral “hedgehog” particles are unique particles that possess a spiky, dendritic, pollen-like architecture and are chiral in form, for example, left- or right-handed. As will be discussed further herein, a particle that exhibits a multiscale chirality may exhibit different chiralities at different scales, for example, thehandedness can be different at different scales, for example, a single hedgehog shaped particle may be right-handed at the sub-nanometer or atomic level, but left-handed at the nanoscale. Accordingly, hedgehog-shaped particles are multi-component particles with binary size regimes, for example, having a micron-scale core on which projections (e.g., needles, spikes, or plates) are connected to the core surface. In certain variations, the projections may be disposed on and thus cover substantially all of the core surface, while in other variations, the projections may only be on select regions of the core surface (for example, only over the exposed surfaces but not on the bottom region when the hedgehog-shaped particle is disposed on a substrate or surface. While the projections may be relatively evenly distributed over the core surface, the projections may have a branching pattern that is stochastic or random.

[0124] A “microparticle” as used herein encompasses “nanoparticles” and “mesoparticles,” as discussed below. In certain variations of the present teachings, a microparticle component has at least one spatial dimension that is less than about 1,000 micrometers (z.e., 1 mm). The term “micro-sized” or “micrometer-sized” as used herein is generally understood by those of skill in the art to mean less than about 100 micrometers. As used herein, a microparticle has at least one spatial dimension that is less than or equal to about 50 micrometers, optionally less or equal to about 40 micrometers, optionally less or equal to about 30 micrometers, optionally less or equal to about 25 micrometers, optionally less or equal to about 20 micrometers, optionally less or equal to about 10 micrometers, optionally less or equal to about 5 micrometers, optionally less or equal to about 1 micrometers.

[0125] The hedgehog particles can also be “nano-sized” or “nanometer-sized” and have at least one spatial dimension that is less than or equal to about 1 micrometer, optionally less than or equal to about 0.5 micrometers (i.e., 500 nm), optionally less than or equal to about 0.4 micrometers (i.e., 400 nm), optionally less than or equal to about 0.3 micrometers (i.e., 300 nm), and in certain variations, optionally less than or equal to about 0.2 micrometers (i.e., 200 nm). It should be noted that so long as at least one dimension of the nanoparticle falls within the abovedescribed nano-sized scale (for example, diameter), one or more other axes may well exceed the nano-size (for example, length and / or width). Notably, nanoscale particles may have a size that is also encompassed by a mesoscale size.

[0126] The hedgehog- shaped particles may be “mesoscale,” which generally refers to particles having a diameter of greater than or equal to about 100 nm to less than or equal to about 1 micrometer. In certain variations, the hedgehog-shaped particle may have a size or diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers, optionally greater than or equal to about 200 nm to less than or equal to about 20 micrometers. Where aplurality of hedgehog particles is present, the plurality of particles may have an average particle size (e.g., average diameter) of greater than or equal to about 200 nm to less than or equal to about 50 micrometers.

[0127] Hedgehog-shaped particles have a core region formed of a first material and a plurality of projections connected to a surface of the core region formed of a second material. The first material and the second material may be the same material composition or may be distinct material compositions. In certain variations of the present disclosure, at least one of the first and second materials forming the core region and the projections comprises a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof. In one variation, at least one of the core regions and the plurality of projections comprises gold. In another variation, both the core region and the plurality of projections comprise gold. In one variation, at least one of the core regions and the plurality of projections further comprises silver, for example, as a coating or layer, disposed over the gold. In other variations, the first or second materials may be a compound comprising a metal and another element, such as sulfur or iodine. By way of nonlimiting example, the first or second materials may be copper sulfide, copper iodide, and the like.

[0128] In certain variations, the copper-based material may comprise a copper sulfide, such as one with an approximate formula of CuC5H10NO2S, which can also be called a copper thiolate salt / copper thiolate coordination polymer. In other variations, the copper-based material may contain copper as well as both iodine and sulfur, e.g., a composition with an approximate formula of CuC5H10INO2S, which may be referred to below as a copper-iodide composition although it also has sulfur. In certain other variations that will be described further herein, the core region may comprise the first material and a second material. By way of example, the core region may be formed of a lamellar structure having periodic or alternating layers of copper sulfide and chiral penicillamine ligands, but without a metallic material. In other variations, the core region and the projections are metallic and do not intentionally comprise other non-metallic elements, aside from impurities, for example, the materials are not chalcogenides comprising non-metallic elements, like sulfur, iodine, selenium, tellurium, and the like.

[0129] The plurality of projections connected to the core region may be protrusions that form the spiky hedgehog-shaped particle having multiscale chirality. In certain aspects, the plurality of projections may have a rod-like needle shape or alternatively, a sheet-like flat shape. Where the projections are rod-like spikes or needles, they may have an axial geometry and are anisotropic with an evident elongated longitudinal axis, which is longer than the other dimensions (e.g., diameter or width). Generally, an aspect ratio (AR) for cylindrical or rod-like shapes (e.g., needle, spike, pillar, etc.) is defined as AR = L / D, where L is the length of the longest axis (herethe maj or longitudinal axis), and D is the diameter of the needle or pillar. Suitable spikes or needles for use in the present technology generally have high aspect ratios, for example, ranging from at least about 100 to in excess of 1,000, for example. In yet other aspects, such needles may have an aspect ratio of 5,000.

[0130] Alternatively, the proj ections may have a planar or platelet-like geometry with two axes that are of similar dimensions (e.g., height and width) and thus, may be plate-like or sheetlike. A planar or platelet-like shape is typically flattened, for example, a plate that may have polygonal (e.g., trapezoidal, or rectangular), an oval (e.g., disc), or irregular shapes. As used herein, sheet, plate, platelet, and flake are used interchangeably. For example, the aspect ratio (AR) of a planar shape (e.g., sheet, plate) may be defined as AR = W / H, where W is the width of the longest axis (here the major lateral axis or dimension along the structure) and H is the height or shortest axis of the structure. Suitable planar structures, like sheets, incorporated as projections on the core region in accordance with certain aspects of the present technology generally have low aspect ratios (AR), for example, ranging from less than or equal to about 20, optionally less than or equal to about 10, optionally less than or equal to about 5, and in certain aspects, optionally less than or equal to about 1. As will be appreciated by those of skill in the art, the sheet-like structures may be attached to the core region via the shortest dimension (e.g., height) so that the sheet extends as a protrusion along the longest dimension (e.g., width).

[0131] The projections are connected to a surface of the core region. As noted above, the entire surface of the core region may have the projections. In certain variations, the projections may be disposed on and thus cover substantially all of the core surface, while in other variations, the projections may only be on select regions of the core surface. While the projections may be relatively evenly distributed over the core surface, the projections may have a branching pattern that is stochastic or random.

[0132] In certain aspects, the projections may be rod-like spikes or needles may be substantially orthogonal to a surface of the core region. By “substantially orthogonal,” it is meant that a longitudinal axis of the elongated structure (e.g., needle or spike) may form an angle with respect to the core surface (where the needle is connected) having an angle of about 90° or another angle that slightly deviates from 90°, for example, deviates up to about 15° (e.g., from about 75° to about 105°).

[0133] In certain variations, the plurality of projections may have an average maximum dimension (e.g., length of a rod-like needle or spike) of greater than or equal to about 75 nm to less than or equal to about 300 nm. Where the projections are rod-like spikes or needles, each mayhave a diameter or an average diameter of greater than or equal to about 1 nm to less than or equal to about 100 nm.

[0134] In certain variations, the core region may have an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers, optionally greater than or equal to about 100 nm to less than or equal to about 20 micrometers.

[0135] Overall, a plurality of hedgehog particles may have an average particle size (including core region and projections) greater than or equal to about 100 nm to less than or equal to about 50 micrometers, optionally greater than or equal to 200 nm to less than or equal to about 50 micrometers. For example, in the case of copper-based hedgehog-shaped particles, an average particle size may optionally be greater than or equal to about 100 nm to less than or equal to about 200 nm, optionally about 500 nm, optionally about 2 micrometers, optionally about 5 micrometers, optionally about 10 micrometers, optionally about 15 micrometers, optionally about 20 micrometers, and in certain variations, optionally be greater than or equal to about 25 micrometers to less than or equal to about 45 micrometers.

[0136] The projections are connected to a surface of the core region. As noted above, the entire surface of the core region may have the projections or only select regions of the core surface. In various aspects, the needles may be substantially orthogonal to a surface of the core region. By “substantially orthogonal,” it is meant that a longitudinal axis of the needle may form an angle with respect to the core surface (where the needle is connected) having an angle of about 90° or another angle that slightly deviates from 90°, for example, deviates up to about 15° (e.g., from about 75° to about 105°).

[0137] In various aspects, each hedgehog-shaped particle comprises a shape-directing moiety that helps to impart chirality and more particularly, helps to ensure that that the hedgehogshaped particle displays multiscale chirality that will be described further herein. The shapedirecting moiety may strongly bind to metal(s) in the particle. Such a shape-directing moiety may be a shape directing moiety, which may be any chiral organic moiety or molecule which binds to the inorganic component (metal(s), metal sulfide, metal iodide) of the particle. A shape directing moiety may include an amino acid-derived chiral moiety. In certain variations, broader shapedirecting moieties may include amino-acid derived moieties, but also other examples, such as Z-cysteine, Z>-cysteine, L- penicillamine, Z>-penicillamine, dopamine, Z-carnosine, Z-glutathione, chiral carboxylic acids, chiral phenols, chiral catechols, phosphines, amines, amyloid peptide monomers, amino-acid derivatives, and combinations thereof. In certain variations, a nanoparticle may include an organic moiety (R), which may be a fragment of an amino acid, like a sulfur-containing amino acid, such as those amino acids containing thiol groups, like cysteine,homocysteine, penicillamine, methionine, and / or combinations thereof or of different amino acids. Thus, a gold hedgehog particle may be represented by AuR, where R is an organic fragment, such as an alkyl group, aryl group, and the like and may be an amino acid-derived chiral moiety that comprises a thiol group. In certain variations, the hedgehog-shaped particle thus comprises an amino-acid derived chiral moiety (with D and / or L enantiomers) that may be selected from the group consisting of cysteine, homocysteine, penicillamine, arginine, lysine, methionine, and combinations thereof. In one variation, the amino acid-derived moiety may comprise cysteine.

[0138] In certain aspects, the hedgehog-shaped nanoparticles are encapsulated and stabilized by a surfactant, such as for example, cetyltrimethylammonium chloride (CTAC) or cetyltrimethylammonium bromide (CTAB), quaternary ammonium surfactants, thus avoiding aggregation during the metal reduction. Other surfactants may also be used, as described further below.

[0139] The hedgehog-shaped particles of the present disclosure are chiral, meaning they provide either left- or right-handed chirality. Chirality can be either L (left-handed) or D (right- handed), or both (racemic). However, the hedgehog-shaped particles exhibit a multiscale chirality. As noted above, a particle that exhibits a multiscale chirality may exhibit different chiralities at different scales and thus, the handedness potentially can be different at different scales (e.g., a single hedgehog shaped particle may be right-handed at the sub-nanometer or molecular level, but left-handed at the nanoscale, and the like). This multiscale chirality enables the hedgehog- shaped particles to specifically capture biomolecules, and their unique interactions make it easier to detect subtle changes in biological assemblies. For example, the projections define a spiky form that creates a scaffold to capture target biomolecules or bioassemblies, such as proteins or exosomes. The multiscale chirality is also important for analyzing biological assemblies, such as exosomes or other extracellular vesicles. Biomolecules, such as the amino acid derived chiral moiety or other organic chiral moieties that serve as a shape directing moiety on the surface of the hedgehog-shaped particles, provide nanoscale chirality, while the projections e.g., spikes) provide nano or microscale chirality. The hedgehog-shaped particle has at least two chiralities on different scales and may have three or more distinct chiralities on three or more scales. As outlined below, the hedgehog particle exhibits distinct chirality for at least two of the scales specified, optionally for more than two of the scales specified.

[0140] By way of non-limiting example, in one variation, a first chirality of the hedgehogshaped particle may be on an atomic scale, for example, occurring at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.10 nm), optionally greater than or equal to about 3 Angstroms (0.3 nm) to less than or equal to about 5 Angstrom 0.5 nm.The smallest chiral object comprises four or more atoms that do not belong to one plane corresponding to the atomic scale. 3 A (Angstroms) chirality is characteristic of the a-carbon atom in organic / biomolecules (e.g., amino acid-derived moiety), such as cysteine when attached to the surface of the core region, while transition metal elements are slightly greater dimension. The hedgehog-shaped particle may also have a distinct second chirality on the nanoscale, for example, at greater than or equal to about 1 nm to less than or equal to about 100 nm, and optionally about 50 nm in certain embodiments. The nanometer level of chirality is believed to be provided by the proj ecti ons / spikes.

[0141] In certain further variations, the multiscale chirality further comprises a third chirality, which may be on a mesoscale. By way of example, the mesoscale chirality may be greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer). For example, the hedgehog-shaped particle itself may provide the third mesoscale, microscale, submillimeter, and / or millimeter scale chirality. The overall geometry of the hedgehog particles can also be chiral and consistently be either left — or right-handed shapes.

[0142] The hedgehog-shaped particle may also have a distinct fourth chirality on the microscale, for example, at greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers).

[0143] The hedgehog-shaped particle may also have a distinct fifth chirality on the submillimeter scale, for example, at greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm).

[0144] Additionally, the hedgehog-shaped particle may also have a distinct sixth chirality on the millimeter scale, for example, at greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters).

[0145] Hedgehog particles may be synthesized by growing a noble metal (e.g., gold, silver) structure radially on an etched seed nanoparticle, or by self-assembly or solvochemical synthesis of metallic, inorganic, or hybrid organic-inorganic material (e.g., gold, copper iodide, copper sulfide-penicillamine) under optimal conditions. The process for making the hedgehog- shaped particles includes using a chiral biomolecule (e.g., cysteine, penicillamine, arginine, lysine, methionine) resulting in a micro or nanoparticle with chiral characteristics. Chirality can be different at different scales within a single particle and can be either L or D, or both (racemic). Hedgehog-shaped particles can be synthesized from different materials, including any inorganic material / metal, and the chiral molecule which induces the formation of spiky chiral particle. In certain aspects, the hedgehog-shaped particles can be made from materials including gold, copper, and silver, to form gold, copper sulfide, copper iodide, copper sulfide iodide, andsilver decorated / coated gold hedgehog-shaped particles. The geometry, size, and chirality can vary based on the biomolecule (chiral shape inducing moiety) and its concentration used in the synthesis reaction.

[0146] As such, free-standing hedgehog- shaped particles may be formed with spiky, dendritic, pollen-like architectures. These structures can display deterministic bifurcation sequences with high regularity and stochastic branching patterns. The unique combination of the spiky form and chirality makes hedgehog-shaped particles particularly advantageous for capturing, analysis, and separation of biomolecules. The spiky form creates a scaffold to capture biomolecules such as proteins, extracellular vesicles, including exosomes, and the like. By way of non-limiting example, chiral hedgehog-shaped particles prepared in accordance with the present disclosure can capture proteins and extracellular vesicles (exosomes). The multiscale chirality is also advantageous for analyzing biological assemblies. The interaction between chiral hedgehog-shaped particles and chiral biomolecules is sensitive and significant for differentiating small changes in biological assemblies. The chirality can be tuned to optimize the efficiency and specificity of capture and analysis.

