Magnetic nanoparticles

Abstract

The invention relates to magnetic nanoparticles. According to an embodiment, a microfluidic device includes: an inlet portion configured to receive a fluid sample; a mixing portion fluidly coupled to the inlet portion and configured to facilitate mixing between the fluid sample and magnetic nanoparticles, the magnetic nanoparticles functionalized to bind to extracellular vesicles and aggregate into vesicular glycans in the fluid sample; a magnetic separation section fluidly coupled to the mixing section and configured to separate clusters of magnetic nanoparticles from the fluid sample; and a magnetic sensor configured to measure a magnetic property of the fluid sample after the fluid sample has passed through the magnetic separation portion. The magnetic nanoparticles may be configured to aggregate in the presence of a corresponding lectin upon binding to an extracellular vesicle carrying a glycan of interest. In a particular embodiment, the magnetic particle includes a magnetic multinuclear coated with a polydopamine.

Description

[0001] This application is a divisional application of the original invention patent application with application number 202080081117.0 (International application number: PCT / SG2020 / 050663, international application date; November 16, 2020, invention name: Magnetic Analysis of Extracellular Vesicle Glycans). Technical Field

[0002] The present disclosure relates to the analysis of extracellular vesicle glycans, and in particular, to a microfluidic device and magnetic nanoparticles for the analysis of extracellular vesicle glycans. Background Art

[0003] Extracellular vesicles (EVs) have recently emerged as an attractive class of circulating biomarkers. These nanoscale membrane-bound vesicles are present in nearly all body fluids and are actively secreted by cells into the circulation. As robust messengers of intercellular communication, EVs possess a rich molecular content, including nucleic acids, proteins, lipids, and a variety of glycans—carbohydrate modifications found free or bound to proteins and lipids. Glycans are compositionally and structurally diverse; depending on the type of sugars and their organized binding, a variety of glycan combinations are possible, resulting in different biophysical properties and functions. In cells, this glycan diversity regulates many processes involved in protein folding, cell recognition, differentiation, and proliferation. Within vesicles, recent studies have demonstrated that EV glycans mediate vesicle biogenesis, intercellular recognition and communication, and disease progression and metastasis. Sensitive and multiplexed analysis of EV glycans may not only allow for a better understanding of EV mechanisms but also translate into new disease diagnostic and therapeutic applications.

[0004] Despite this potential, the study of EV glycans has lagged significantly behind that of proteins and nucleic acids found in vesicles. As with cellular studies, this lag is primarily due to a lack of suitable analytical techniques. Conventional assays use mass spectrometry for carbohydrate structural analysis. While this approach enables comprehensive measurements, it requires extensive processing (e.g., EV isolation, sample lysis, enzymatic digestion, and reduction), large amounts of sample, and advanced instrumentation and data analysis to make the assay applicable for EV-specific glycan analysis. In contrast, lectin microarrays offer a streamlined alternative. The approach relies on carbohydrate-binding proteins called lectins; after the lectins are immobilized on a sensor chip, fluorescently labeled EVs are applied and washed before the binding signal is measured. However, for EV-specific measurements, the assay not only requires purified EV preparations but also has limited sensitivity. Due to the weak interaction between lectins and glycans (dissociation constant, K d =10 -3 –10 -6M), which further limits the performance of the assay in detecting rare targets in rare and complex specimens. Summary of the invention

[0005] According to the first aspect of the present disclosure, a microfluidic device for analyzing extracellular vesicle glycans is provided. The microfluidic device comprises: an inlet portion, the inlet portion is configured to receive a fluid sample; a mixing portion, the mixing portion fluid is connected to the inlet portion and is configured to promote mixing between the fluid sample and magnetic nanoparticles, the magnetic nanoparticles are functionalized to bind to extracellular vesicles and aggregate into vesicle glycans in the fluid sample; a magnetic separation portion, the magnetic separation portion fluid is connected to the mixing portion and is configured to separate magnetic nanoparticle clusters from the fluid sample; and a magnetic sensor, the magnetic sensor is configured to measure the magnetic properties of the fluid sample after the fluid sample has passed through the magnetic separation portion.

