Integrated disc-fluidics for high-throughput sample processing

Abstract

The present invention relates to an integrated centrifugal device and method for analyte processing, particularly for the separation, enrichment, labeling, and analysis of nanovesicles such as extracellular vesicles (EVs). The device leverages a compact, rotatable substrate to execute multi-step assay protocols that may include chromatographic separation, centripetal fluid transport, immuno-capture, and protein labeling, all under low-speed centrifugal conditions. The device architecture enables automated, hands-free operation without requiring ultracentrifugation or specialized personnel. Fluidic modules including sedimentation, chromatography, displacement, and multiple assay chambers may be functionally integrated through a series of valves, channels, and immiscible liquid-based displacement mechanisms, facilitating precise sample handling, minimal loss, and multiplexed analysis.

Description

[0001] INTEGRATED DISC-FLUIDICS FOR HIGH-THROUGHPUT SAMPLE PROCESSING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 668,818, filed on July 9, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] BACKGROUND OF THE INVENTION

[0005] The assessment of circulating biomarkers (liquid biopsy) is a powerful approach to gaining molecular insights into patients’ tumors through repeated yet minimally invasive sampling. One target for such analyses is extracellular vesicles (EVs), membrane-bound particles released by cells. These vesicles carry biomolecules (e.g., proteins, nucleic acids, and lipids) reflective of parent cells, thereby serving as surrogate biomarkers. Analyzing tumor-derived EVs thus can offer a valuable window into the dynamic status of tumors, potentially improving cancer diagnostics and treatment monitoring. Indeed, research has shown that EV analyses can achieve high accuracy in cancer diagnostics, including early-stage diseases, identify tumor origins based on the molecular signature, and inform therapeutic responses earlier than clinical imaging.

[0006] Despite the promising reports on EVs' potential as biomarkers, few EV-based tests have been translated into clinical practice. Challenges exist in the extensive and often manual nature of sample preparation required for EV analyses. EVs exist within complex, heterogeneous matrices; EV purification is essential for accurate downstream assays. This need is particularly pronounced with blood, the most used specimen type. Blood plasma contains lipoprotein particles (LPPs) that are similar to EVs in size and density but are more abundant (>10 million times). Removing LPPs and thereby enriching EVs is recognized as essential for improving the accuracy of downstream analyses and validating EV biomarkers. However, isolating EVs is technically demanding, frequently involving rounds of ultracentrifugation or chromatography steps. A separate workup then follows to prepare the isolated EVs for analytical measurements, extending the overall workflow and potential for yield losses. Innovative microfluidic devices have shown promise in facilitating such preparations, but their adoption for routine uses has been limited due to operational complexity and low throughput. In short, for EV diagnostics to realize their clinical potential, there remains a compelling need to develop new platforms that address technical challenges in sample handling: high-purity EV isolation, high- throughput processing, and automation.

[0007] SUMMARY OF THE INVENTION

[0008] We have developed a device with on-disc integrated capabilities for sample processing.

[0009] In one aspect, the invention provides a device including a rotatable substrate having an inlet in fluid communication with a first channel via a first valve; a chromatography chamber, optionally including chromatography separation media, that is in fluid communication with the first channel; an eluant chamber in fluid communication with the chromatography chamber via a second valve; first and second eluate chambers in fluid communication with the chromatography chamber; and a displacement chamber in fluid communication with the second eluate chamber via a third valve, wherein the displacement chamber is located centrally relative to the second eluate chamber. In some embodiments, the device further includes a sedimentation channel in fluid communication with the inlet and having a proximal end located centrally relative to a distal end, wherein the first channel intersects the sedimentation channel between the proximal and distal ends. In some embodiments, the sedimentation channel is slanted at an angle of at least 10° relative to a centripetal force generated by rotating the device, e.g., about 60°. In some embodiments, the device includes a sample chamber between the inlet and the sedimentation channel.