[0147] Moreover, the hedgehog-shaped particles exhibiting multiscale chirality according to the present disclosure are capable of interacting with one or more target analytes, such as biomolecules, without necessitating the presence of a targeting moiety or ligand being present. Previous chiral gold nanoparticles required the use of a targeting ligand or moiety to interact with biomolecules, such as cancer exosomes, and further lacked multiscale chirality. As such, one or more target analytes, such as target biomolecules, are capable of associating with the plurality of hedgehog-shaped particles, for example, in an active or sensing region of a device, in the absence of any added targeting moiety. By associating with the target analytes, the hedgehog-shaped particles can indicate the presence of one or more target analytes, analyze one or more target analytes, or separate one or more target analytes in a fluid sample. The hedgehog-shaped particles provided in accordance with various aspects of the present disclosure are unique because they possess and exhibit multiscale chirality.

[0148] Thus, in certain variations, dendrimer-shaped hedgehog-shaped gold particles are contemplated with distinct stochastic branching and intense chiroptical activity. Unlike typical molecular or nanostructured dendrites, gold nanodendrimers are two-dimensional, with branches radially spreading within one plane. They are also chiral, with mirror asymmetry propagating through multiple scales, as described above. This complex architecture has been quantitatively described by image informed graph theory (GT) models accounting for both regular and disordered structural components of the nanodendrimers.

[0149] By way of further background, most cells secrete extracellular vesicles (EVs) of nanoscale dimensions that carry informative cargo-containing proteins, lipids, and nucleic acids. It appears EVs play essential roles in cell-to-cell communication. Furthermore, the cargo and membrane proteins of small EVs, such as cancer-derived exosomes, reflect biological activity and status of malignant cells they are secreted by, playing an important role in cancer progression and metastatic destination. Recently, it has been shown that exosomes may carry mutated proteins reflective of their cellular origin, prompting ongoing studies of these exosomes as prominent biomarkers for cancer diagnosis. Thus, cancer-cell secreted nanoscale small extracellular vesicles (sEVs); known as exosomes, represent a rapidly emerging family of biomarkers for cancer detection. While being high in information content, the current protocols for profiling sEVs require complex procedures and equipment involving exosome purification, which prevents their utilization in timely diagnosis of malignancies.

[0150] Another area where detection and analysis of extracellular vesicles / components of extracellular vesicles is important is in detecting sepsis. Sepsis is characterized by a dysregulated immune response to an infection and its clinical syndrome is highly complex and heterogeneous, involving nearly every organ system. It is a life-threatening condition that has become the leading cause of death worldwide, surpassing heart disease and cancer, with nearly 1 in 3 hospital admissions ending in death. In addition, there is profound variability in physiologic and laboratory variables among patients and pathogens, and many variables have the potential for rapid change over time for a given patient. EVs derived from activated or apoptotic cells during sepsis carry altered cargoes and play a role in regulating signal transduction and altering the phenotype of neighboring cells in an autocrine or paracrine manner. The number of EVs significantly increases in sepsis and positively correlated with its severity.

[0151] Studies have shown that the protein profiles carried by EVs in the body fluids of sepsis patients are altered and may be associated with disease progression. EVs originating from various cell types, including activated macrophages, monocytes, and neutrophils, have been found to have altered protein profiles. Moreover, EVs carry different types of nucleic acids, such as mRNA, miRNA, IncRNA, and circRNA, which change during sepsis progression. The development and application of omics technology has provided insights into the dynamic expression profiles of EV cargo in sepsis. Altered expressions are associated with disease risk, severity, and prognosis. All these factors play a role in immune regulation, microvascular dysfunction, and organ dysfunction. Stratifying patients with sepsis based on biochemical and immunological profiles is required for personalizing treatment. While omic technologies are also gaining traction in clinical settings for diagnostics, prognostics, and treatment monitoring,challenges remain in data interpretation, standardization, regulatory approval, and reimbursement, hindering widespread clinical adoption. A novel diagnostic approach that can be complementary to other tools and decrease the time and cost significantly for interpreting data from sepsis related EVs would be advantageous.

[0152] In various aspects, the present disclosure provides a device for detecting, analyzing, and / or separating one or more target analytes that may be biological in nature, and / or a method of separation of one or more of such target analytes such as a target analyte. The device may be used in proteomics, diagnostics, and / or therapeutics, by way of non-limiting example. The device may be capable of processing a fluid sample, such as a biological fluid sample obtained from a subject, by use of any device that will be described herein. In certain aspects, the present disclosure relates to a method of separating one or more target analytes in a biological fluid sample obtained from a subject in such a device. The method comprises passing a biological fluid sample through a device comprising an active region having a plurality of hedgehog-shaped particles disposed thereon. Each respective hedgehog shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, as in any of the variations described previously above. At least one of the core regions and the plurality of projections comprise a material selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and combinations thereof. Each respective hedgehog-shaped particle also comprises a shape directing organic chiral moiety, such as an amino acid derived chiral moiety. The plurality of hedgehog- shaped particles is capable of associating with a target analyte optionally present in the biological fluid sample. The method further comprises separating the target analyte from the biological fluid sample by capturing the target analyte in a respective hedgehog-shaped particle in the active region.

[0153] In one aspect, the device comprises a microfluidic channel comprising the active region and the passing the biological fluid sample is through the microfluidic channel.

[0154] As noted above, in certain aspects, the device may be microfluidic device that comprises at least one microfluidic channel, for example, having at least one dimension on the microscale as described above in the context of the hedgehog- shaped particles. In certain aspects, a microfluidic channel may be a groove or enclosed capillary optionally having a volume of less than or equal to about 30 microliters (pL). In certain variations, each microfluidic channel may have a volume of greater than or equal to about 4 pL to less than or equal to about 30 pL. The microfluidic channel may have an inner diameter of less than or equal to about 1 mm, for example, having an inner diameter of greater than or equal to about 5 micrometers to less than or equal to about 1,000 micrometers (1 mm).

[0155] In another aspect, the device comprises a fluid receptacle comprising the active region and the passing the biological fluid sample is through the fluid receptacle. The active region may be a layer formed of the plurality of hedgehog-shaped particles through which the biological fluid sample may pass. Notably, while the fluid receptacle may have a volume in the microfluidic range, it is not limited to such small volumes and may be process much larger samples. In this manner, the device may be a column, such as a spin-column or tube, as will be described further below. In certain variations, the spin-column may be filled with chiral gold hedgehog nanoparticles that will interact with proteins on EVs, capturing and holding them in place, thus separating them from the fluid sample. This approach can thus streamline EV capture by reducing processing time, minimizing sample volume, and enhancing the cost-effectiveness of EV-based diagnostics and biomarker identification.

[0156] After separating the one or more target analytes in the active region, the separated analytes may be later collected, for example, by passing an elution medium or liquid or elution buffer through the device. For example, the elution medium may be introduced into the fluid receptacle, so that it then passes through the layer of the hedgehog- shaped particles where they are released and eluted from the layer. After the elution, they may be collected and further analyzed or purified. In certain further aspects, the plurality of hedgehog-shaped particles may be washed one or more times by being exposed to a washing medium or liquid or washing buffer.

[0157] Active materials according to certain aspects of the present disclosure may include a plurality of hedgehog-shaped particles that exhibit multiscale chirality. Such active materials may be incorporated into a device that comprises an active region or sensing region having a plurality of hedgehog-shaped particles that exhibit a multiscale chirality. The active or sensing region may include a substrate formed of an inorganic material or a polymeric material and is desirably transmissive to electromagnetic radiation, such as circularly polarized light, in a target range of wavelengths (e.g., red light or NIR). Thus, the substrate on which the microfluidic channel may be formed may comprise a material that is transparent to certain predetermined wavelengths of light, such as a silicon dioxide material (e.g., fused silica or glass or borosilicate), quartz and polymers (e.g., polycarbonate, or acrylates). The substrate may be coated, for example, with polydimethylsiloxane (PDMS). Further, the substrate may be treated, for example cleaned and / or etched (with chemicals, plasma, electron beam, or high intensity lasers, for example). In certain aspects, the substrate may be a microchip.

[0158] The active or sensing region comprising the hedgehog-shaped particles may be formed within a portion of fluid receptacle, which may be at least one microfluidic channel or an open volume that is configured to receive a biological fluid sample for analysis that may containa target analyte(s) that may be a bioactive / biomolecule target analyte. The hedgehog-shaped particles are those as described above. As will be described further herein, one or more target analytes of biological origin in the fluid sample associate with the plurality of hedgehog-shaped particles in the active or sensing region. This association is achieved in the absence of any added targeting moiety to indicate the presence of the one or more target analytes therein. For example, the one or more analytes may be biomolecules or biological assemblies that may indicate a presence of cancerous cells, mutated proteins, products of sepsis, cardiovascular disease, neurological disease, autoimmune disease, and the like in the biological fluid sample.

[0159] The fluid sample may be any sample that is a liquid that potentially contains one or more target analytes of a biological origin, such as biomolecules or bioassemblies. In certain aspects, the fluid sample is a biological fluid sample from a subject or organism. In certain variations, the biomolecule target analyte originates from a cell and is present in a biological fluid sample, like blood or plasma, obtained from a subject to be treated, from another subject, or from another species. Further, the biomolecule target analyte may originate from a eukaryotic cell, e.g., from an animal, such as a mammal. By way of non-limiting example, the mammal may be a human, domesticated companion animal, such as a cat or dog, or livestock, such as a cow, horse, sheep, goat, and the like. The biological fluid sample may include bodily fluids, such as blood, serum, plasma, saliva, cerebrospinal fluid, urine, and the like. In certain aspects, the biological fluid sample comprises blood or plasma.

[0160] The one or more target analytes may be biological in nature, including biomolecules, assemblies of biomolecules, or may be a bioactive material such as a cellular component (having a size smaller than a cell), protein, or other biological materials derived therefrom, including by way on non-limiting example, nucleic acids, such as DNA, RNA (e.g., mRNA, miRNA, IncRNA, and circRNA, tRNA, rRNA, snRNA, siRNA), carbohydrates, lipids, proteins, polypeptides, amino acids, liposomes, hormones, prostaglandins, and the like. In general, extracellular vesicles (EVs) or exosomes are biological assemblies comprising multiple biomolecules and other substances. In certain aspects, a target analyte may be an extracellular vesicle (or a biomolecule or moiety associated with the extracellular vesicle), for example, selected from the group consisting of an exosome, a micro vesicle, an apoptotic body, and combinations thereof. As discussed above, exosomes are extracellular vesicles secreted by cells that can mirror cellular information from cells of origin. Extracellular vesicles (EVs), particularly exosomes, are increasingly recognized for their potential in diagnostics, therapeutics, and research as non-invasive biomarkers. However, current EV capturing technologies are slow, costly, andrequire large sample volumes, which limits their efficiency. Additionally, the identification of EV biomarkers demands high purity, making the process time-consuming and expensive.

[0161] In certain variations, the biomolecule target analyte may comprise phosphatidylserine (PS), which is an anionic phospholipid maintained on the inner-leaflet of a cell membrane and may be externalized in malignant cells and their exosomes and / or formed during apoptosis. In certain other aspects, a biomolecule of interest may comprise a protein or polypeptide, such as a mutated protein. Alternatively, or additionally, the biological target analyte may comprise another type of cell-derived material, such as tumor / extracellular vesicle specific proteins. The target analyte may be liposomes or proteins, such as tetraspanin proteins (e.g., CD81, CD9, CD63, CD133, and CD56) or any other cancer-associated proteins such as epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and the like. In certain variations, the target analyte may include bioactive molecules, and / or the conjugates or the assemblies of such biomolecules, can have other biological activity or relevance beyond cancer-associated proteins or markers. This can include lipoproteins (e.g., Apolipoprotein A, including ApoA-I, ApoA-II, ApoB, and ApoE) or any other proteins, biomolecules, and the specific assemblies of thereof, including, but not limited to, those associated with neurodegenerative or cardiovascular diseases. The mutations of apolipoproteins are responsible for disrupted lipid transport and relate to the number of deaths associated with cardiovascular and neurodegenerative diseases). Therefore, a facile separation of these proteins is particularly important for further biomedical research and diagnosis.

[0162] In some examples, such an analyte, biostructure, or molecule can have a relevant biological activity, such that the ability to detect, analyze, purify, or separate is related to its biological importance.

[0163] As noted above, mutated proteins or cell secreted extracellular vesicles, like nanoscale small extracellular vesicles (sEVs), known as exosomes, are biomarkers for detection of conditions like cancer, sepsis, and autoimmune disease. As will be described further herein, in certain variations, hedgehog-shaped particles are disposed in an active or sensing region, such as on a surface of a microfluidic channel of a microfluidic device or within a layer in an active region through which a fluid sample may pass. The fluid sample is thus processed by contacting the hedgehog-shaped particles which rapidly isolate and detect one or more biomolecules, such as cancer associated exosomes directly from blood plasma, by way of non-limiting example. Exosomes from lung cancer patients can be distinguished from those from healthy donors by chiroptical spectroscopic, or polarization rotation, signatures of biomolecular components of exosomes enhanced by hedgehog- shaped particles that exhibit a multiscale chirality.

[0164] In certain aspects, methods for detecting the presence of one or more target analytes from a biological fluid sample obtained from a subject are contemplated. The method generally includes providing an active region (e.g., sensing region), which may be part of a fluid channel, which in certain variations, may be a microfluidic channel in a microfluidic device, including a plurality of hedgehog-shaped particles that exhibit a multiscale chirality. The active region is configured to receive a biological fluid sample. In certain aspects, the method further comprises passing a biological fluid sample through a microfluidic channel that includes the active region. A presence of one or more target analytes in the fluid sample may be detected when one or more target analytes associate with the plurality of hedgehog-shaped particles in the active region in the absence of any added targeting moiety. By associating with hedgehog-shaped particles, the presence of one or more target analytes is indicated. In certain aspects, if one or more target analytes are captured, they may be removed from the hedgehog-shaped particles in the active region by passing a treatment fluid where the target analyte(s) are extracted and thus may be further processed / analyzed. As noted above, the treatment fluid may be an elution medium / liquid or buffer, such as a buffer solution comprising PBS / EDTA. By way of non-limiting example, captured analytes / biomolecules, such as proteins, can be rapidly released from hedgehog-shaped particles by dissolving the material in buffer solutions, for example, comprising PBS / EDTA optionally at a pH around 7.4. In certain aspects, if one or more target analytes are present, further analysis may be conducted, including profiling the biomolecule(s) or biological assemblies, such as cancer-associated exosomes or sepsis-associated extracellular vesicles, from the biological fluid sample.

[0165] In certain other variations, the device for detecting, analyzing, and / or separating one or more target analytes may be an isolation column that includes a fluid receptacle configured to hold a fluid sample, such as a centrifuge spin-column or other isolation column, defining an open volume for receiving the fluid sample. See for example, FIGS. 31 A-31C, where a centrifuge spin-column device 30 includes a fluid receptacle 32, as well as an active region 34 that comprises the hedgehog-shaped particles according to any of the embodiments described previously above. The active region 34 may be defined by a layer within the fluid receptacle 32. After introducing the fluid sample 36 into the open region of the fluid receptacle 32, at least a portion of the fluid sample 36 can pass through the active region 34 (e.g., layer) of the plurality of hedgehog-particles so that they may interact with any of the target analytes present therein. In this manner, one or more target analytes associate with the plurality of hedgehog-shaped particles in the active region 34 (e.g., layer) and do so in the absence of any added targeting moiety.

[0166] The device 30 may be a cylindrically shaped component or a column for passing a fluid sample therethrough. In certain variations, like those shown in FIGS. 31A-31C, the device 30 may be a spin-column that may be used in a centrifuge. However, the device 30 may also be a column that receives the fluid sample and processes it without any spinning, but rather by gravity feed and / or by injection of the fluid sample, for example, under pressure via a syringe feed where gravity assists with separation as the fluid passes through the device. By way of non-limiting example, in the variation shown in FIGS. 31A-31C, the spin-column device 30 may include two components. It should be noted that a design and shape of the column may vary from that shown in FIGS. 31A-31C, which are provided as one example.