[0006] In an embodiment, the magnetic sensor is a giant magnetoresistive sensor.

[0007] In an embodiment, the mixing portion comprises an array of micropillars.

[0008] In an embodiment, the magnetic separation portion comprises a serpentine channel covered with magnetic separation strips.

[0009] In an embodiment, the microfluidic device comprises the magnetic nanoparticles functionalized to bind to extracellular vesicles in the fluid sample provided in the inlet portion.

[0010] In embodiments, the microfluidic device comprises a lectin selected to bind to a target extracellular vesicle glycan in the fluid sample.

[0011] According to a second aspect of the present disclosure, a system for analyzing extracellular vesicle glycans in a fluid sample is provided, wherein the system comprises a plurality of microfluidic devices, wherein different lectins are provided in corresponding inlet portions of the plurality of microfluidic devices to bind to different corresponding target extracellular vesicle glycans in the fluid sample.

[0012] According to a third aspect of the present disclosure, there is provided a magnetic nanoparticle comprising a magnetic multicore covered in a polymer coating, wherein the polymer coating is functionalized to bind to an extracellular vesicle.

[0013] The polymer coating can be functionalized to bind to a plurality of extracellular vesicles. The polymer coating can be functionalized with polydopamine.

[0014] The polymer coating can be functionalized with recognition elements selected to bind to specific extracellular vesicles expressing the corresponding biomarker.

[0015] The magnetic nanoparticles can be configured to aggregate in the presence of a corresponding lectin after binding to extracellular vesicles carrying target glycans.

[0016] The diameter of the magnetic nanoparticles may be selected to correspond to the diameter of the target extracellular vesicles. The diameter of the magnetic nanoparticles may be in the range of 20 nm to 1000 nm.

[0017] According to a fourth aspect of the present disclosure, a method for analyzing extracellular vesicle glycans in a fluid sample portion. The method comprises the following steps: mixing the fluid sample with magnetic nanoparticles and lectins, the magnetic nanoparticles being functionalized to bind to extracellular vesicles in the fluid sample, the lectins being selected to bind to target extracellular vesicle glycans in the fluid sample portion; magnetically separating nanoparticle clusters, the nanoparticle clusters being formed by multivalent binding of the lectins and the target extracellular vesicle glycans; measuring the magnetic properties of the remaining fluid sample portion; and determining the characteristics of the extracellular vesicle glycans in the fluid sample based on the magnetic properties of the remaining fluid sample portion.

[0018] According to a fifth aspect of the present disclosure, a method for analyzing extracellular vesicle glycans in a fluid sample is provided. The method comprises the following steps: for a corresponding fluid sample portion of the fluid sample, performing a method for analyzing extracellular vesicle glycans in a fluid sample portion to determine the characteristics of each of a plurality of corresponding target extracellular vesicle glycans in the fluid sample according to the corresponding magnetic properties of the corresponding remaining fluid sample portion.

[0019] The identity of each of the corresponding target extracellular vesicle glycans in the fluid sample can be determined based on the corresponding magnetic properties of the corresponding remaining fluid sample portion, and includes: identifying at least one conserved glycan signature.

[0020] The conserved glycan signature is measured using lectin RCA120 and / or WGA.

[0021] According to a sixth aspect of the present disclosure, a method for distinguishing patient prognosis is provided, wherein the method comprises: analyzing extracellular vesicle glycans in a corresponding fluid sample.

[0022] The corresponding target extracellular vesicles and associated glycans can be selected to distinguish the patient prognosis of colorectal cancer and gastric cancer.

[0023] The magnetic nanoparticles can be functionalized with the antibody CD24 to bind to cancer extracellular vesicles.