[0010] In some embodiments, the chromatography separation media includes a size-exclusion stationary phase and / or an ion-exchange stationary phase, e.g., cation exchange stationary phase. In some embodiments, the chromatography separation media includes a size-exclusion stationary phase disclosed centrally relative to an ion-exchange stationary phase, e.g., cation exchange stationary phase.

[0011] In some embodiments, the displacement chamber includes a liquid immiscible with water that has a density greater than water, e.g., greater than or equal to 1 .3 g / cm3.

[0012] In some embodiments, the device further includes a third eluate chamber in fluid communication with the chromatography chamber, wherein a fourth valve is disposed between the second and third eluate chambers and a fifth valve is disposed between the second and first eluate chamber, wherein the first eluate chamber is disposed azimuthally further from the chromatography chamber, the third eluate chamber is disposed azimuthally closest to the chromatography chamber, and the second eluate chamber is disposed azimuthally between the first and third eluate chambers.

[0013] In some embodiments, the device further includes a second channel in fluid communication with the displacement chamber via a sixth valve; an immobilization chamber including a solid support in fluid communication with the second channel; a first assay chamber in fluid communication with the immobilization chamber via a seventh valve; and a waste chamber in fluid communication with the immobilization chamber via a liquid permeable barrier. In some embodiments, the device further includes one more additional assay chambers, e.g., a second, third, fourth, fifth, and sixth assay chambers, in fluid communication with the immobilization chamber via eighth, ninth, tenth, eleventh, and twelfth valves.

[0014] In some embodiments, the device further includes a third channel in fluid communication with the immobilization chamber via a thirteenth valve; and first and optional second labeling chambers in fluid communication with the third channel via fourteenth and optional fifteenth valves. In some embodiments, the device includes one or more additional labeling chambers, e.g., a third, fourth, fifth, sixth, seventh, and eighth labeling chambers in fluid communication with the third channel via sixteenth, seventeenth, eighteenth, nineteenth, and twentieth valves. In some embodiments, the fourteenth to twentieth valves, if present, are capillary valves.

[0015] In another aspect, the invention provides a method for sample processing by providing a device as described herein; rotating the device to transport a portion of a sample into the chromatography chamber; opening the second valve and rotating the device to transport an eluant in the eluant chamber into the chromatography chamber, wherein eluate flows into the first and second eluate chambers to provide first and second eluates; and opening the third valve and rotating the device to transport a liquid immiscible with water that has a density greater than water from the displacement chamber into the second eluate chamber thereby displacing the second eluate into the displacement chamber.

[0016] In another aspect, the invention provides a method for sample processing by providing a device as described herein, rotating the device to transport a sample including particulate components and liquid, e.g., whole blood, into the sedimentation channel, wherein particulate components of the sample sediment towards the distal end leaving liquid at the proximal end; opening the first valve and rotating the device to transport the liquid into the chromatography chamber; opening the second valve and rotating the device to transport an eluant in the eluant chamber into the chromatography chamber, wherein eluate flows into the first and second eluate chambers to provide first and second eluates; opening the third valve and rotating the device to transport a liquid immiscible with water that has a density greater than water from the displacement chamber into the second eluate chamber thereby displacing the second eluate into the displacement chamber; opening the sixth valve and rotating the device to transport the second eluate into the immobilization chamber, wherein particulate components, e.g., EVs, in the second eluate bind to the solid support; opening the seventh valve and rotating the device to transport a labeling reagent from the first assay chamber to the immobilization chamber, wherein the labeling reagent binds to the particulate components, EVs; and opening the thirteenth valve and rotating the device to transport first and second aliquots of the solid support into the first and second labeling chambers.

[0017] In some embodiments, the method further includes oscillating after opening the sixth valve and rotating the device to transport the second eluate into the immobilization chamber and before opening the seventh valve and rotating the device to transport a labeling reagent from the first assay chamber to the immobilization chamber, and / or after opening the seventh valve and rotating the device to transport a labeling reagent from the first assay chamber to the immobilization chamber and before opening the thirteenth valve and rotating the device to transport first and second aliquots of the solid support into the first and second labeling chambers. In some embodiments, the first and second labeling chambers contain first and second antibodies. In some embodiments, the method includes oscillating the device after opening the thirteenth valve and rotating the device to transport first and second aliquots of the solid support into the first and second labeling chambers.