[0167] The first component 40 is an extraction component for solid-liquid extraction that includes the active region / layer 34 comprising the plurality of hedgehog-shaped particles. The spin-column device 30 also includes a second component 42 in the form of a removable collection tube in which the first component 40 (extraction component) seats in a fluid-tight manner to retain fluids during spinning while being processed in a centrifuge. The spin-column device 30 may also have a cap 44 that can be reversibly closed over an open terminal end of the second component 42 / collection tube and thus over the first component 40 / extraction component to provide a fluid-tight seal prior to spinning in a centrifuge. The components of the spin-column device 30 may be formed of polymeric materials or glass materials. For example, the components may be formed of polypropylene, which is advantageously autoclavable. In certain aspects, one of the components may be formed by additive manufacturing / three-dimensional printing.

[0168] The upper extraction component 40 includes the fluid receptacle 32, which defines an open region configured to receive the fluid sample 36 when it is introduced to the device 30. The open region 32 has an open end and defines a first diameter that is less than a second diameter of the tube 42 in which the extraction component 40 seats. The extraction component 40 may have distinct diameters, for example, graduated and reduced diameters from an upper region to a lower region. Thus, the extraction component 40 may have a third diameter that is less than the first diameter of the open region. The extraction component 40 may also optionally have a fourth diameter less than the third diameter at a terminal end of the extraction component 40. The terminal end of the extraction component includes an opening that defines an outlet 46 for the fluid to exit the extraction component 40 after passing through the extraction component 40. As described above, the open region of the extraction component 40 comprises a layer of the hedgehog-shaped particles as the active region 34. The open region 32 of the extraction component 40 also comprises a filter 38 that defines a first side adjacent to and / or on which the hedgehog-shaped particle layer / active region 34 is disposed. The filter 38 may also define a secondside opposite to the first side and adjacent to the outlet 46. The filter 38 may be any filtration media suitable for use in such biological fluid processing applications, such as a paper filter or the like. The outlet 46 of the extraction component 40 may be in fluid communication with a fluid containment region 48 of the second component / collection tube 42 for collecting the processed fluid after it passes through the outlet 46.

[0169] As noted above, the fluid containment region 48 may be a separate removable component, such as a tube 42, which may be replaced during the separation process, for example, the filtrate (e.g., lysate) that has passed through the active region / layer 34 of hedgehog-shaped particles and the filter 38 can be contained in the collection tube 42 and later removed. The one or more analytes retained in the active region / layer 34 of the hedgehog-particle after the separating from the fluid sample 36 may be analyzed in situ or further processed. In certain variations, the collection tube 42 may be a first tube and one or more additional tubes may be used. After removing the initial fluid containment / collection tube 42 having the filtrate in the fluid containment region 48, the extraction component 40 may be seated in the additional tube, where a product comprising the separate target analyte(s) may be eluted by passing a removal / elution medium / buffer into the open region 32, through the active region / layer 34, through the filter 38, and through the outlet 46 for collection of the eluent as product in the fluid containment region 48 of the additional tube 42, where the eluent / product can be further processed and analyzed. As noted above, before or after the elution / collection step, one or more washing steps with a washing medium / buffer may occur, which may include collecting the spent washing medium in the collection tube that may likewise be removed from the extraction component and / or discarded.

[0170] Thus, in certain variations, the present disclosure contemplates a kit that includes a separation device comprising a column with an active region comprising the plurality of hedgehog-shaped particles. The kit may include a washing medium, such as a washing buffer, to eliminate unwanted serum proteins, and an elution medium or buffer for EV isolation while maintaining integrity of the EVs. The kit may include a filter (as part of the separation or extraction device or separate), along with one or more containers comprising a washing medium, one or more containers comprising an elution medium (as described previously above comprising PBSZEDTA, by way of example), and at least one additional tube. By way of example, washing buffers may include one or more of the following components: phosphate buffered saline (PBS), tris buffer, tris-HCl, ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl), monosodium phosphate (NaEEPC ) and small amounts of detergent buffer(s). These may be provided in an aqueous medium. The washing medium / buffer serves to remove non-specifically bound biomolecules. In the kit, I would recommend including one pre-made washing buffer tosimplify use, particularly in clinical settings. The treatment or elution medium to release the target analytes from the chiral hedgehog-particles may comprise a buffer solution including an aqueous carrier, PBS, and EDTA. The kit may include additional components typically contained in kits for biological sample analysis and / or for use in centrifuges, as recognized by those of skill in the art.

[0171] In certain aspects, the fluid receptacle (e.g., open region of the extraction component) has a volumetric capacity of greater than or equal to about 100 microliters. As will be appreciated, the fluid sample passes through the fluid receptacle having a predetermined volumetric capacity and thus a greater amount of fluid sample may be introduced overtime. While the separator device may be on a microscale, in certain variations, it is advantageously not on a microscale and thus can process large volumes of fluid samples rapidly (as microfluidic devices have slower processing times). Thus, in certain variations, the capacity or volume of the fluid receptacle / open region that receives the fluid sample may have a volume of greater than or equal to about 1 milliliter, optionally greater than or equal to about 2 milliliters, optionally greater than or equal to about 3 milliliters, optionally greater than or equal to about 4 milliliters, optionally greater than or equal to about 5 milliliters, optionally greater than or equal to about 10 milliliters, and in certain variations, optionally greater than or equal to about 15 milliliters. In certain aspects, the volumetric capacity for receiving the fluid sample may be greater than or equal to about 100 milliliters. For example, in one variation, the fluid receptacle has a volume of greater than or equal to about 1 milliliter to less than or equal to about 100 milliliters.

[0172] In the variations like that shown in FIGS. 31 A-31C, the separation device may be a spin-column that may be used in a centrifuge system. Thus, the separation device is sized to be received in a conventional centrifuge. The spin-column is configured to seat within the centrifuge. The separation of the one or more target analytes occurs by associating with the plurality of hedgehog- shaped particles where they are separated from the remainder of the fluid sample, which may be collected in the fluid containment region. The separating process occurs more rapidly by spinning in a centrifuge. In certain variations, the centrifuge may be a traditional centrifuge with slower speeds than ultracentrifugation, which takes longer for processing and is more expensive equipment.

[0173] In certain variations, the spinning is in a centrifuge and conducted at a speed of less than or equal to about 10,000 rpm (revolutions per minute), optionally less than or equal to about 5,000 rpm, optionally less than or equal to about 4,000 rpm, optionally less than or equal to about 3,000 rpm, optionally less than or equal to about 2,000 rpm, optionally less than or equal to about 1,000 rpm, optionally less than or equal to about 500 rpm, optionally less than or equal toabout 250 rpm, and optionally less than or equal to about 100 rpm. In other variations, the spinning in the centrifuge for the separation process may be conducted at a speed of greater than or equal to about 50 rpm to less than or equal to about 3,000 rpm.

[0174] In certain other variations, methods for separating one or more target analytes from a biological fluid sample obtained from a subject are contemplated. The method generally includes providing an active material that may be in an active region (e.g., sensing region). The active material includes a plurality of hedgehog-shaped particles that exhibit a multiscale chirality. The active material disposed in an active region may be part of a microfluidic channel in a microfluidic device. The active region comprising the active material may also be disposed within a fluid receptacle or open region. The active region is configured to receive a biological fluid sample. The method further comprises passing a biological fluid sample so that it contacts the active material, for example, in certain variations, passing a biological fluid sample through a microfluidic channel that includes the active region with the plurality of hedgehog-shaped particles that exhibits a multiscale chirality. In other aspects, the passing is through a fluid receptacle that contains the active material, for example, as a layer through which the fluid sample passes. One or more target analytes in the fluid sample may be captured and separated from the fluid sample when the target analytes associate with the plurality of hedgehog-shaped particles in the absence of any added targeting moiety. By associating with hedgehog-shaped particles, one or more target analytes are removed from the passing fluid sample. In certain aspects, if one or more target analytes are separated, they may be removed from the hedgehog-shaped particles in the active region by passing a treatment fluid where the target analyte(s) are extracted and may be further processed / analyzed. By way of non-limiting example, captured analytes / biomolecules, such as proteins, can be rapidly released from hedgehog-shaped particles by dissolving the material in buffer solutions, for example, comprising PBSZEDTA optionally at a pH around 7.4. For example, further analysis of the target analyte(s) may be conducted, including profiling the biomolecule(s) or biological assemblies, such as cancer-associated exosomes or sepsis-associated extracellular vesicles.

[0175] Optically convenient visible light range resonances of some hedgehog-shaped particles that exhibit a multiscale chirality enable rapid and versatile profiling of various EVs. The light may also be polarized light, such as s-type or p-type linear polarization, left and right circularly polarized light, elliptically polarized light, any combination of the foregoing, or any other suitably polarized light sequences. Light (polarized or unpolarized) may be directed at the sensing region comprising hedgehog-shaped particles and then properties of the light transmitted or reflected may be measured. Generally, visible light has a wavelength visible ranging from about390 to about 750 nm, with a red color having a wavelength in a range of about 625 nm to about 750 nm, while infrared radiation (IR) includes near infrared radiation (NIR) ranging from about 750 nm to about 1.4 micrometers (gm). In certain aspects, the emitted light is measured at greater than or equal to about 520 nm to less than or equal to about 1.4 micrometers, optionally from greater than or equal to about 520 nm to less than or equal to about 750 nm, and in certain variations, optionally from greater than or equal to about 625 nm to less than or equal to about 750 nm.

[0176] As discussed herein, in certain aspects, hedgehog-shaped particles that exhibit a multiscale chirality can be integrated into a microfluidic platform that make possible rapid and robust detection of biomolecules, such as cancerous exosomes, directly from plasma without prior purification steps, which is highly desirable for clinical settings. Such microfluidic devices may be formed on a chip. Where the microfluidic device is on a chip, it can be mounted to a conventional spectrometer and its measurement is simple and completed rapidly. In certain nonlimiting aspects, the microfluidic device may comprise a single microfluidic channel that improves the sensitivity and speed of detection by 14 times and 10 times, respectively, compared to traditional techniques. However, multiple channel complexes or arrays of microfluidic devices are also contemplated. Optically convenient near-infrared resonances of chiral nanoparticles enable in perspective the low-cost glass / plastic-based microfluidics that represents an attractive pathway for rapid and versatile profiling of various extracellular vesicle.

[0177] The hedgehog-shaped particles that exhibit a multiscale chirality are capable of rapidly isolating and profiling target analytes of a biological origin, such as biomolecules or vesicles, like cancer-associated exosomes, directly from blood plasma using their own unique chiral signal. For example, exosomes from lung cancer patients can be distinguished from those from healthy donors by chiroptical spectroscopic signatures of biomolecular components of exosomes enhanced by hedgehog-shaped particles that exhibit a multiscale chirality. Furthermore, mutation / deletion of epidermal growth factor receptor also demonstrates an ability for in-depth mutation profiling in addition to cancer diagnostics.

[0178] In one variation, a clinical application and product may be a diagnostic chip for identifying sepsis phenotypes by chiral Raman Optical Activity extracellular vesicle detection chip (cROAev) shown in FIGS. 1A-1D. While chiral Raman spectroscopy is a lesser-known analytical method, in this application, the detection regime may be considered to be similar to a well-known analytical tool, surface plasmon resonance (SPR) Raman, with the benefits of chirality information being coupled to regular signal and no need for a specialized surface.

[0179] It is hypothesized that the unique polarization-resolved signal from sepsis-related EVs can be more efficiently identified if the resulting signal from their interaction with hedgehogshaped nanoparticles is determined. A diagnostic chip may be utilized with circularly polarized Raman spectroscopy, especially circularly polarized Raman Optical Activity (cROA), technology to analyze extracellular vesicle (EV) biomolecules and detect alterations associated with sepsis. cROA not only helps determine the chiral properties of molecules, but it also reveals insights into their interactions with other molecules, the effects of the surrounding environment, and their dynamic behavior. This comprehensive understanding of EV biomolecules is essential for identifying alterations associated with sepsis phenotypes. cROA is a much more powerful detection method than circular dichroism (CD) spectroscopy where changes in the optical properties of the chiral particles are measured upon biomolecule and exosome binding. By capturing sepsis-specific EVs and analyzing their biomolecular interactions with hedgehogshaped gold particles that exhibit a multiscale chirality (chiral substrate), the chip provides a rapid, non-invasive, and highly sensitive method for identifying sepsis phenotypes in patients and monitoring their response to treatment, which can improve patient care in sepsis management.

[0180] Such a device includes two components: a microfluidic chip and a sensing or detection region comprising hedgehog-shaped particles that exhibit a multiscale chirality. The microfluidic chip comprises an inlet, an outlet, and a sensing region where hedgehog- shaped particles that exhibit a multiscale chirality are deposited (FIG. 1A). In the production of the detection chamber, a uniform coating on the glass surface of the device may be formed by using a layer-by-layer (LBL) assembly method. This approach is used for simplicity and the fact that it is not affected by the shape of the substrate. LBL also aligns the asymmetric planar NPs parallel to the substrate. These chips are designed to fit into a holder of a cROA instrument, positioning the sensing region directly in the beam path (FIG. IB). When exosomes are captured by the hedgehog- shaped (FIG. 1C), they strongly alter the characteristic bisignate cROA peaks of the hedgehog-shaped particles based on the strength and nature of the interactions, resulting in a spectroscopic change (FIG. ID). Changes to the surface expression of the EVs (e.g., proteins, lipids) will change the cROA signal allowing for differentiation and identification of biomarkers. Similarly, the cROA signal for the EVs themselves will change due to enhanced signatures from plasmonic coupling of the Raman active bands of the biomarkers interacting with the particles. This change in spectral signature can serve as a secondary mode of biomarker identification. While standalone analysis with a dedicated instrument is contemplated in this embodiment, the nature of the measurement means that any laser-based chip reading diagnostic system could also bemodified to collect this information using off-the-shelf components (e.g., fiber optic collector, Raman detector, circular polarization filter).

[0181] This technology thus uses multiscale chirality nanoparticles to specifically capture target analytes of a biological origin, such as biomolecules or biological assemblies. The unique interactions make it easier to detect subtle changes in biological assemblies. Biomolecules, such as amino acids or other organic moieties on the surface, provide atomic or nanoscale chirality, while the nanoparticle spikes provide nanoscale or microscale chirality. As described above, the particles may have independent chiralities defined on three or more distinct scales. Such technology could allow for a more accurate and less invasive test for cancer progression.

[0182] Nanostructured hedgehog-like gold particles with various branching generations may be formed when gold is reduced in presence of cysteine. These hedgehog particles are flat with spikes predominantly confined to one plane. An additional structural distinction is that the particles are chiral with multiscale mirror asymmetry determined by / . / J-en anti omers of cysteine. Their branching segments can have preferentially left- or right-handed twists, leading to optical activity and polarization rotation. The complex structure of the particles can be quantified using graph theory (GT), capable of an accurate description of their architecture combining order and disorder.

[0183] In certain other aspects, the present disclosure contemplates a method of separating one or more target analytes from a biological fluid sample obtained from a subject. The method optionally comprises introducing a biological fluid sample into a column comprising a fluid receptacle and having a layer comprising a plurality of hedgehog-shaped particles. Each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, according to any of the variations previously described above. The plurality of hedgehog-shaped particles is capable of associating with one or more target analytes optionally present in the biological fluid sample. The method includes separating the one or more target analytes from the biological fluid sample by capturing the one or more target analytes in a respective hedgehog-shaped particle in the active region by passing the fluid sample through the column, so that a remainder of the biological fluid sample not captured by the hedgehog-shaped particles in the active region passes through the layer for collection e.g., in a containment region / collection tube below).