[0024] The prognostic glycan signature may be measured using a lectin selected from the group consisting of Jacalin, ConA, RCA120, PHA-E, STA, LEL, WGA and DSL. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In the following, embodiments of the present invention will be described as non-limiting examples with reference to the accompanying drawings, in which:

[0026] Figure 1A Magnetic labeling of extracellular vesicles according to an embodiment of the present invention is illustrated;

[0027] Figure 1B illustrates the aggregation of magnetically labeled extracellular vesicles according to an embodiment of the present invention;

[0028] Figure 1C Magnetic measurement of a supernatant according to an embodiment of the present invention is illustrated;

[0029] Figure 2 An assay kit for performing extracellular vesicle glycan analysis according to an embodiment of the present invention is shown;

[0030] Figure 3A A scanning electron micrograph of a mixer of an assay cartridge according to an embodiment of the present invention is shown;

[0031] Figure 3B A photograph showing a giant magnetoresistive sensor of an assay cartridge according to an embodiment of the present invention;

[0032] Figure 4 A measurement kit for extracellular vesicle glycan analysis according to an embodiment of the present invention is shown;

[0033] Figure 5 shows a cross-sectional view of an embedded GMR sensor according to an embodiment of the present invention;

[0034] 6A to 6H Steps in a method of manufacturing a sensor for use in an embodiment of the present invention are shown;

[0035] FIG. 7A to FIG. 7D The analysis method of extracellular vesicle glycans according to an embodiment of the present invention is illustrated;

[0036] Fig. 8A This is a transmission electron micrograph of smaller Fe3O4 particles. Figure 8B is a transmission electron micrograph of a multi-core particle used in an embodiment of the present invention;

[0037] Fig.9A and Fig. 9B The GMR output signal and the GMR output signal change are shown for two different magnetic nanoparticles;

[0038] Fig. 9C A schematic diagram showing the functionalization of nanoparticles using polymers is shown;

[0039] Fig. 10A A comparison of EV labeling efficiency of different functional nanoparticles is shown;

[0040] Fig. 10B The results of the study on the kinetics of EV binding are shown;

[0041] Fig. 10C The results of the study show the specificity of magnetic aggregation;

[0042] Fig. 10D The results of the study showing the correlation between magnetic signals and EV glycan concentrations;

[0043] Fig.11 Shown are the results of studies using size-matched PMPs to measure EV-bound glycans; and

[0044] FIG. 12A to FIG. 12C Analysis of cancer extracellular vesicles to determine prognosis is shown. DETAILED DESCRIPTION

[0045] The present disclosure relates to magnetic analysis of extracellular vesicle glycans. The underlying platform of embodiments of the present invention integrates three functional steps: magnetic labeling of extracellular vesicles (EVs); aggregation of labeled EVs; and magnetic measurement. Reference will now be made to Figures 1A to 1C Describe these functional steps.

[0046] Figure 1A Magnetic labeling of extracellular vesicles according to an embodiment of the present invention is illustrated. EV 110 is magnetically labeled with magnetic nanoparticles 120. The size of the magnetic nanoparticles 120 matches that of the EV 110. The magnetic nanoparticles 120 include a magnetic multicore 122 formed of a plurality of magnetic particles and a functional shell 124. The functional shell 124 is coated on the magnetic nanoparticles 120 to improve binding to the EV 110. The functional shell 124 may include, for example, polydopamine or an antibody.

[0047] Figure 1B The aggregation of magnetically labeled extracellular vesicles according to an embodiment of the present invention is illustrated. Figure 1BAs shown, a targeted lectin 130 is added to promote the aggregation of labeled EVs. In the presence of specific glycan moieties 112 on extracellular vesicles 110, the addition of targeted lectin 130 causes multivalent binding and aggregation of EVs 110 and their associated magnetic nanoparticles 120. This aggregation is doubly selective for both EV biophysical properties and specific glycans. This forms aggregates 140 of EVs. Aggregation is only in response to specific glycans, not to free-floating glycosylated proteins. As more EV glycan-specific aggregates 140 are formed, they precipitate due to the external magnetic gradient 150 and are consumed from the solution to leave a supernatant.

[0048] Figure 1C Magnetic measurements of the supernatant according to an embodiment of the present invention are illustrated. The remaining magnetic nanoparticles 120 in the supernatant 160 are measured in real time using a giant magnetoresistance (GMR) sensor 170. The output of the GMR sensor 170 is coupled to a voltmeter 172. The GMR signal 180 can be directly related to the EV glycan profile. This can be used to analyze specific glycan moieties found on EVs.