[0018] In some embodiments, the method further includes quantifying the number of particulate components in the first or second aliquot. In some embodiments, the method includes analyzing protein expression in the first or second aliquot.

[0019] The invention also provides the above methods performed with the omission of the sedimentation step, e.g., on a device not including a sedimentation channel and / or performed with the capture of soluble components rather than particulate components.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0022] Definitions

[0023] The term “about,” as used herein, refers to + / - 10% of a value. The term “azimuthally,” as used herein refers to a relationship between elements where the elements are spaced along an arc within the substrate.

[0024] The term “centrally,” as used herein refers to a direction closer to the center of rotation of the device.

[0025] The term “fluidically connected,” as used herein, refers to a direct connection between at least two device elements, e.g., a channel, chamber, etc., that allows for liquid to move between such device elements without passing through an intervening element.

[0026] The term “in fluid communication with” or “in fluid communication,” as used herein, refers to a connection between at least two device elements, e.g., a channel, chamber, etc., that allows for fluid to move between such device elements with or without passing through one or more intervening device elements. For the purposes of the invention, an element that is “in fluid communication with” another element “via a valve” refers to a fluid connection controlled by the state of the valve. Thus, one element separated from a second element by a valve is in fluid communication with the second element via the valve whether the valve is opened or closed.

[0027] The term “radially,” as used herein refers to a direction closer to the periphery of the device.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0030] FIGs. 1(a) - 1(d) present a schematic overview of a SpinEx approach. FIG. 1(a) shows that SpinEx streamlines the sample processing for EV analyses. It takes the whole blood as an input, separates plasma, and enriches EVs. Subsequently, enriched EVs are captured on microbeads and fluorescently labeled. All procedures are performed on a single device. HDL, high-density lipoprotein; (V)LDL, (very) low-density lipoprotein; HA, heterophilic antibody; RBC, red blood cell; WBC, white blood cell; StAv, streptavidin; Abs, antibodies. FIG. 1(b) shows a schematic of a SpinEx disc. An integrated centrifugal device was designed to perform SpinEx processing steps: plasma separation from the whole blood (4 min), EV enrichment (4 min), EV capture on beads (20 min), and EV protein labeling (40 min). A single disc contained two processing units, each capable of labeling EVs for eight different protein markers. FIG. 1(c) shows a photograph of a SpinEx disc prototype. A compact disc-sized device (diameter of 11 .4 cm) is comprised of machined plastic layers bonded together through a thin (35 pm) adhesive layer. The disc incorporated various functional elements such as chromatography columns, diaphragm valves activated by rotation (torque-activated), and membranes. FIG 1(d) shows a SpinEx disc operation within a dedicated desktop spinner system. This system precisely controls the disc's rotation parameters (angle, speed, duration) and allows for the real-time monitoring of the disc's operation through a camera.

[0031] FIGs. 2(a) - 2(d) show SpinEx operation for EV enrichment from whole blood. FIG. 2(a) illustrates the sequential steps involved, while the bottom row shows corresponding photographs of a disc section during actual operation. The disc was preloaded with a buffer, chromatography column resins, and mineral oil (density, 1 .6 g / mL). i) A whole blood sample is loaded into the disc through an inlet, ii) Spinning the disc (1800 rpm) separated plasma from the whole blood within 4 min. The isolated plasma is then transferred to the on-disc chromatography column, iii) Plasma and an elution buffer flew through the chromatography column while the disc spun at a lower speed (600 rpm, 4 min). This step enriched EVs into a designated elution fraction (blue shade), iv) The enriched EVs were moved to the inner region of the disc and transferred to an adjacent chamber at the same radial position for protein labeling. FIG. 2(b) shows fluidic configuration to centripetal EV transfer. A dense oil was positioned at a smaller radial distance than an aqueous solution. Upon disc rotation, the oil displaced the aqueous solution toward the disc center (centripetal transport). FIG. 2(c) shows computational simulations revealed a rapid (<2 sec) fluidic exchange upon disc rotation (1800 rpm). FIG. 2(d) an experimental validation. Colored aqueous buffer was loaded in the outer chamber ((£)) and then centripetally transferred to the inner chamber (@). Optical density measurements showed no significant difference (P = 0.06, two-sided paired t-test) before and after the transfer. Data are displayed as mean ± s.d. from technical triplicates.