[0184] In certain variations, the column is a spin-column and the separating occurs by spinning the spin-column in a centrifuge. As described above, the spinning in the centrifuge maybe conducted at a speed of less than or equal to about 10,000 rpm or any of the other speeds previously specified. In certain aspects, the spinning in the centrifuge may be conducted at a speed of greater than or equal to about 50 rpm to less than or equal to about 3,000 rpm. In certain aspects, the separating occurs in less than or equal to about 60 minutes, optionally less than or equal to about 45 minutes, optionally less than or equal to about 30 minutes, optionally less than or equal to about 20 minutes, optionally less than or equal to about 15 minutes, optionally less than or equal to about 10 minutes, and in certain variations, optionally less than or equal to about 5 minutes. These separation times are advantageous, especially as compared to conventional ultracentrifugation separation and other techniques that typically take many hours (e.g., greater than 4 and typically greater than 8 hours) for achieving comparable separation.

[0185] The one or more target analytes may be any of those described previously above, including by way of example, those selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof. In certain aspects, the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins for diagnostic purposes. One advantage of the devices provided according to certain aspects of the present disclosure are that they are marker-agnostic. In this manner, the one or more target analytes may comprise extracellular vesicles (EVs) and the separating that occurs is advantageously marker-agnostic and thus capable of separating heterogenous extracellular vesicle (EV) populations from the biological fluid sample.

[0186] In certain aspects, a separation efficiency for removing the one or more target analytes from the fluid sample is high, for example, greater than or equal to about 70% of an initial amount of the one or more target analytes present in the biological fluid sample are separated from the biological sample (after the separating occurs), optionally greater than or equal to about 75%, optionally greater than or equal to about 80%, optionally greater than or equal to about 85%, and in certain variations, optionally greater than or equal to about 90% of the target analytes are separated from the fluid sample.

[0187] In certain further aspects, the method further comprises, after the separating, introducing a washing liquid (as described above) into the fluid receptacle so that it passes through the layer comprising a plurality of hedgehog-shaped particles. This washing may be conducted one or more times and can lead to a higher purity level of the captured target analytes in the active region comprising the hedgehog-shaped particles. For example, where a volumetric capacity of the fluid receptacle is a relatively small volume, multiple wash cycles may be used. However,with larger capacity volume fluid receptacle / columns, a single wash (e.g., of about 1 mL) is sufficient to remove non-specifically bound biomolecules from the hedgehog-shaped particles. In certain aspects, the captured target analyte(s) may be further collected after the separating. In such embodiments, the method may include introducing an elution liquid into the fluid receptacle, so that it passes through the layer comprising a plurality of hedgehog-shaped particles and elutes the one or more target analytes from the layer for collection.

[0188] In this manner, a diagnostic platform for early disease detection is contemplated in certain aspects of the present disclosure. Based on the initial proteomics analysis, washing protocol can improve the specificity of the isolation process. For example, this can be achieved by reducing non-specific protein binding while effectively capturing extracellular vesicles (EVs), as shown by BCA analysis. Multiple washes of the device may be conducted to minimize nonspecific protein contamination. Afterward, a lysis buffer may be added to lyse the captured EVs for biomarker extraction. A BCA protein assay quantified the protein content before and after the washing steps and after lysing the EVs. Elutions from different devices show a decrease in protein concentration with each wash. Two to three washes appears to effectively remove most nonspecific binding proteins, while retaining EVs in the device. The captured EVs may then be lysed with a radioimmunoprecipitation assay (RIP A) buffer to collect EV biomarkers.

[0189] With reference to FIG. 32, a kit according to certain variations of the present disclosure where separation may be conducted in conjunction with a centrifuge is provided. In one variation, the one or more target analytes are extracellular vesicles (EVs), such that the kit is an EV capture kit. The kit includes a separation device according to certain aspects of the present disclosure in the form of an EV capture column (e.g., a spin-column) that includes an extraction component seated in a first removable collection tube having a reversibly sealable cap for forming a fluid tight seal. The kit may also include one or more additional removable / disposable collection tubes, at least one sealed container comprising washing medium (e.g., washing buffer) and at least one sealed container comprising an elution medium (e.g., elution buffer). In certain aspects, the EV capture column may be provided in the kit with the hedgehog-shaped particles already processed and ready for use. In other aspects, the kit may further comprise a container with a suspension of the hedgehog-shaped particles in a liquid medium, such that the hedgehog- shaped particle medium may be introduced into the extraction component of the capture column, where it is introduced into the centrifuge and spun for about a minute to create an active layer comprising the hedgehog-shaped particles (e.g., over a filter) while the remainder of the liquid is removed. The extraction component / spin-column is ready for introduction and processing of a fluid sample.

[0190] Next, in step 2, a biological fluid sample is introduced into a fluid receptacle of the spin-column (above the active region / layer of hedgehog-shaped particles and the filter). The spin- column / EV capture column may then be introduced and seated in a centrifuge and spun for about 1 minute. This is optionally followed by introducing washing medium (e.g., PBS) into the fluid receptacle. The extraction component may be seated in a tube, which may be sealed and placed into the centrifuge for additional spinning, for example, for 1 minute. As shown, the separating occurs such that a large portion of the EVs are retained in the active region / layer comprising the plurality of hedgehog-shaped particles, while filtrate (e.g., serum proteins and the remainder of the contents of the biological sample) passes through the active region / layer and filter.

[0191] Where the separated EVs are to be further processed and / or analyzed, they may be released from the active region / layer comprising the plurality of hedgehog-shaped particles. In one optional variation, shown as Step 3.1, EV proteins may be released in an example of a process that involves incubation and centrifuge spinning for protein lysis and release. For example, serum proteins that are undesirable can be flushed and removed. In one variation, a radioimmunoprecipitation assay (RIP A) buffer is used for cell lysis, where it may be introduced into fluid receptacle and incubated at 4°C for 30 minutes, for example. Then, the column spin- column / EV capture column may be spun for about 1 minute in the centrifuge, so that the lysed EV proteins or biomarkers are released for downstream analysis. As appreciated by those of skill in the art, the collection tube may be removed for further processing of the EV proteins and replaced with an additional tube that may be provided in the kit.

[0192] The kit can be used as part of an innovative diagnostic platform capable of swiftly isolating EVs from patient samples in under 30 minutes, as compared to conventional methods that typically take over 4 hours. The comprehensive kit contains the EV isolation column as described above incorporating the chiral hedgehog-shaped particle technology, a washing medium or buffer to eliminate unwanted serum proteins, and an elution buffer for EV isolation while maintaining EV integrity. This user-friendly system allows for complete and rapid agnostic biomarker isolation (e.g., within 30 minutes), facilitating early identification of disease biomarkers for quicker intervention and monitoring of disease progression and treatment response.

[0193] In an alternative and optional Step 3.2, the extracellular vesicles (EVs) may be eluted from the active region / layer comprising the plurality of hedgehog-shaped particles. In this example, the extraction component of the spin-column / EV capture column may be seated in a new collection tube. The elution buffer is introduced into the fluid receptacle of the extraction component of the spin-column / EV capture column, where it may be rest for about 2 minutes so that it passes through the active region / layer to elute the EVs into the containmentregion / collection tube. In this manner, EVs may be collected in the collection tube at a high purity and the separating process may be as short as 5 minutes. As noted above, greater than or equal to about 70% of the initial target analytes, here EVs, optionally 90% or greater of the initial target analytes are separated and recovered from the initial fluid sample. These EVs are thus efficiently captured while having full integrity for downstream analysis and use, which is needed for diagnostic and therapeutic applications. These methods and devices thus facilitate efficient isolation of intact, functional EVs at scale, which may allow EV-based drug delivery system protocols for therapeutic agent loading, and ensuring quality control methods that maintain consistent EVs preparations. In this manner, devices prepared in accordance with certain aspects of the present disclosure feature rapid isolation time, preserved EV integrity, excellent reproducibility, and compatibility with small sample volumes — ideal for clinical applications with time and sample constraints.

[0194] FIGS. 33A-33D show another variation of a device in the form of a syringe-fed isolation column 50 for detecting, analyzing, and / or separating one or more target analytes from a fluid sample in accordance with certain variations of the present disclosure. FIG. 33A shows three components prior to and after assembly (FIG. 33B) to form a device that is an isolation column, and more specifically a syringe-fed column 50, having an active region comprising a layer with a plurality of hedgehog-shaped particles according to the present disclosure. A first component 60 (also labeled as 1) is a separation or extraction component similar to that shown in FIGS. 31A-31C and as described previously above, which includes an active region 62 comprising a layer with a plurality of hedgehog-shaped particles. The active region 62 is disposed above an outlet 66 through which portions of the fluid sample may pass after exiting the active region 62.. The filter 64 may be disposed below the outlet 66, so that fluids passing out of the first extraction component 60 pass through the outlet 66 and then through the filter 64 (as best seen in FIG. 33C) that is downstream of the outlet 66 In other variations, while not shown, the filter 64 may be disposed in the first extraction component 60 and thus may be disposed above an outlet 66 for the fluid. The isolation / syringe-fed column 50 further includes a fluid retainment component in the form of a collection tube 70 (also labeled as component (3)) having a first end 72 that is closed or sealed along a lower region and a second end 74 along an upper region that is open. In this manner, the first extraction component 60 is disposed in an open internal region of the collection tube 70. A second component (2) of the isolation column includes a sleeve 80 that may be disposed within the collection tube 70 (third component (3)) and in which the first extraction component 60 (first component (1)) seats and is disposed (as best seen in FIG. 33C). The sleeve 80 defines in a lower region a first end 82 that may be open or closed / sealed and asecond end 84 along an upper region that has at least one opening. A lower sleeve region 88 below the filter 64 may allow the fluids may pass out an opening in the first end 82 of the sleeve 80 so that it exits the sleeve 80 and transfers into the first end 72 of the collection tube 70 (alternatively, the lower sleeve region 88 may contain fluid). The sleeve 80 may define a portion of an interlock region 86 configured to receive a tip region 102 of a syringe 100 (best seen in FIGS. 33D and 33E). The interlock region 86 may form a fluid tight seal when the tip region 102 of the syringe 100 (as best seen in FIGS. 33D and 33E) is secured in position to deliver a fluid sample, so that a fluid may be introduced into a fluid receptacle within the extraction component 60 without any leakage. In certain aspects, while not limiting, the interlock defined between the syringe 100 and the sleeve 80 may be in the form of threads, so that the syringe tip 102 may be screwed into the terminal second end 84 of the sleeve 80 and secured for fluid delivery. However, other interlocks known in the art are also contemplated.

[0195] FIGS. 33B and 33C show the isolation column 50 with the sleeve 80 and collection tube 70 assembled, but without any syringe 100. FIG. 33D shows a syringe 100 secured by screwing the syringe tip 102 into the second end 84 of the sleeve 80. FIG. 33E shows the syringe 100 and sleeve 80 secured together and disposed within the collection tube 70 and ready for injection of the fluid sample from the syringe 100 into the extraction component 60 disposed within the sleeve 80. In this manner, a separation process of a fluid sample may be conducted by capturing target analytes in the active region layer 62 comprising the hedgehog-shaped particles and filtering the target analytes from the remainder of the fluid sample. As noted above, in certain variations, the filter 64 may be disposed within the extraction component 60, while in other variations, the filter 64 may be disposed within the sleeve 80 and thus downstream of the extraction component 60 with the active region layer 62 and outlet 66.

[0196] As will be appreciated by those of skill in the art, other methods for injection and introduction of the fluid sample under pressure could be used in alternative variations. Thus, the fluid sample may be introduced by any process where it is pressurized as it is introduced to the device. In this variation shown, the fluid passes both by pressurization of the stream provided by the syringe, as well as being gravity fed through the extraction component as it passes through the filter and enters the collection tube. In this embodiment, while the processing time may be longer than the embodiment using centrifugation, no external equipment like a centrifuge is required, making it particularly suitable for field and portable applications. In this variation, a kit may include the components of the isolation column as described above in the context of the spincolumn isolation column, including the extraction component with the active region comprising the plurality of chiral hedgehog- shaped particles and filter (or a filter may be provided as aseparate component), as well as one or more collection tubes, but may further include the sleeve component and one or more syringes. As noted above, in certain variations, the filter may be a separate component in the kit to be placed by the end user or may be seated in the sleeve instead of in the extraction component having the active region layer.

[0197] As will be appreciated, the present disclosure in certain variations contemplates both a medical device that is marker-agnostic for EV isolation for diagnosis and therapeutics. The devices and methods of separation can be applied to multiple therapeutic applications, including sepsis detection, cancer diagnostics, neurodegenerative disease monitoring, infectious disease identification, and autoimmune condition management, by way of non-limiting example. Thus, devices prepared in accordance with certain aspects of the present disclosure can rapidly isolate intact extracellular vesicles from patient samples for early detection of sepsis, cancer, and autoimmune diseases, enabling clinicians to identify disease-specific biomarkers and intervene earlier than conventional methods allow. Additionally, these isolated EVs can be utilized for drug delivery or targeted therapy applications, potentially reducing mortality rates and improving treatment outcomes across multiple high-impact clinical applications.

[0198] Examples

[0199] Hydrogen tetrachloroaurate trihydrate (HAuCh 3H2O), cetyltrimethylammonium bromide (CTAB, > 99%), cetyltrimethylammonium chloride (CTAC, 25 wt. % in water), potassium iodide (KI, > 99%), sodium hydroxide, (NaOH, >98.0%), D-cysteine (D-cys, 99%), L- cysteine (L-cys, 98%), and L-ascorbic acid (AA, > 99.9 %) were obtained from Sigma-Aldrich (Milwaukee, WI). Milli Q water was used to prepare solutions, unless otherwise stated. Ultrathin carbon film on lacey carbon support film, 400 mesh, copper (Prod # 01824) was purchased from Ted Pella (Redding, CA).

[0200] Gold nanoprism seeds are a first step in the hedgehog-shaped particle preparation. 211.8 pL of cetyltrimethylammonium chloride (CTAC) solution, 25% CTAC by weight, was added to 9,338 mL of deionized water. 75 uL of 10 mM KI was stirred into the solution. In a separate small container, 80 pL of 25.4 mM HAuC14 was mixed with 20.3 pL of 0.1M NaOH. This mixture was injected into the KI-CTAC solution, turning the clear solution yellow orange. 80 pL of 64 mM ascorbic acid and the solution was quickly mixed. Then, 10 pL of 0.1M NaOH was immediately added to the solution and the mixture was slightly shaken. The intensity of shaking is a factor in the synthesis. With intensive mixing, roundish particles are obtained instead of nanoprisms. After 10 to 15 minutes, the solution became dark blue; a red or purple solution would indicate the formation of undesired small, round nanoparticles. After the particles formed, they were centrifuged at 10,000 RPM for 5 minutes. The supernatant was drained and replacedwith approximately 1.5 mL of 5mM CTAC. This washing procedure was repeated two more times. Once the seeds are washed, they remain stable for as long as several months if kept in dark at 4°C.