[0049] To address the challenges mentioned in the background section, we believe that magnetic measurements have distinct advantages. First, magnetic nanomaterials can be size-controlled and multifunctional to improve interaction kinetics (i.e., solution-phase reactions). Second, magnetic measurements are fast and require minimal sample processing, as background sensitivity from native biological samples is negligible. Therefore, we developed an all-magnetic platform for direct analysis of EV glycans in native clinical biofluids. The technology utilizes rationally designed polycore magnetic particles (PMPs) to convert EV glycan features into magnetic signals. For specific signal conversion, the platform enables EV glycan-lectin interactions in solution phase to induce dual-selective aggregation of PMPs—which is specific for both EV biophysical properties and their glycan composition, but unresponsive to free-floating glycoproteins—resulting in in situ changes in magnetic signals. The resulting magnetic signal is quantified in real time by a built-in magnetoresistive sensor for direct analysis of EV glycans. When implemented on a miniaturized microfluidic platform, the technology enables rapid, wash-free, and multiplexed analysis of EV glycans in complex biofluids (<30 min, 1 μl native sample). We further applied the developed platform to examine primary clinical samples. This technology not only revealed the glycan signatures of cancer EVs in different biological contexts, but also enabled the differentiation of prognostic signatures of patients through direct EV glycan profiling.

[0050] Figure 2200 is a test kit for performing extracellular vesicle glycan analysis according to an embodiment of the present invention. The test kit 200 is configured to be received by a common measurement box. Figure 4 A measuring box is described.

[0051] like Figure 2 As shown, the assay cartridge includes four key compartments: an inlet 210 for sample loading and magnetic labeling, a mixer 220 including an array of micropillars 222 for improving local mixing and aggregate formation, a serpentine sorter 230 for increasing the contact time with an external magnetic field for depleting larger multinuclear magnetic particle (PMP) aggregates, and an embedded giant magnetoresistance (GMR) sensor 240 for direct measurement of the magnetic supernatant. The assay cartridge can be recyclable and can be reused for analysis of different samples.

[0052] The test box 200 is provided with grooves extending along each side to allow insertion into the measurement box. The test box 200 includes microfluidic channels that form an inlet 210, a mixer 220, a serpentine sorter 230, a sensing area overlapping with the GMR sensor 240, and an outlet 250. The serpentine sorter 230 includes a channel aligned with a magnetic separation strip on a common measurement box to consume larger PMP aggregates. The outlet 250 is provided with an outlet valve 252 that can be removed to allow the sample to flow from the inlet 210 through the components of the test box 200. A pair of electrodes 242 are provided on one of the grooves to allow electrical connection to the GMR sensor 240 when the test box 200 is inserted into the measurement box.

[0053] Standard soft lithography was used to fabricate the microchannels in the mixer and their associated microcolumns. We used SU-8 negative resist (SU8-2025, Microchem) to prepare the mold. The photoresist was spin-coated onto a silicon wafer at 2000 rpm for 30 seconds and baked at 65°C and 95°C for 2 minutes and 5 minutes, respectively. After UV light exposure, the resist was baked again and then developed under stirring. Before subsequent use, the developed mold was chemically treated with trichlorosilane vapor in a desiccator for 15 minutes. Polydimethylsiloxane polymer (PDMS) and cross-linking agent were mixed in a ratio of 10:1 and cast onto the SU-8 mold. After curing at 65°C for 4 hours, the PDMS layer was cut from the mold and assembled onto the GMR sensor.

[0054] Figure 3A A scanning electron micrograph of a mixer of an assay cartridge according to an embodiment of the present invention is shown. Figure 3A As shown, the mixer 220 includes a plurality of micro-pillars 222 to enhance lectin mixing and aggregation. The micro-pillars 222 are cylindrical. Figure 3A The scale bar in the figure represents 50 μm.