[0201] For the synthesis of the hedgehog- shaped particles, the seeds were first diluted with 5 mM CTAC from 1.5 mL to around 4 mL. This stock of seeds can be used for several subsequent syntheses of hedgehog. 1.6 mL of 0.01 M cetyltrimethylammonium bromide (CTAB) solution, was added to 7.9 mL of deionized water. Then, 400 pL of 10 mM HAuCL was stirred in and the solution was allowed to rest for 10 minutes, during which it turned from pale yellow to orange. All solutions used for the procedures described above were made in deionized water, because in millipore water, the reaction did not run out. In rapid succession, stirring between each step, 950 pL of 40 mM ascorbic acid, necessary volume of 4 mM L- or D-cysteine (for example, to obtain the final Cys concentration 3.6 pM, 10 pL of Cys was used), and 200 pL of the previously synthesized seeds were added to the solution. The solution was left for 90 minutes (without stirring) before centrifuging at 10,000 RPM for 5 minutes, after which the hedgehog was resuspended in 1 mM CTAB or 5 mM CTAC wash solution.

[0202] For time-dependent series of CD and UV-Vis spectra, the particles after synthesis were washed twice by centrifugation to get rid of the growth solution and prevent the alteration of the particle shapes. After that process, samples at 1, 3 and 5 minutes were not stable in dispersion due to decrease of electrostatic repulsion between the particles.

[0203] Chiral sensing experiment. After synthesis, the L-XNDs were washed 5 times with 1 mM CTAB to remove the majority of cysteine molecules from the surface. Then 3.6 uM of L- or D-Cys were added to the bare L-XNDs and after 30 min the UV-vis spectra were measured.

[0204] The morphology was analyzed by scanning transmission electron microscopy (STEM) using JEOL 3100 R05, operated at 300 kV, and Thermo Fisher Talos F200X G2 S / TEM, operated at 200kV. The electron tomography studies at room temperature were carried out on a Thermo Fisher Talos F200X G2 S / TEM equipped a Gatan One View bottom mount camera for high-speed 4k x 4k and an X-FEG high-brightness Schottky type field emission gun (FEG) operating at 200 kV. A series of 2D projection images were recorded by tilting the specimen from -70° to 70° at nominal magnification of 150,000 using Fischione Tomography holder Model 2020 with high-tilt angles (x = ±70°). A tomography reconstruction software package Gatan GMS 3 was used to align the tilt series and calculate three-dimensional tomograms using a weighted back projection algorithm. Circular Dichroism (CD) and UV-Vis spectra were obtained by a JASCO J- 815. Raman spectra were recorded by a Renishaw Invia Raman spectrometer equipped with a green laser (k=532 nm).

[0205] In certain variations, a synthesis method provided by certain aspects of the present disclosure allows geometry of nanohedgehog particles to be changed and provides a reliable pathway to depict their structure based on electron microscopy images. Seed-mediated growth when the growth of linear segments is catalyzed by the pre-made metal particles of smaller size, is convenient for the controllable growth of chemical structures with spiky architecture. Specifically, trigonal prisms of gold with sides of 60-70 nm are used as seeds dispersed in the solution of HAuCh in presence of L-Cysteine (Cys) or D-Cys. Ascorbic acid (AA) served as the reducing agent, while cetyltrimethylammonium chloride (CTAC) or bromide (CTAB) helped dispersibility of the produced particles as labile surface ligands. The branching spikes radiated predominantly from the vertices of the prisms and had a length of about 200-300 nm (FIGS. 2A, 2C). The investigation of the effect of branching and similar topological parameters on the properties of the particles was further facilitated by their flatness. The branching segments grow predominantly in a plane of the original prism most likely due to the tendency of gold NPs to attach in epitaxial manner to each other. Also, strong electrostatic repulsion of the NPs from the central part of the prism due to the highly charged layer of cetyltrimethylammonium cations, made peripheral attachment of the NPs energetically more favorable than that for prism center. Thus, the particles acquire a shape reminiscent of a snowflake, as was observed by TEM tomography and atomic force microscopy (AFM, FIGS. 2A-2D).

[0206] When Cys surface ligands were enantiopure, the spikes revealed dominant twists. L-Cys promoted formation of the right-handed spikes (FIGS. 2C-2D); when D-Cys is used the left-handed spikes dominate (FIGS. 2A-2B). These spiky nanostructures will be referred to as complex chiral nanohedgehog particles (abbreviated as XNDs, where X stands for both complex and chiral) because (1) unlike PAMAM and similar hedgehog with strict bifurcation sequence, these particles contain both order and disorder; (2) unlike polymer brushes, spherical amino acids, “hairy” polymers, microparticle fractals, etc., the linear segments and overall dimensions of these spiky particles are nanoscale; (3) unlike the majority of nanoscale spiky structures, these particles are mirror asymmetric. Helical patterns of the spikes appear due to chirality transfer from Cys to metallic gold. Gold NPs possess surfaces with intrinsic chirality due to the presence of the chiral terraces, or kink sites. Usually these are faces with high Miller-index gold surfaces ({hkl }, h k 4- 1 4- 0), such as {321 } or {521 } facets. L- and D-Cys bind to the R- or S-type facets enantioselectively, thus promoting the dissymmetric crystal growth and resulting in a chiral morphology of linear segments and spikes.

[0207] The extinction spectra of XNDs combining the contributions of both absorption and scattering, displayed two peaks at 500 nm and 1010 nm (FIG. 2E). The two bands correspondto plasmonic oscillation along the short and long axes of the spikes, also known as longitudinal and transversal plasmons. The peaks are strongly broadened compared to gold nanorods due to the cross-spike plasmon coupling and stochastic organization of the nanohedgehog.

[0208] All particles of XNDs as geometrical objects display multiscale chirality. One level of chirality is at scale of about 3 A and is characteristic of the a-carbon atom in Cys attached to the surface of gold. Another level of chirality is at the scale of about 50 nm characteristic of the pitch of the spikes. The two scales of chirality are reflected by the different peaks in the CD spectrum. The angstrom scale chirality of the amino acids is responsible for the appearance of the peak at 267 nm (FIG. 3H). The nanoscale chirality is responsible for the CD peaks at 520 nm and 1030 nm. The nanohedgehog made with L-Cys (z.e., L-XNDs) displayed a positive transversal and a negative longitudinal circular CD peak, with the opposite handedness for nanohedgehog made with D-Cys (i.e., L-XNDs, FIG. 2E). The markedly improved signal-to-noise ratio for the CD peaks compared to the UV-vis peaks is due to the constructive interference of and stronger coupling between the plasmons with the same handedness in the neighboring spikes.

[0209] Besides the angstrom (amino acid) and nanometer (spike) levels of chirality, the overall mesoscale geometry of the XND particles can also be chiral and have a consistent preference toward left- or right-handed shapes. However, the CD spectra of the XNDs alone are insufficient for conclusive assessment of the mesoscale mirror asymmetry. To understand its contribution to the overall optical asymmetry of the particles the nanohedgehog were etched using the 6 pM HAuCL solution for 2 hours. As expected, the branched structure of the nanohedgehogshaped particles was gradually destroyed, and the optical activity concomitantly diminished. Nonetheless, the g-factor spectra of the etched L-XND retained a weak but distinct positive peak at 550 nm corresponding to the non-bifurcating remainder of the spikes. Combined with the UV- vis extinction spectra, these data indicate that the 3D arrangement of spikes should be chiral corresponding to the third level of chirality at 200 to 300 nm scale.

[0210] Growth Stages of hedgehog-shaped particles that exhibit a multiscale chirality (XNDs). L-XNDs morphology changes for different durations of the synthesis, t, sheds light on the growth stages of the nanohedgehog (FIGS. 3 A-3F). UV-Vis, CD and g-factor spectra obtained for 10 min < t < 90 min, displayed the similar trend to increase and then plateau for t > 60 min (FIGS. 3G-3I). The maxima in all spectra continuously shifted to the red, which indicates an increase in the inter-spike coupling with increasing the size of nanohedgehog. The half-width of the spectra did not increase over time compared to the initial particles as one would expect for plasmonic structures with gradually increasing stochasticity and randomness. In fact, the amplitude of the g-factor spectra consistently increased for t >10 min, which indicates theconsistent increase of mirror asymmetry at nanometer scale responsible for the 520 nm plasmonic peaks, which should be attributed to the growth of linear segment of the nanohedgehog and terminal spikes. Both gradually develop the twist reflected in the CD and g-factor spectra (FIGS. 3G-3I).

[0211] TEM images of the particles at the different stages of the growth in FIGS. 4A-4F confirm the spectroscopic observations in FIGS. 3G-3I for t > 10 min. The TEM images also afford overcoming the limitation of colloidal stability and enable the access to particle morphologies for t < 10 min. For t = 1 min, many small spheroidal NPs with the size of 10-20 nm are observed in addition to the 60-70 nm prismatic NPs serving as seeds (FIG. 4A). Simultaneously, the distinct change of the shape of the seed particles due to nucleation and selfassembly of the NPs from dispersions on the corners of the prisms is observed, forming proto spikes (FIG. 4B). NPs continue to self-assemble on proto-spikes for t = 3-5 min (FIGS. 4C-4D). Further, the re-crystallization of the constitutive gold NPs leads to the linear segments of the nanohedgehogs (FIG. 4E). They no longer appear as coalesced round particles but become monolithic elongated nanostructures with helical shapes. The initially formed spheroidal NPs are consumed in the self-assembly process. For 10 < t < 20 minutes, the particles increase in size, the length of their spines becomes longer (FIGS. 3 A-3B). Interestingly, the particle morphology does not show large geometric changes 20 < t < 90 min (FIGS. 3B-3F) but the optical activity continues to increase (FIGS. 3H-3I).

[0212] In the absence of cysteine, under the same synthetic conditions, achiral (or to be exact racemic) NPs with polyhedron shapes are formed.

[0213] As the concentration of cysteine (z.e., [Cys]) is raised from 0.125 to 36.2 pM (FIG. 5) while maintaining [HAuCh] = constant 0.37 mM, the size of the particles, the length of the spikes and the number of branching generations rises with [Cys], One approach to the quantitative relations between the organization of XNDs and is to count the number of spikes, the number of bifurcation points. For example, in the range of concentrations of [Cys] from 0.125 to 1.8 pM, the length of the spikes increases from a 75-100 nm to 250-300 nm while the number of bifurcation points is approximately 10. However, [Cys] has non-trivial influence on the optical activity (FIGS. 6E). With an increase of [Cys], the maximum of extinction, its spectral position, the maximum of chiroptical g-factor and its spectral position, all reveal strong non-linear dependence following the trend of initial rise to a maximum followed by the precipitous drop. For example, the size of the particles continuously increases with the increase of [Cys] but no optical properties correlate with it over the entire range of the concentrations. XNDs obtained at higher [Cys] also showed greater number of the bifurcation points that can scatter light and can be chiral, but the number ofbifurcation points increases continuously with [Cys], Considering the contributions of the particle geometry in 50-60 nm and 200-300 nm range to chiroptical activity, the maximum of g-factor at 0.5 pM could be interpreted as the result of either elongation of twisted spikes that reach the scale of 50-60 nm or the entire particle reaching the scale of 200-300 nm. Looking at the angstrom level of chirality, DFT calculations suggested that the binding of Cys to certain facet of gold depends on the degree of coverage of the NP surface, making it thermodynamically more favorable for Cys to enantioselectively attach to a chiral facet at low coverage and to achiral (110) facet at high coverage, leading to a decrease in optical activity as well as the appearance of outgrowths at high [Cys],

[0214] FIGS. 6A-6F show control on morphology with seed mediated growth. FIGS. 6 A show particles after additional growth with a seed concentration of 40 pM (FIGS. 6A-6B) and 0.04 pM (FIGS. 6C-6D), respectively. Optical activities or g-factor (FIG. 6E) and extinction spectra (FIG. 6F) of the particles before and after additional growth are shown. Various gold hedgehog-shaped nanoparticles having multiscale chirality prepared in accordance with certain aspects of the present disclosure are shown capturing extracellular vesicles in FIG. 7. FIG. 8 likewise shows gold hedgehog-shaped nanoparticles having multiscale chirality capturing extracellular vesicles, where the image on the left has a scale bar of 1 micrometer and the inset shows a magnified single extracellular vesicle.

[0215] TEM images of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles are shown in FIG. 9, including cumulative, potassium, sodium, and phosphorus. FIG.9 also includes spectra from area 1 in an intensity (kCounts) versus energy (keV).

[0216] FIG. 10 shows TEM images of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles, including TEMS of cumulative, gold, potassium, phosphorus, and sodium. FIGS. 11 and 12 show images of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure capturing extracellular vesicles (FIG. 11) and liposomes (FIG. 12), including TEMS of gold and phosphorus or gold, phosphorus, and silver.

[0217] Silver-coated gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure are shown in FIGS. 14 and 15 capturing extracellular vesicles (FIG. 13) and liposomes (FIG. 14), including TEMS of gold and combined gold, silver, and phosphorus.

[0218] FIG. 15 shows comparative circular dichroism (CD) and g-factor spectra for peaks of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the presentdisclosure with no extracellular vesicles or two distinct extracellular vesicles. A 2.5 nm red shift occurs when the extracellular vesicles are associated with the gold hedgehog-shaped particles, as shown in the CD data. A table shows comparison of gold hedgehog-shaped nanoparticles with and without extracellular vesicles, including zeta potential, mobility, and conductivity values for comparison.

[0219] Gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure having different amino acid-derived shaped directing moieties and show the comparative circular dichroism (CD) (FIG. 16 A) and g-factor spectra for peaks (FIG. 16B). More specifically, a gold hedgehog-shaped nanoparticle having 100% cysteine as an amino-acid derived chiral moiety is compared to a gold hedgehog-shaped nanoparticle having 50% cysteine to 50% methionine as an amino-acid derived chiral moiety. The gold hedgehog-shaped particles having 50% cysteine to 50% methionine amino acid-derived chiral shape moieties have a higher and distinct peak for both CD and g-factor.

[0220] FIGS. 17A and 17B show HAADF images of gold hedgehog-shaped nanoparticles prepared in accordance with certain aspects of the present disclosure having different amino acid- derived shaped directing moieties. FIG. 17A has a scale bar of 100 nm and shows a gold hedgehogshaped nanoparticle having 100% cysteine as an amino-acid derived chiral moiety. FIG. 17B has a scale bar of 50 nm and shows a gold hedgehog-shaped nanoparticle having 50% cysteine to 50% methionine as an amino-acid derived chiral moiety.

[0221] All the structural characteristics of XNDs considered above, namely, the overall diameter of the particles, the number of spikes per particle, spike length, the number of bifurcation points, and geometry of gold surface in presence of cysteine and / or methionine, have a collective effect on XND spectra. Albeit tempting, none of these factors should or can be considered independently of others when the structure is complex and combines some reproducible motifs and some stochasticity. Furthermore, as exemplified by three contributions to XND chirality at angstrom, nanometer, and submicron scales, the relationship between XND geometry and optical properties should be treated based on the parameters describing the structure of the nanohedgehog particles as a with structural descriptors at multiple scales.

[0222] Simplicity of this synthetic process makes possible inclusion of other noble metals leading to a large family of inorganic nanostructured spiky (hedgehog-shaped) particles.

[0223] The present disclosure shows that various nanostructured hedgehog-like particles with various branching generations are formed, for example, when gold is reduced in presence of cysteine or with copper-based materials. These hedgehog-shaped particles may be considered to be flat with spikes predominantly confined to one plane. Their additional structural distinction isthat they are chiral with multiscale mirror asymmetry determined by L / D-enantiomers of an amino-acid derived moiety, such as cysteine. Their branching segments can have preferentially left- or right-handed twists, leading to optical activity and polarization rotation. The complex structure of the particles can be quantified using graph theory (GT), capable of an accurate description of their architecture combining order and disorder. It is found that GT parameters, Euclidean distance closeness (EDC) and spike-branch factor (SBF), can successfully describe their complex structural patterns and are determined by the concentration of cysteine used in the synthesis. Furthermore, the macroscale optical properties, namely the amplitude of circular dichroism peaks and optical asymmetry factor, are dependent on SBF and EDC, providing the pathway for property-centric design of the complex particles.

[0224] Hedgehog-shaped particles are thus demonstrated as suitable for use in chiral sensing with very high enantioselectivity, based on the interaction of enantiomeric molecules with an intrinsically chiral gold surface. The hedgehog-shaped particles have a high surface area along with intrinsically chiral facets. Particles with the highest degree of branching were found to be most effective for chiral sensing.

[0225] In another variation, hedgehog-shaped particles formed in accordance with certain aspects of the present disclosure comprise copper.

[0226] In one example, complex biofunctional hedgehog-shaped particles with hierarchical assemblies of atomically thin copper sulfide chiral nanosheets (NSs) stabilized by penicillamine (Pen) surface ligands can be formed that are capable of reversible, controllable, and traceable chirality transfer through multiple scales are provided. Penicillamine used in all experiments is structurally a mercapto-derivative of an amino acid, valine. Once incorporated into CuS nanosheet, it enables synthesis of chiral hedgehog shaped particle of different multiscale chirality, size, morphology, and spikiness. Not limited to penicillamine, introduction of a tertiary thiol group in all 20 natural amino acids, to be similarly employed in such hybrid supramolecular systems, is believed to be synthetically straightforward and would enable to expand the concept presented in this disclosure to a wide library of possible chiral, spiky particles.

[0227] Compared to the biological nanostructures of similar dimensions, inorganic NSs reduce degrees of freedom which makes it easier to identify modes of chirality transfer. Additionally, they also improve their contrast and stability under electron beams. It is observed that, under controllable conditions, such nanosheets self-assemble into complex supraparticles (SPs) (FIGS. 18A-18C). Unlike other hybrid materials with lamellar structure, such as M0S2 and graphenes, or “hard” chiral NPs, these hybrid NSs undergo reversible chemical and spatial rearrangements driven by forces typically affecting biomolecules. As a result of reversiblechemistry of Cu-S bonds and adjustable electrostatic interactions bridging the NSs layers (FIGS. 18A-18B), SPs architecture can be controlled by multiple factors, yielding the library of optically active and biofunctional hedgehogs.

[0228] The chirality of used ligand defines the multilevel morphology of SPs. When enantiopure D- or L-Pen is employed in a surfactant-controlled reaction with Cu2+cations, under neutrality conditions of Pen (5 = 0, no protonation or deprotonation), complex spiky SPs of 15-20 micrometer in diameter emerge. In contrast, racemic mixtures (rac-Pen) promote the growth of approximately 15 micrometer spherical SPs composed of merged sub-micrometer needles (FIG. 18C). For brevity, abbreviations of D-SPs, L-SPs, and rac-SPs are used for SPs made of CuS-D- Pen, CuS-L-Pen, and CuS-rac-Pen, respectively. All these particles precipitate as fine yellow powders and can be resuspended in any common solvent without decomposition, which indicates the covalent character and stability of CuS network within NSs. The unique structural difference between spiky SPs and achiral supraspheres comes from the absence of sufficiently competitive hydrogen bonds counteracting with electrostatic repulsion that otherwise dominates. Further, the spikiness and other submicron features of SPs can be progressively tuned by using D- / L-Pen mixtures of different enantiomeric excess, temperature, and surfactants, resulting in SPs of varying mesoscale morphologies, similarly to the reported bowtie assemblies with chirality continuum.

[0229] It is found, however, that the pH-dependent degree of protonation of Pen (5) is one parameter that helps determine the structure and assembly states of particles, enabling adjusting their size, shape, spikiness, and porosity (FIG. 18C). The zwitterionic nature of the ligand allows a gradually increase in the Coulombic charge of assembling CuS-Pen building blocks by protonation of the amine group at low pH values (5 > 0) or decrease it by deprotonation of carboxylic acid at higher pH (5 < 0, where 5 refers to the net charge of Pen; 5 = +1.0 for fully protonated Pen, 5 = 0 for zwitterionic, and 5 = -1.0 for fully deprotonated Pen, FIG. 18B). As a result of electrostatic repulsion between charged CuS-Pen clusters, no SPs assemble at pH = 1.4 or pH = 7.4, when is 5 = +1.0 or -1.0, respectively. Instead, soluble nanoclusters (NCs) remain in the system, as indicated by transmission electron microscopy (TEM) and high-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM), and the disappearance of scattering peaks above about 700 nm in the UV / Vis absorption spectra, otherwise accompanying the dispersions of sub-micron particles.

[0230] A gradual decrease of Pen charge from 5 = +1.0 yields flat hexagonal microcrystals that start to emerge at 5 = +0.5. Upon further deprotonation to 5 = +0.3, the NSs start to assemble in different directions since the decreased electrostatic repulsion does not restrict the growth alongthe two-dimensional plane anymore. When the electrostatic repulsion oscillates between 5 = ±0.25, the particles are composed of hundreds of thin spikes, since the short-range repulsion still does not allow for full aggregation into dense solids but is sufficient for the self-separation of weakly charged spikes. At 5 = -0.3, most sub-micron features vanish to yield uniformly spherical SPs, because the excess of a base neutralizes +0.3 equivalents of hydrochloric acid (HC1) released in the reaction accompanying the self-assembly, hence no Pen moi eties bear any net charge. When 5 = -0.35, microspheres become twisted as the negatively charged nanosheets partially repel each other. At 5 = -0.4, the particles grow porous as the base prevents their complete assembly and leaves empty spaces between merged NSs. Eventually, the electrostatic repulsion emerging at 5 = -0.5 restricts the particle growth to nanoplatelets of <2 micrometers thin. For 5 < -0.6, only approximately 3 nm nanoclusters are stable.

[0231] X-ray diffraction (XRD, FIG. 18D) reveals the identical poly crystalline arrangement of D- and L-SPs. TEM and HAADF-STEM images confirm the hierarchical structure of SPs comprising multiple layers of atomically thin CuS nanosheets stabilized by the bilayers of Pen ligands. High-resolution (HR)-TEM and selected area electron diffraction (SAED) patterns prove the hexagonal packaging of CuS, while the basal spacing between NSs calculated from XRD is d = 1.28 nm (29 = 6.89°), which is in a perfect agreement with molecular dynamics (MD) simulation of complementary stacking nanosheets (FIGS. 18F-18G). Noteworthy, MD reveals the NSs’ left-handed twist perpendicular to the nanosheet plane in D-SPs, and right-handed in L-SPs, respectively. Those twists are induced by chiral Pen ligands organized into Moire patterns with oppositely oriented amine and carboxylate groups (FIGS. 18E-18F), and macroscopically observable in SEM images (FIG. 18H). In contrast, MD-simulated racemic system of alternating D- and L- nanosheets shows the cancellation of analogous twists, which explains the growth of thin straight needles in rac-SPs instead of continuous chiral microprisms, and thus-resulting poorer crystallinity observed by XRD. The free energies obtained from MD reveal stronger binding between opposite Pen enantiomers (-12.2 kcal / mol) than homochiral Pen pairs between NSs (- 10.0 kcal / mol). This further supports the experimental disruption of asymmetry in rac-SPs and their low complexity, which typically requires a delicate balance between attraction / repulsion forces rather than strong binding.

[0232] It was anticipated that SPs shown in FIG. 18C could be disassembled into primary NSs and NCs by tuning the pH to sufficiently high or low values by adding a base or an acid to their suspensions, respectively. Beside the electrostatic repulsion of Pen ligands associated with as-controlled protonation degree, copper (I) thiolate polymers are known to be decomposed into well-defined nanoclusters, such as Cu4(SR)e2", by breaking Cu-S bonds. To verify this, spiky D-SPs are titrated, synthesized at 5 = 0, with 2M KOH and HC1 solutions in a presence of cetyltrimethylammonium chloride (CTAC) as protecting surfactant. SEM images indicate that both acid and base gradually disassemble SPs into small flat fragments of stacked NSs; at sufficiently high absolute 5 values, all NSs disintegrate into approximately 3 nm NCs. Interestingly, under acidic conditions, SPs disassemble into well-defined micro- and nanoprisms, whose size depend on the 5 values, what can barely be seen in base-decomposed particles. Such uneven digestion of SPs and NSs by base can by rationalized by the fact that thiols are prone to oxidation at high pH values, followed by strong stabilization of once-oxidized Cu(II) atoms by free NH2 groups

[0233] The pH-dependent multiscale chirality of different D- / L-SPs, NSs, and NCs is confirmed by circular dichroism (CD) spectroscopy in ultraviolet, visible, and infrared regions (FIGS. 22A-22B). As an example, typical D- and L-SPs display symmetric peaks between 200 and 300 nm due to electronic transitions in Pen ligands, as well as excitation peaks around 350 nm from conjugated semiconducting CuS network, and broad signals between 500-1,300 nm attributed to Rayleigh and Mie scattering. The optical anisotropy g-factors of SPs are relatively low, i.e., below 5 x 10^, due to high rigidity of interlocked nanosheets, as the (CusSsjn honeycomb structure is intrinsically achiral - thus the dissymmetry of NSs is defined by ligand-induced anisotropic distortion. In contrast, CD spectra of free NCs and exfoliated NSs display multiple nodes in the visible region, and g-factors increase an order of magnitude. This suggests that the geometry of free Cu-Pen NCs and atomically thin NSs adapts better to the chirality of solvated ligands than densely packed ones, leading to a larger distortion of Cu-S bonds. Such effect is observed in the MD-simulated 2.5 nm NSs. As a result, the twisting angle y between 2.5 nm NSs assemblies is y= ±11°, while between 5 nm NSs it decreases to y= ±4°. Eventually, the simulated CD transitions in NSs are in nearly perfect agreement with the experimental ones, which confirms the structure of modeled NSs. This confirms the multiscale chirality of particles.

[0234] In this example, copper sulfide-amino acid nanosheets self-assemble into a repertoire of complex chiral materials. Tuning of the repulsive and attractive interactions between the nanosheets enables spanning a previously inaccessible continuum of assemblies, including biosimilar hedgehogs, with progressively controllable morphology, handedness, and chiroptical characteristics. Hierarchical nanoscale organization of such assemblies allows for their reversible disassembly and “on demand” reassembly into diverse hedgehogs, controllable by pH, temperature, and chemical additives. This remarkable ability to manipulate the structural characteristics of complex systems over multiple scales, coupled with their biocompatibility, enables the utilization of such networks in the separation of elusive biomolecules.

[0235] In further variations, hedgehog-shaped particles formed in accordance with certain aspects of the present disclosure comprise copper with different compositions. For example, chiral hedgehog- shaped particles are formed in accordance with the present disclosure that comprise copper sulfide or copper iodide having multiscale chirality. In certain examples described herein, the copper-based particles have a similar chemical composition, for example, a copper sulfidepenicillamine structure, of an approximate formula, CuC5H10NO2S. It can be alternatively called a copper thiolate salt / copper thiolate coordination polymer, and the like. An overall morphology, spikiness, and chirality of these particles are controlled by synthesis temperature, presence of surfactants, pH value (— > delta value), chirality and enantiomeric excess of penicillamine (or any other chiral ligand, e.g., glutathione, and the like). Other examples include copper iodidepenicillamine particles that contain both iodine and sulfur, e.g., a composition with approximately CuC5H10INO2S. As the properties of the particles are more similar to copper iodide itself, rather than copper sulfide, it is designated copper iodide-penicillamine, herein.

[0236] By way of example, copper iodide-penicillamine hedgehog-based particles are shown in FIGS. 19A-19B. SEM images in FIG. 19A of D-chiral copper iodide-penicillamine and in FIG. 19B of L-chiral copper-iodide penicillamine (Cui-Pen) hedgehog-shaped particles are shown. The introduction of iodine allows an increase in the chemical stability and a decrease in the size of the spiky particles from about 20 micrometers to 4 micrometers and less.

[0237] FIGS. 20A-20C show TEM images of various chiral hedgehog-shaped supraparticles (SPs) comprising copper sulfide having multiscale chirality prepared with lefthanded penicillamine (L-SPs) (Z-copper sulfide-penicillamine (L-CuS-Pen)) where the supraparticles comprising copper formed have left-handed chirality. More specifically, these hedgehog-shaped particles are prepared by post-synthesis process. A hedgehog particle diameter was adjusted to about 200 nm by selective etching of 20-micron (D-CuS-Pen) particles by adding a weak base, sodium carbonate.

[0238] In further examples, Z>-Penicillamine (98%) and Z-penicillamine (99-101%) were purchased from Sigma-Aldrich and further purified by recrystallization. Copper (II) chloride dihydrate (>99.99% metal basis), potassium iodide (99%), cetyltrimethylammonium chloride (CTAC, 25 wt. % solution in water), cetyltrimethylammonium bromide (CTAB, >98%), hydrochloric acid (37%), potassium hydroxide (-86%, pellets), sodium hydroxide (reagent grade, >98%, pellets, anhydrous), sodium hydrogen carbonate (99.7+%), poly(sodium 4- styrenesulfonate) (PSS, Mw-70,000, 30 wt. % solution in water), l-butyl-3-methylimidazolium hydrogen sulfate ([BMIMjfHSCU], >95%), choline chloride (>98%), poly(diallyldimethylammonium chloride) (PDADMAC, Mw<1 00,000; 100,000-250,000, and200,000-350,000), (3-chloro-2-hydroxypropyl)trimethylammonium chloride (3-CHTMAC, 60 wt. % in water), Z-cysteine (Z-Cys, 97%), Z-glutathione, reduced (Z-GSH, >98%), phosphate buffered saline (PBS) tablets, bovine serum albumin fluorescein isothiocyanate conjugate (BSA), cytochrome C from bovine heart (CytC, >95% based on Mw12,327), glycerol, ethylene glycol, methanol, ethanol 99.5+%, isopropanol, and acetonitrile were purchased from Sigma-Aldrich and used without further purification. Ethylenediaminetetraacetic acid di sodium salt dihydrate (EDTA, 99.9%) and Gibco™ Fetal Bovine Serum, certified (FBS), were purchased from Fisher Scientific. Freshly deionized water (18.2 mfi cm ') was employed in all experiments, deoxygenated, and decarbonized by saturation with nitrogen prior to use.

[0239] Synthesis of CuS-Pen SPs. 2 mL of solution prepared by mixing 10 mL of deionized water, 10 mL of 25% aqueous CTAC solution, and 2 mL of 100 mM CuCh solution was poured into a 4 mL glass vial. The vial was incubated in a 50°C water bath for 10 min. Then, 386 pL of D-, L-, or rac-Pen solution (85 mg • mL1, 6.5 equiv. regarding the molarity of Cu) was added at once using an automatic pipette. The vial was immediately shaken for 2-3 seconds and placed back in the water bath for the self-assembly. No stirring was applied. The reaction was complete when the yellowish precipitate settled and the supernatant became nearly transparent, which typically took 10-20 min. In the reaction, Cu2+ions are first reduced to Cu+, which is immediately complexed by thiol to produce yellowish Cu(I) complex, according to the reaction equation,

[0240] 2 Cu2++ 4 PenSH - 2 Cu!S(Pen) + (PenS)2+ 4 H+.

[0241] This process is followed by continuous supram olecul ar polymerization of the complex, during which the covalent Cu-S bonds are formed and propagate along a two- dimensional plane to form the CuS nanosheets stabilized by Pen ligands. Simultaneously, hydrogen bonds between the sheets result in three-dimensional self-assembly into complex particles. The growth process is self-limited by electrostatic interactions, e.g., yielding particles of approximate 20 micron in diameter at 5 = 0.