[0055] Figure 3B A photograph of a giant magnetoresistance sensor of an assay cartridge according to an embodiment of the present invention is shown. Figure 3B As shown, the GMR sensor 240 includes a plurality of parallel strips 244 connected to a wider connecting portion 246 . Figure 3B The scale bar in FIG. 2 represents 50 μm. The cross section of the GMR sensor 240 is shown in FIG. Figure 5 As shown. The GMR film used in our experiments has a bottom spin valve structure. The sensor chip is made by depositing a thin film of spin valve with a fixed magnetization setting in a selected direction on a 4-inch silicon wafer. Each chip sensor consists of 8 detection areas including 4 spin valve strips with an electrically active area of ​​500μm×5μm.

[0056] Figure 4 A measurement box for performing extracellular vesicle glycan analysis according to an embodiment of the present invention is shown. The measurement box 400 includes a plurality of slots 410 to accommodate the test box 200. The test box 200 can be cut into the slots 410 on the measurement box 400. The magnetic separation strip 420 extends along the measurement box 400 and overlaps with the serpentine sorter 230 of the test box 200. Each slot 410 is provided with a portion of an electrical contact 430, which is arranged to contact the electrode 242 of the GMR sensor 240 connected to the corresponding test box 200 to allow the measurement of the signal from the GMR sensor 240. For multiplexed measurements, four independent test boxes can be loaded into the measurement box, so that four different glycan assays can be performed in parallel. The measurement box not only aligns the external magnetic field with the serpentine sorter, but also provides electrical contacts for simultaneous GMR measurements, thereby simplifying the assay process.

[0057] Figure 5 FIG. 4 shows a cross-sectional view of an embedded GMR sensor according to an embodiment of the present invention. Figure 5 As shown, the GMR sensor 240 includes a 3 nm tantalum layer 502, which is laid on a 2 nm iron-nickel alloy layer 504. The 2 nm iron-nickel alloy layer 504 is laid on a 1.5 nm cobalt-iron alloy layer 506. Below the 1.5 nm cobalt-iron alloy layer 506 is a 2.3 nm copper layer 508. The 2.3 nm copper layer 508 is laid on a 2 nm cobalt-iron alloy layer 510. Below the 2 nm cobalt-iron alloy layer 510 is a 0.8 nm ruthenium layer 512. The 0.8 nm ruthenium layer 512 is laid on a 2 nm cobalt-iron alloy layer 514. Below the 2 nm cobalt-iron alloy layer 514 is an 8 nm iridium-manganese layer 516. The 8 nm iridium-manganese layer 516 is laid on a 2 nm iron-nickel alloy layer 518. Below the 2 nm iron-nickel alloy layer 518 is a 5 nm tantalum layer 520.

[0058] 6A to 6HSteps in a method of manufacturing a sensor for an embodiment of the present invention are shown.The sensor is fabricated on a 4-inch silicon (Si) wafer.

[0059] like Fig. 6A As shown, a GMR film 610 is deposited by thin film deposition with a fixed magnetization setting in a selected direction on a silicon wafer 600. After applying a photoresist 620, deep ultraviolet (DUV) lithography is performed to define the sensor stripes.

[0060] like Figure 6B As shown, the pattern is transferred to the wafer via deep reactive ion etching. This leaves a stripe pattern formed by the GMR film 610.

[0061] like Figure 6C As shown, after removing the electron beam (e-beam) resist, the lead pattern is covered on the spin valve sensor strips using optical lithography by applying photoresist 630.

[0062] like Fig.6D As shown, a thick copper (Cu) layer 640 (100 nm) is then deposited on the wafer by thermal evaporation.

[0063] like Fig. 6E As shown, the electrical leads and pads of the spin valve sensor are formed after photoresist stripping.

[0064] like Fig. 6F As shown, photolithography is performed to define the pad area by applying photoresist 660 .

[0065] like Figure 6G As shown, a thin SiO2 protective layer 670 (50 nm) is deposited on the sensor and leads by plasma-enhanced chemical vapor deposition (PECVD).