[0242] Purification of supraparticles (SPs) is conducted as follows. The reaction mixture was cooled down to room temperature. The supernatant was discharged with a pipette, the particles were resuspended in 4 mL of water, and left undisturbed until precipitated again. Washing with water was repeated two more times, followed by analogous washing with 4 mL of 99% ethanol. Purified SPs were directly resuspended in water for characterization - if it required their use in dispersed state. In some cases, purified SPs can be prone to oxidation. To avoid decomposition, SPs dispersions can be protected from oxidation by adding approximately 150 pL of fresh Pen solution in diluted CTAC or can be kept under inert atmosphere at mildly acidic pH.Alternatively, SPs dispersed in high-viscosity organic solvents, such as glycerol or polyethylene glycol). Alternatively, SPs can be dried under gentle N2 flow, followed by storage under vacuum for several hours. Dried SPs powders are then relatively stable under air and, in most cases, can be stored at least for a year.

[0243] In another example, synthesis of SPs with alternative thiols is conducted. Other copper sulfide or copper-thiolate SPs, such as CuS-cysteine, CuS -glutathione, can be synthesized using analogous procedure as for standard Cu-Pen SPs described above. Briefly, 2.00 mL of Cu / CTAC precursor solution is reacted with 386 pL of 0.55 M aqueous thiol solution in a 50°C water bath until precipitate settled and supernatant became transparent (<10 seconds up to 3 hours). In circumstances where thiol is solubilized poorly in water, e.g., certain thiolated acids, a minimum amount of dimethyl sulfoxide (DMSO) can be added to aqueous suspension to solubilize it before reaction.

[0244] In yet another example, SPs are synthesized with other surfactants. The synthesis procedure is identical to that described above for forming standard SPs, except different surfactants are used instead of CTAC. Briefly, 2.00 mL of solution containing 16.7 mM of CuCL and 0.40 M of surfactant of choice: CTAB; PDADMAC of varying molecular weights; choline chloride; 3-CHTMAC; SDS; PSS, and [BMIM][HS04]. The surfactant was reacted with Pen solution (85 mg • mL1) at 50°C. SDS solution was initially acidified with 0.5 equiv. HC1 to decrease the pH to approximately 4.0; otherwise, the high pH of SDS would prevent the formation of any microparticles. Synthesis of SPs in a presence of PSS was carried out using 0.20 M surfactant solution containing 58 pL 2.00 M KOH solution. All other surfactants were used without changing their original pH values.

[0245] Protein extraction from serum using CuS-Pen SPs. 2.00 mL of SPs dispersions (D- , L-, and rac- synthesized at variable protonation degrees, +0.3 > 5 > -0.4) were purified by removal of supernatant, washing twice with water, then washing with anhydrous ethanol (2 mL) and again with water. Washing with ethanol assures the removal of residual CTAC, which can form micelles and mono- and bilayers stacking to the SPs surface in aqueous systems, which would otherwise interfere with protein adsorption process. The supernatants were discharged, and the remaining SPs (7.0 mg, 28 pmol) were mixed with 2 mL of fetal bovine serum (FBS) solution, followed by gentle shaking for 15 minutes. SPs were either decanted or centrifuged at low g (<1000 rpm, 5 min) and washed three times with water (in a first procedure (I)) or with water, IM KC1 solution, and water again (procedure II). Analogously to procedure I, procedure III was also performed using 3.00 mL of SPs and 1.00 mL of FBS and water washing. Smaller protein-to-SPs ratio and larger available chiral surface allowed to observe the differences in binding of weak-affinity proteins (compared to procedure I, were FSPS / FFBS = 1 : 1). Procedure I was carried out to separate both high-affinity proteins and weakly bound proteins due to the presence of ‘hard’ and ‘soft’ corona on SPs surface, while procedure II was performed to remove the most external ‘soft corona’ protein components at high ionic strength.

[0246] Washed SPs-protein conjugates were then disassembled by dissolution in 800 pL of phosphate buffered saline (PBS) solution, containing 75 mM EDTA, adjusted to pH = 7.2 with 1 M NaHCOs. The vials were gently mixed until the red photoluminescence vanished (approximately 1-2 min) and the solutions became transparent, which indicated the complete SPs disassembly. Protein concentrates were then immediately frozen at -80°C for the proteomics analysis.

[0247] SEM samples were prepared by drop-casting 10 pL of aqueous SPs / NSs / LC dispersions on 5 x 5 mm silicon wafers (TedPella), followed by removing the excess of a solvent with a Kim wipe, and drying at room temperature. The samples were then sputter coated with an approximate 5 nm film of gold on the wafer to minimize the local charging under focused electron beam. SEM imaging was performed on FEI Helios Nanolab at 5.0 kV accelerating voltage and 0.4 nA beam current under secondary electrons detection mode. In case of excessive sample charging (which typically occurred when rigid SPs spikes had minimal contact surface area with conductive silicon wafer) the voltage was decreased to 3.0 kV at 0.2 nA beam current, with multiple fast scan accumulation, without significantly affecting final image resolution.

[0248] TEM samples were prepared by immersing the carbon-coated copper grids (TedPella, 150 mesh) in aqueous dispersions / solutions, placed on a Kim wipe to remove the excess of the solvent, and drying at room temperature. Bright-field TEM images and diffraction experiments were acquired using Thermo Fisher Talos F200X operating at 200 kV equipped with a Gatan One View camera. A HAADF detector was used to acquire Z-contrast STEM images in which the intensity is proportional to the atomic number of the column over which the electron probe is placed.

[0249] Powder X-ray diffraction (XRD) measurements of purified, dried SPs samples were performed using Rigaku SmartLab diffractometer equipped with a copper Kasource (wavelength of 1.5406 A) in a Bragg-Bentano, 0-20 configuration. The acquisition of scattered beams was carried out using a D / Tex detector with 20 angle varying from 2° o to 60° in a step of 0.01° and slowest possible accumulation time, to maximize the signal-to-noise ratio. Background subtraction was performed using PDXL software.

[0250] CD measurements in ultraviolet (UV), visible (Vis), and near-infrared (NIR) range were performed using JASCO J-1700 and J-815 spectrometers equipped with one photomultipliertube detector in a 200-800 nm range and two InGaAs NIR detectors in an 800-1,600 nm range. Typical scanning parameters for SPs were as follows: scanning speed, 500 nm min"1; data pitch, 0.1 nm; bandwidth, 5 nm (NIR bandwidth, 10 nm), digital integration time, 0.25 s. In certain cases, due to the low signal-to-noise ratio of tightly packed SPs dispersions, approximately 20 accumulations were collected with a careful check of the background between the runs. Higher- anisotropy g-factors of NCs and NSs permitted acquisition of quality spectra at with 2-3 accumulations. The g-factors were calculated according to the equation g=CD / (32,980*Abs), where CD is the signal obtained from CD / DC channel and Abs is the absorbance calculated from ABS and DC channels of the spectrophotometer.

[0251] VCD measurements were performed on purified and vacuum-dried samples dispersed in heavy water (D2O) at 35 mg mL-1concentration, using JACSO FVS-6000 VCD spectrometer. 100 pL of such suspensions were sandwiched between two barium fluoride (BaF2) crystals separated by a 50 pm Teflon spacer. An MCT-V detector was used to acquire infrared and VCD data in the range of 2,000 to 850 cm1with a resolution of 4 cm1and a total of 100 and 2000 accumulations, respectively. The sandwiched dispersions between BaF2 crystals were rotated along the a axis coinciding with the direction of the beam at a constant speed to avoid settling of particles. Corresponding infrared and VCD were plotted as A and AA / A, respectively, with exclusion of the 1,300 to 1, 100 cm1range which corresponds to strong absorption from D2O.

[0252] CPLE samples were prepared by dispersing freshly synthesized SPs in water, or, preferably, another achiral solvent of high viscosity, such as ethylene glycol, due to rapid precipitation of particles in water during data collection. The CPL handedness, intensities, and emission maxima were nearly identical regardless of employed solvent, as the luminescence originated from the rigid microparticle, and was not affected by the surface solvation effects. CPLE, PL, and excitation spectra were collected using JASCO J-300 spectrometer under constant HT voltage at 600 V, 4,000 pm excitation and emission pinhole, scanning speed, 500 nm min"1; data pitch, 1.0 nm; bandwidth, 5 nm (NIR bandwidth, 10 nm), digital integration time, 1.0 s, 4 accumulations. Samples of low-CPLE maximum intensity, z.e., <20 mdeg, were measured at 200 nm min1scanning speed to improve the signal-to-noise ratio.

[0253] Optical microscopy samples were prepared by depositing approximately 10 pL of SPs / NSs suspensions between two standard microscopy glass slides (25 x 75 x 1.0 mm and 22 x 22 mm, Fisherbrand). SPs could be dispersed both in water or high-viscosity organic solvents, such as ethylene glycol or glycerol, while the latter typically improved the image quality and limited the particles’ movement. However, the images of samples titrated with a base or acid were all taken in water, to avoid the chemical interactions or differences in solvation betweendisassembled NSs and glycol or glycerol. Confocal microscopy images were acquired using Nikon AIR and Leica 8 microscope with 405 nm (Nikon) and 365 nm (Leica) excitation lasers. Three- dimensional z-stack images were obtained after superimposing multiple frames captured at different z-heights (thus varying focal points), with an interval of Az = 0.5 pm.

[0254] High-throughput proteomics were conducted with enriched protein solutions were prepared as described above. The samples were analyzed using optimized protocols. Briefly, cysteine thiol residues were reduced with 10 mM DTT (45°C for 30 min) and alkylated with 65 mM 2-chloroacetamide, under darkness, for 30 min at room temperature. Overnight digestion with 1 ug sequencing grade, modified trypsin was carried out at 37°C with constant mixing (ThermoMixer). Digestion was stopped by acidification and peptides were desalted using SepPak C18 cartridges using manufacturer’s protocol (Waters Corp.). Samples were completely dried using vacufuge. Resulting peptides were dissolved in 9 pL of 0.1% formic acid / 2% acetonitrile solution. Two pL of the resulting peptide solution were resolved on a nano-capillary reverse phase column (EasySpray PepMap C18, 2-micron, 50 cm, #ES903, Thermo Scientific) using a 0.1% formic acid / acetonitrile gradient at 300 nL / min over a period of 90 min. Eluent was directly introduced into Q Exactive HF mass spectrometer (Thermo Scientific, San Jose CA) using an EasySpray source. MSI scans were acquired at 60K resolution (AGC target=3xl06; max IT = 50 ms). Data-dependent collision induced dissociation MS / MS spectra were acquired on 20 most abundant ions following each MSI scan (NCE -28%; AGC target 1x105; max IT 45 ms).

[0255] Various chiral hedgehog-shaped supraparticles (SPs) comprising copper, such as copper sulfide or copper iodide, have a multiscale chirality. When prepared with right-handed penicillamine (D-Pen), the copper sulfide-penicillamine supraparticles are designated (D-SPs) and confirmed to have left-handed nanoscale chirality. Analogously, the copper sulfide-penicillamine (L-SPs) prepared with left-handed penicillamine (L-Pen), have right-handed nanoscale chirality. Likewise, FIGS. 21A-21B show various chiral hedgehog-shaped supraparticles (SPs) comprising copper sulfide and penicillamine, prepared at different pH or delta values, having multiscale chirality. All particles exhibit multiscale chirality. However, all SPs prepared with right-handed penicillamine (D-SPs), have left-handed chirality in at least one scale, while all SPs prepared with left-handed penicillamine (L-SPs) have right-handed chirality in at least one scale. As an example, copper sulfide-penicillamine particles shown in FIG. 21 A are right-handed at the atomic / molecular level (D-Pen) but are left-handed at the nanoscale. Additional hedgehog-shaped particles formed in accordance with certain aspects of the present disclosure comprise copper sulfide-penicillamine SPs of different chiroptical properties shown in FIGS. 22A-22C.

[0256] FIG. 22 A shows the CD spectra, in a form of anisotropy g-factor, of D- and L-SPs prepared at different temperatures. FIG. 22B shows the vibrational circular dichroism (VCD) spectra of the same D- and L-SPs of 5 value of 0 formed with right-handed penicillamine (D-SPs) and left-handed penicillamine (L-SPs), prepared at different temperatures. FIG. 22C shows the circularly polarized light emission (CPLE) spectra of D- and L- SPs at a 5 value of 0, prepared at different temperatures. Synthesis temperature allows to control the multi-scale chirality of the particles. This is observed by SEM (FIG. 25) and by changes of CD, VCD, and CPLE spectra (FIGS. 23A-23C). Strong VCD signals originating from ordered packaging of Pen ligands at the nanoscale appear above T=40°C, while SPs synthesized below that temperature lack long-range order of chiral CuS-Pen units. This can be observed by weak CPLE characteristics of SPs obtained at lower temperatures. Synthesis temperature allows control of the multi-scale chirality of the particles. This is observed by SEM (FIG. 25) and by changes of CD, VCD, and CPLE spectra (FIGS. 23A-23C).

[0257] FIG. 23 shows typical copper sulfide-penicillamine hedgehog-shaped supraparticles formed with right-handed penicillamine (D-SPs), left-handed penicillamine (L- SPs), and racemic penicillamine (D / L-SPs) at scales indicated in each SEM image. The particles are prepared at 50° C in the presence of CTAC, as described above. Note that in the absence of chirality, i.e., when racemic Pen is used (D / L-SPs), the particles lack characteristic spiky morphology.

[0258] FIG. 24 shows the SEM images of copper sulfide-penicillamine hedgehog-shaped supraparticles (SPs) synthesized at different enantiomeric excess (X), at different temperatures, and captured at different magnifications. The fraction X denotes the chirality of used Pen; X = +100 for pure D- enantiomer, X = -100 for pure L- enantiomer, and X = 0 for racemate (D / L-Pen).

[0259] FIGS. 25A-25B and 26A-26B show copper sulfide-penicillamine hedgehogshaped supraparticles formed with right-handed amino acid (D-SPs) prepared in accordance with certain aspects of the present disclosure capturing biomolecules in the form of exosomes. In FIG. 25B, the exosomes were not trapped due to variation in the procedures. The exosome solution was added after the deposition of Sis on a microscopy slide. This was done to show that, under specific conditions, how the exosomes are adsorbed on the SPs surface can be controlled. FIGS. 26A-26B show the assemblies of GFP-labeled exosomes adsorbed on the spikes of D-SPs.

[0260] Further, copper sulfide-penicillamine hedgehog-shaped supraparticles formed with left-handed amino acid (L-SPs) prepared in accordance with certain aspects of the present disclosure can also capture biomolecules in the form of bovine serum albumin (BSA), as shown in FIG. 27. Organic layers are visible as more of the protein is adsorbed between thespikes / platelets, and BSA is used as a model protein (e.g., biomolecule) to show capture and association. The L-SPs were washed with water. Similarly, FIG. 28 shows the capture of serum proteins with copper sulfide-penicillamine hedgehog-shaped supraparticles formed with lefthanded amino acid (L-SPs) by adsorption. The material shown in FIG. 28 was prepared as described above in the context described for Procedure I.

[0261] Copper sulfide-penicillamine hedgehog-shaped supraparticles formed with either right-handed (D-SPs), left-handed amino acid (L-SPs), and racemic penicillamine (rac-Pen, also referred to as D / L-) prepared in accordance with certain aspects of the present disclosure have demonstrated the ability to capture a variety of biomolecules, including specific serum proteins shown in FIGS. 29A-29C. In particular, FIGS. 30A-30C prove that certain biomolecules can selectively interact with spiky particles of different chirality and spikiness and can be used for chirality-controlled separation / extraction / purification of biomolecules using chiral hedgehogshaped spiky particles.