[0066] Finally, if Figure 6H As shown, the pad region 690 is exposed by stripping of the photoresist 660 .

[0067] FIG. 7A to FIG. 7D The method for analyzing extracellular vesicle glycans according to an embodiment of the present invention is illustrated. Figure 4 The measurement box 400 shown above refers to Figure 2 The method is performed using the described test kit 200.

[0068] like Fig. 7AAs shown, magnetic labeling was initially performed. Extracellular vesicles and magnetic nanoparticles were introduced into the inlet 210 of the assay cartridge 200. Lectins were then introduced into the mixture to induce EV glycan-specific aggregation. For on-chip analysis of EV glycans, the assay cartridge was mounted on a common measurement cartridge. In brief, in the open inlet of the assay cartridge, we mixed 1 μl of sample with 1 μl of PMP (1 mg / ml, functionalized with PDA for direct labeling or antibodies for clinical studies) and incubated the mixture for 5 minutes for magnetic labeling. 2 μl of BSA (2% in PBS) was then added to the mixture to block any nonspecific binding sites in situ. Subsequently, we added an excess of lectin (Vector Laboratories) to the mixture to promote EV glycan-specific aggregation and ensure good availability of lectin binding sites even in the presence of competing free-floating glycoproteins.

[0069] like Figure 7B As shown, the mixture flows through the mixer 220 of the test box 200, which includes a micro-pillar array to enhance multivalent lectin binding and aggregation. All fluid flows are actuated by syringe pumps (Harvard Instruments) that apply negative pressure at the common measurement box.

[0070] Then, if Figure 7C As shown, the mixture flows through the serpentine channel 230 of the test box 200. When the mixture flows through the serpentine channel 230, it is subjected to the magnetic field applied by the magnetic separation strip 420 of the measuring box 400. The magnetic field causes the larger aggregates to settle. The smaller magnetic particles are still suspended in the supernatant. The external magnetic field strength applied is 300mT and the duration is 5 seconds.

[0071] Then, if Fig.7D As shown, the magnetic content of the supernatant is measured in real time by the on-chip GMR sensor 240 and can be correlated to the EV glycan composition.

[0072] Specifically, the mixture was first introduced across an array of microfluidic pillars to enhance local mixing and aggregation, and then into a serpentine channel to effectively separate larger aggregates. We then measured the magnetic signals of the smaller PMPs remaining in the supernatant via an embedded GMR sensor.

[0073] To develop an assay platform, we evaluated functional PMPs coated with different polymers for magnetic labeling of EVs. Specifically, we prepared multi-core magnetic nanomaterials that can induce strong GMR signals.

[0074] Fig. 8A This is a transmission electron micrograph of smaller Fe3O4 particles. Figure 8Bis a transmission electron micrograph of a multi-core particle used in an embodiment of the present invention. Figure 8B As shown, the multi-core particles have a diameter in the range of 20 nm to 50 nm.

[0075] Fig.9A and Fig. 9B The GMR output signal and the GMR output signal change are shown for two different magnetic nanoparticles. The change in the GMR signal (ΔGMR) is determined as (V ef -V Fe / V ref ), where V ref is the inherent voltage signal output of the GMR sensor, V Fe It is the signal output at a certain Fe concentration.

[0076] Fig. 9C Schematic diagram of nanoparticle functionalization with polymers is shown. Multicore particles were grafted with dopamine (DA) to prepare PMP-PDA. For functionalization with other polymers, the multicores were modified with MATMS to introduce surface reactive double bonds before functionalization with 3-acrylamidophenylboronic acid (AAPBA), acrylic acid (AA), and polyethylene glycol methacrylate (PEGMA) to prepare PMP-P(AAPBA), PMP-PAA, and PMP-P(PEGMA), respectively.

[0077] To evaluate the labeling efficiency of EVs by these functional PMPs, we incubated equal amounts of each nanomaterial with EVs of known concentrations. After magnetic isolation of the PMPs, we measured the remaining EV counts as well as the molecular content in the supernatant to determine the corresponding labeling efficiency. Across both analyses, PDA-coated PMPs exhibited the highest labeling efficiency (>90%), even for EVs obtained from different cell sources and complex biological fluids (i.e., serum and urine). We attribute this high performance to the unique adhesion properties of PDA, which enables a variety of interactions (e.g., covalent and electrostatic) with different EV surface biomolecules. When evaluated at different EV concentrations, the PDA coating showed not only efficient labeling but also rapid binding. 7 Complete magnetic labeling was also achieved in just 2 min at 100 EVs / ml. Therefore, we selected PMPs coated with PDA as the optimal nanomaterial for subsequent assay development.

[0078] Fig. 10AComparison of EV labeling efficiency of different functional nanoparticles is shown. PMPs were grafted with different polymers to study their interaction with EVs. All materials were prepared to achieve comparable hydrodynamic diameters. Equal amounts of the respective PMPs were incubated with known concentrations of EVs obtained from human renal cancer cells (A498). PMPs with PDA coating showed the highest labeling efficiency and were used for subsequent assay development.

[0079] Since EVs are multivalent targets with multiple surface biomolecules, we investigated the role of PMP size in inducing nanoparticle aggregation. We prepared PMPs of different sizes and measured the aggregation of these PMPs when treated with equal concentrations of EVs and the lectin RCA120.

[0080] Fig. 10B Figure 2 shows the results of a study on the kinetics of EV binding. We prepared various concentrations of EVs (obtained from A498 cells; high, 10 9 / ml; medium, 10 8 / ml; low, 10 7 The labeling efficiency was determined by counting vesicles in the supernatant.

[0081] Fig. 10C The results of the study on the specificity of magnetic aggregation are shown. We measured the average hydrodynamic diameter of PMP under different experimental conditions (+, presence; -, absence). PMP only aggregates in the presence of both EV (obtained from A498 cells) and lectin (RCA120, which binds to galactose-associated carbohydrates common in EV). This aggregation can be competitively inhibited by adding galactose. In the absence of EV or targeted lectin, PMP remains dispersed, just like untreated particles.

[0082] Fig. 10D Results of a study showing the correlation of magnetic signals with EV glycan concentrations. When treated with increasing amounts of EV glycans, PMPs aggregated more extensively, as shown by the increase in the hydrodynamic diameter of the clusters (top). These magnetic aggregates were consumed by an external magnetic field, resulting in a decrease in the amount of residual PMPs in the supernatant (middle). We measured the magnetic signal of the supernatant using a GMR sensor. The GMR signal is displayed as a heat map and is positively correlated with the amount of EV glycans (bottom). All measurements were performed three times and the data are shown as mean ± standard deviation.

[0083] Since EVs are multivalent targets with multiple surface biomolecules, we investigated the role of PMP size in inducing nanoparticle aggregation. We prepared PMPs of different sizes and measured the aggregation of these PMPs when treated against equal concentrations of EVs and the lectin RCA120.

[0084] Fig.11 Results of studies of size-matched PMPs for measuring EV-bound glycans are shown. We prepared PMPs of different sizes, each coated with PDA, and incubated the nanomaterials with equal concentrations of EVs (A498 cells) in the presence of the lectin RCA120. The diameter of the aggregates was normalized relative to the diameter of the untreated PMPs. The 190-nm PMPs showed the highest signal.

[0085] FIG. 12A to FIG. 12C Analysis of cancer extracellular vesicles to determine prognosis is shown.

[0086] Fig. 12A A schematic diagram of the analysis is shown. A biological fluid (e.g., urine, serum, ascites) 1210 is incubated with PMPs functionalized with antibodies 1220 to target specific cancer antigens 1230. This enhances magnetic measurements of putative cancer EVs using PMPs. Without further washing, the sample is treated with lectins 1240 to achieve EV glycan-specific aggregation 1250 and analysis.