[0262] As an example, the incubation of different copper sulfide-penicillamine SPs with fetal bovine serum (FBS), separation of SPs by low-g centrifugation, allows to selectively capture specific proteins, which is an ultrafast and convenient method of extraction and purification of biomolecules. This method does not require the use of complex and time-consuming techniques, such as multi-step electrophoresis or chromatographic methods. Further, the claimed method does not involve expensive equipment, such as ultra-centrifugation. Eventually, the method is scalable due to the simplicity of manual operations required.

[0263] Trapped biomolecules, such as proteins, can be rapidly released from the spiky SPs by dissolving the material in PBS / EDTA buffer solutions, optionally at pH around 7.4. As an example, high-throughput proteomics analysis (FIGS. 29A-29C) of such concentrates reveals high affinity of chiral D- and / .-SPs to specific apolipoproteins (ApoB, ApoE, ApoA-I), which are separated from much more abundant components, such as albumin and glycoproteins (FIGS. 29 A- 29B). In sharp contrast, the lack of spikes in rac-SPs (FIG. 29C) results in large differences in trapped biomolecules. The chirality of spiky SPs allows concentration of selected apolipoproteins to >40% content, compared to 0.1-0.7% of their physiological concentrations in serum, in a single step in less than 10 minutes, in a scalable manner. However, rac-SPs are four times less effective in isolation of Apo proteins, significantly binding albumin, myosin, and alpha-2-HS-glycoprotein (FIG. 29C). These differences suggest that while chiral, spiky SPs have affinity to the certain proteins due to comparable nanoscale binding effects, the substantially different nanoconfinement of non-spiky, achiral SPs disrupts the assembly of certain proteins. Not being limited to aforementioned examples including copper sulfide-penicillamine particles, the precise control ofnanoscale chirality of other possible chiral SPs can promisingly become a fast and convenient methodology to separate specific biomolecules from other complex systems.

[0264] The EV isolation platform using chiral gold particles in a spin-column / EV isolation column has been shown to reduce EV isolation time from a biological sample to just 3 minutes in certain cases, while maintaining at least 74% capture efficiency in a prototype. This represents a dramatic improvement over traditional ultracentrifugation methods that take from 4 to 18 hours (typically greater than 8 hours). Further, another experiment developed an optimized spin columnbased device having only a 1 -minute sample preparation time. Enhanced purity through improved washing protocols, confirmed by BCA analysis has been shown. EV capture has been further validated using Western blot analysis for specific biomarkers, including Flotillin-1, CD81, and TSG101.

[0265] Certain variations of the present technology are marker-agnostic, allowing effective isolation of heterogeneous EV populations without relying on specific surface markers. This feature enables comprehensive biomarker analysis, enhancing the versatility of the platform. Laboratory testing indicates that certain variations of the present technology consistently achieve 90% capture efficiency across various sample types, which is a marked improvement over traditional methods that typically only reach 30 to 60%. Additionally, the platform contemplated by certain variations of the present disclosure can isolate EVs from as little as 100 microliters (pL) of biological fluid, making it particularly suitable for clinical applications and small animal studies where sample volume is limited.

[0266] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

Claims

CLAIMSWhat is claimed is:

1. A device for detecting, analyzing, and / or separating one or more target analytes, the device comprising: an active region comprising a plurality of hedgehog-shaped particles that exhibits a multiscale chirality, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, wherein one or more target analytes associate with the plurality of hedgehog- shaped particles in the active region in the absence of any added targeting moiety.

2. The device of claim 1, wherein the shape directing organic chiral moiety is an amino acid derived chiral moiety comprising a thiol group.

3. The device of claim 1, wherein the shape directing organic chiral moiety is an amino acid derived chiral moiety selected from the group consisting of cysteine, homocysteine, penicillamine, arginine, lysine, methionine, and / or combinations thereof.

4. The device of claim 1, wherein the plurality of projections is substantially orthogonal to a surface of the core region and extend over an entire surface of the core region.

5. The device of claim 1, wherein the plurality of projections has a rod-like needle shape or a sheet-like flat shape.

6. The device of claim 1, wherein a branching pattern of the plurality of projections is stochastic.

7. The device of claim 1, wherein the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

8. The device of claim 1, wherein an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

9. The device of claim 1, wherein at least one of the core regions and the plurality of projections comprises gold.

10. The device of claim 1, wherein at least one of the core regions and the plurality of projections further comprises silver disposed over gold.

11. The device of claim 1, wherein the material comprises copper (Cu) and is selected from the group consisting of: copper sulfide, copper iodide, and combinations thereof.

12. The device of claim 1, wherein the core region and the plurality of projections are formed of the material that is the same composition.

13. The device of claim 1, wherein the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm.

14. The device of claim 1, wherein the multiscale chirality further comprises at least one third chirality selected from the group consisting of: a mesoscale chirality of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer); a microscale chirality of greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers); a sub-millimeter scale chirality of greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm); a millimeter scale chirality of greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters); and combinations thereof.

15. The device of claim 1, wherein the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins.

16. The device of claim 1, wherein the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

17. A microfluidic device for detecting, analyzing, and / or separating one or more target analytes, the microfluidic device comprising: a microfluidic channel having an inlet and an outlet that receives a fluid sample, the microfluidic channel comprising at least one active region having a surface comprising a plurality of hedgehog-shaped particles that exhibits a multiscale chirality and associates with one or more target analytes, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiralmoiety, wherein the one or more target analytes associate with the plurality of hedgehog-shaped particles in the absence of any added targeting moiety.

18. The microfluidic device of claim 17, wherein the microfluidic channel is formed on a microchip.

19. The microfluidic device of claim 17, wherein the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids and combinations thereof.

20. The microfluidic device of claim 17, wherein the shape directing organic chiral moiety is an amino acid derived chiral moiety is selected from the group consisting of: cysteine, homocysteine, penicillamine, arginine, lysine, methionine, and / or combinations thereof or of different amino acids.

21. The microfluidic device of claim 17, wherein the plurality of projections has a rodlike needle shape or a sheet-like flat shape and are substantially orthogonal to a surface of the core region and extend over an entire surface of the core region.

22. The microfluidic device of claim 17, wherein the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

23. The microfluidic device of claim 17, wherein an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

24. The microfluidic device of claim 17, wherein the material is selected from the group consisting of: gold, silver disposed over gold, copper sulfide, copper iodide, and combinations thereof.

25. The microfluidic device of claim 17, wherein the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm.

26. The microfluidic device of claim 17, wherein the multiscale chirality further comprises at least one third chirality selected from the group consisting of: a mesoscale chirality of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer);a microscale chirality of greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers); a sub-millimeter scale chirality of greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm); a millimeter scale chirality of greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters); and combinations thereof.

27. A method of separating one or more target analytes in a biological fluid sample obtained from a subject, the method comprising: passing a biological fluid sample through a device comprising an active region having a plurality of hedgehog-shaped particles disposed thereon, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, wherein the plurality of hedgehog- shaped particles is capable of associating with one or more target analytes optionally present in the biological fluid sample; and separating the one or more target analytes from the biological fluid sample by capturing at least a portion of the one or more target analytes in a respective hedgehog-shaped particle in the active region.

28. The method of claim 27, wherein the device comprises a microfluidic channel comprising the active region and the passing the biological fluid sample is through the microfluidic channel.

29. The method of claim 27, wherein the device comprises a fluid receptacle comprising the active region and the passing the biological fluid sample is through the fluid receptacle.

30. The method of claim 29, wherein the device is a spin-column for a centrifuge and the separating further comprises spinning the spin-column in the centrifuge.

31. The method of claim 29, wherein the device is a column and the passing further comprises passing the biological fluid sample through the active region comprising a layer of the plurality of hedgehog-shaped particles and a filter.

32. The method of claim 31, further comprising introducing the biological fluid sample into the device by pressurizing the biological fluid as it is introduced to the device.

33. A method of detecting one or more target analytes in a biological fluid sample obtained from a subject, the method comprising: passing a biological fluid sample through a microfluidic channel comprising an active region having a plurality of hedgehog-shaped particles disposed thereon and directing circularly polarized light at the microfluidic channel while the biological fluid sample is disposed therein to measure a first level of at least one of magnitude of circular dichroism or peak wavelength, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and an amino-acid derived chiral moiety, wherein the plurality of hedgehog-shaped particles is capable of associating with a target analyte optionally present in the biological fluid sample; and comparing the first level of at least one of magnitude of circular dichroism or peak wavelength to a baseline level of at least one of magnitude of circular dichroism or peak wavelength in the microfluidic channel in the absence of the biological fluid sample, wherein a difference between the first level and the baseline level indicates a presence of the target analyte in the biological fluid sample.

34. The method of claim 33, further comprising measuring the baseline level of at least one of magnitude of circular dichroism or peak wavelength by directing circularly polarized light at the microfluidic channel in the absence of the biological fluid sample.

35. A device for detecting, analyzing, and / or separating one or more target analytes, the device comprising: a fluid receptacle configured to receive a fluid sample comprising one or more target analytes, the fluid receptacle comprising a layer of a plurality of hedgehog-shaped particles that exhibits a multiscale chirality, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, wherein one or more target analytes associate with the plurality of hedgehog-shaped particles in the layer in the absence of any added targeting moiety.

36. The device of claim 35, wherein the device is a spin-column and the fluid receptacle defines an open region configured to receive the fluid sample, the layer comprising the plurality of hedgehog-shaped particles is disposed adjacent to the open region, a filter having afirst side disposed adjacent to the layer comprising the plurality of hedgehog-shaped particles and a second side disposed adjacent to an outlet, and a fluid containment region disposed adjacent to the fluid outlet.

37. The device of claim 36, wherein the fluid receptacle, the layer comprising the plurality of hedgehog-shaped particles, the filter, and the outlet define an extraction component and the fluid containment region is a tube in which the extraction component seats and the device further comprises a cap configured to reversibly seal the device.

38. A centrifuge system comprising a centrifuge configured to receive the device of claim 37, wherein the spin-column is configured to seat in the centrifuge and to separate the one or more target analytes associated with the plurality of hedgehog- shaped particles from the remainder of the fluid sample collected in the fluid containment region.

39. A kit comprising the device of claim 37 and further comprising: a first container comprising a washing medium, a second container comprising an elution medium, and at least one additional tube.

40. The device of claim 35, wherein the device is a syringe-fed column and the fluid receptacle defines an open region configured to receive the fluid sample from a syringe and the layer comprising the plurality of hedgehog-shaped particles is disposed adjacent to the open region and the syringe-fed column further comprises a filter and a fluid containment region that is a tube.

41. A kit comprising the device of claim 40, wherein the kit further comprises: a first container comprising a washing medium, a second container comprising an elution medium, at least one additional tube, and at least one syringe.

42. The device of claim 35, wherein the fluid receptacle has a volume of greater than or equal to about 100 microliters.

43. The device of claim 35, wherein the fluid receptacle has a volume of greater than or equal to about 1 milliliter.

44. The device of claim 35, further comprising a syringe in fluid communication with the fluid receptacle, the syringe configured to inject the fluid sample into the fluid receptacle.

45. The device of claim 35, wherein the plurality of projections has an average maximum dimension of greater than or equal to about 75 nm to less than or equal to about 300 nm and the core region has an average diameter of greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

46. The device of claim 35, wherein an average particle size of the plurality of hedgehog-shaped particles is greater than or equal to about 100 nm to less than or equal to about 50 micrometers.

47. The device of claim 35, wherein at least one of the core regions and the plurality of projections comprises gold or silver disposed over gold.

48. The device of claim 35, wherein the material comprises copper (Cu) and is selected from the group consisting of: copper sulfide, copper iodide, and combinations thereof.

49. The device of claim 35, wherein the core region and the plurality of projections are formed of the material that is the same composition.

50. The device of claim 35, wherein the multiscale chirality comprises a first chirality at greater than or equal to about 1 Angstroms (0.01 nm) to less than or equal to about 10 Angstrom (0.1 nm) and a second chirality at greater than or equal to about 1 nm to less than or equal to about 100 nm, and at least one third chirality selected from the group consisting of: a mesoscale chirality of greater than or equal to about 100 nm to less than or equal to about 1,000 nm (1 micrometer); a microscale chirality of greater than or equal to about 1,000 nm (1 micrometer) to less than or equal to about 100,000 nm (100 micrometers); a sub-millimeter scale chirality of greater than or equal to about 10,000 nm (10 micrometers) to less than or equal to about 1,000,000 nm (1,000 micrometers or 1 mm); a millimeter scale chirality of greater than or equal to about 1,000,000 nm (1 millimeter) to less than or equal to about 10,000,000 nm (10 millimeters); and combinations thereof.

51. The device of claim 35 wherein the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins.

52. The device of claim 35 wherein the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

53. A method of separating one or more target analytes from a biological fluid sample obtained from a subject, the method comprising: introducing a biological fluid sample into a column comprising a fluid receptacle and having a layer comprising a plurality of hedgehog-shaped particles, wherein each respective hedgehog-shaped particle comprises a core region and a plurality of projections connected to a surface of the core region, wherein at least one of the core region and the plurality of projections comprise a material selected from the group consisting of: gold (Au), silver (Ag), copper (Cu), and combinations thereof and a shape directing organic chiral moiety, wherein the plurality ofhedgehog- shaped particles is capable of associating with one or more target analytes optionally present in the biological fluid sample; and separating the one or more target analytes from the biological fluid sample by capturing the one or more target analytes in a respective hedgehog- shaped particle in the active region by passing through the column so that a remainder of the biological fluid sample passes through the layer for collection.

54. The method of claim 53, wherein the column is a spin-column and the separating occurs by spinning the spin-column in a centrifuge.

55. The method of claim 54, wherein the spinning in the centrifuge and conducted at a speed of less than or equal to about 10,000 rpm.

56. The method of claim 54, wherein the spinning in the centrifuge is conducted at a speed of greater than or equal to about 50 rpm to less than or equal to about 3,000 rpm.

57. The method of claim 53, wherein the column comprises the fluid receptacle defining an open region configured to receive the fluid sample, the layer comprising the plurality of hedgehog-shaped particles disposed adjacent to the open region, a filter having a first side disposed adjacent to the layer comprising the plurality of hedgehog-shaped particles and a second side disposed adjacent to an outlet, and a fluid containment region disposed adjacent to the fluid outlet.

58. The method of claim 57, wherein the fluid receptacle, the layer comprising the plurality of hedgehog-shaped particles, the filter, and the outlet define an extraction component and the fluid containment region is a tube in which the extraction component seats.

59. The method of claim 53, wherein the separating occurs in less than or equal to about 30 minutes.

60. The method of claim 53, wherein the separating occurs in less than or equal to about 10 minutes.

61. The method of claim 53, wherein the one or more one or more target analytes indicates a presence of cancerous cells or mutated proteins.

62. The method of claim 53, wherein the one or more target analytes is selected from a group consisting of: exosomes, extracellular vesicles (EVs), phosphatidylserine (PS), tetraspanin proteins, epithelial cancer adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), liposomes, proteins, nucleic acids, DNA, RNA, lipids, and combinations thereof.

63. The method of claim 53, wherein the one or more target analytes comprises extracellular vesicles (EVs) and the separating is marker-agnostic for separating heterogenous extracellular vesicle (EV) populations from the biological fluid sample.

64. The method of claim 53, wherein greater than or equal to about 70% of an initial amount of the one or more target analytes present in the biological fluid sample are separated from the biological sample after the separating.

65. The method of claim 53, further comprising after the separating, introducing a washing liquid into the fluid receptacle so that it passes through the layer comprising a plurality of hedgehog-shaped particles.

66. The method of claim 53, further comprising after the separating, introducing an elution liquid into the fluid receptacle so that it passes through the layer comprising a plurality of hedgehog-shaped particles and elutes the one or more target analytes from the layer for collection.

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