[0087] Fig. 12B Analysis of simulated clinical specimens is shown. Samples were prepared by spiking EVs from renal cancer (A498) and brain cancer (GLI36) into vesicle-depleted urine and serum, respectively. Results labeled as iMAGE were obtained using methods and devices according to embodiments of the invention. EVs were magnetically labeled and measured using PMPs functionalized with anti-CD63 antibodies. In both samples, the platform showed good correlation with the glycan signature of pure cancer EVs (R2>0.85). All signals were normalized to the corresponding pure EV measurements. For comparison, Fig. 12BResults from enzyme-linked immunosorbent assay (ELISA) are shown. EVs were adsorbed to ELISA plates (Thermo Scientific) and blocked in PBS containing 1% BSA for ELISA measurements. After washing, biotinylated antibodies (anti-CD63, 1 μg / ml, BD Biosciences) or biotinylated lectins (2 μg / ml, Vector Laboratories) were added to the blocking solution and incubated at room temperature for 2 hours. After incubation with FITC-conjugated streptavidin (BD Biosciences), the fluorescence signal was measured (Tecan).

[0088] Fig. 12C Analysis of 21 lectin markers in clinical cancer ascites is shown (n=11). Patient samples were analyzed via anti-CD24 functionalized PMPs. Analysis allowed the clinical samples to be divided into two distinct groups by hierarchical clustering of patient specimens (rows) and their lectin signals (columns). This EV glycan-based classification was highly consistent with independent clinical assessment of patient prognosis. Specifically, lectin markers could be classified as poor prognostic markers (P<0.0001, two-way ANOVA) or non-discriminating markers. All measurements in (BC) were performed three times with a sample-matched control group and data are shown as mean values. au arbitrary units.

[0089] For clinical analysis, ascites samples were used directly. To be able to selectively measure glycan features on cancer EVs, we incubated ascites samples with PMPs functionalized with anti-CD24 antibodies and then introduced different lectins to induce glycan-specific aggregation. For all measurements, we included a lectin-free control group matched to the patient samples. Analyses were performed relative to this control group to account for nonspecific aggregation from sample matching. Clinical assessments of patient characteristics were determined independently. Specifically, patient prognosis was determined by overall survival from the time of ascites collection. Patients were considered to have a good prognosis when the overall survival was more than ten months. Conversely, patients were determined to have a poor prognosis if the overall survival was less than five months. All measurements were performed without knowledge of these clinical assessments.

[0090] The scientific applications of embodiments of the present invention are potentially broad. With its sensitive and specific detection, we anticipate that the platform can be easily extended to measure other EV surface markers, including established and new EV modifications (e.g., ubiquitination). Its solution-phase magnetic actuation further improves assay performance and simplifies probe selection (e.g., low-affinity binders) to identify weak but real interactions, and can be easily integrated with other technologies to facilitate the development of molecular assays. Such studies will not only improve the characterization of biomarkers, but also confer new application potential. As demonstrated in the present disclosure, the identification of conserved EV glycan features can make it possible to develop new glycan-based methods for EV enrichment that is ubiquitous in complex biofluids.

[0091] Although the foregoing description has described exemplary embodiments, those skilled in the art will appreciate that many changes are possible to the embodiments within the scope and spirit of the invention.

Claims

1. A magnetic nanoparticle comprising a magnetic multicore covered in a polymer coating, wherein the polymer coating is functionalized to bind to an extracellular vesicle.

2. The magnetic nanoparticle according to claim 1, wherein The polymer coating is functionalized to bind to a plurality of extracellular vesicles.

3. The magnetic nanoparticle according to claim 2, wherein The polymer coating is functionalized with polydopamine.

4. The magnetic nanoparticle according to claim 1, wherein The polymer coating is functionalized with recognition elements selected to bind to specific extracellular vesicles expressing the corresponding biomarker. 5 . The magnetic nanoparticle according to claim 1 , wherein the magnetic nanoparticle is configured to aggregate in the presence of a corresponding lectin after binding to an extracellular vesicle carrying a target glycan.

6. The magnetic nanoparticle according to any one of claims 1 to 5, the diameter of the magnetic nanoparticle being selected to correspond to the diameter of the target extracellular vesicle.

7. The magnetic nanoparticle according to any one of claims 1 to 5, having a diameter in the range of 20 nm to 1000 nm.

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