[0032] FIGs. 3(a) - 3(g) show characterization of EV enrichment by SpinEx. FIG. 3(a) shows plasma separation from whole blood. Two-channel configurations were compared: a straight channel (0 = 0°) and a slanted channel (0 = 60°) angled relative to the centrifugal force. The height ( / ?) of the separated plasma layer was monitored as the disc spun at 1800 rpm. The slanted channel achieved faster plasma separation, reaching a stable height (plateau) within 4 min. Data are displayed as mean ± s.d. from technical triplicates. The dotted lines indicate the calculated / l values based on a theoretical model. FIG. 3(b) shows on-disc chromatography for EV enrichment. The column performed a two-step enrichment process. The size exclusion captured particles (e.g., HDLs, proteins) that are smaller than EVs; the ion exchange preferentially retained (V)LDLs that are less negatively charged than EVs. The eluting fluid progressively moved through the collection chambers, with the earlier elute eventually filling a chamber farther away from the column outlet. FIG. 3(c) shows fluidic simulations indicated that particles exiting the column initially enter the chamber closest to the outlet and then flow into neighboring chambers. Accounting for this flow pattern, the disc operation was optimized to collect the EV-fraction (F2) in the middle chamber. The far-side chamber received the initial buffer elute (F1 ), and the chamber closest to the column outlet was filled with the late elution (F3) containing (V)LDLs. FIG. 3(d) shows use of SpinEx to enrich EVs from human plasma samples spiked with cancer cell-derived EVs. Transmission electron microscopy revealed that EVs were enriched in the F2 fraction (shown here), compared to the F1 and F3 fractions (see FIG. 15). The red arrow points to a representative EV. FIG 3(e) shows single-particle imaging corroborated EV 26 enrichment in the F2 fraction. Samples were labeled with fluorescent antibodies against representative markers: ApoAl (HDL), CD63 (EV), and ApoB100 (LDL). Images for F1 and F3 fractions are shown in FIG. 15. FIG. 3(f) shows particles positive for ApoAl, CD63, or ApoB100 were counted in single-particle fluorescent images (field of view, 58 x 58 / jm2). The number of CD63- positive particles was the highest in the F2 fraction, while ApoB100-positive particles were abundant in the later fraction (F3). Data are displayed as mean ± s.d. from technical duplicates. FIG. 3(g) shows three methods of EV isolation were compared: conventional column-based size exclusion chromatography (SEC), disc with SEC resin only (disc-SEC), and SpinEx. The amounts of human serum albumin, ApoB100, ApoAl, and CD63 were quantified before and after the separation operations. SpinEx showed the highest depletion of (V)LDLs. HDL and albumin depletion, as well as EV recovery, were similar between the three methods. Data are displayed as mean ± s.d. from technical duplicates. FIGs. 4(a) - 4(e) show a design of EV-capture and labeling module. FIG. 4(a) shows a schematic of the EV-processing section in a disc. This segment contained reagents for EV biotinylation (TFP-biotin), capture (microbeads), protein targeting (1 ° Ab), and fluorescent tagging (StAv, 2° Ab). The output was eight EV aliquots, each stained for a different target protein marker. TFP, 2,3,5,6-tetrafluorophenyl; 1 ° Ab, primary antibody; 2° Ab, secondary antibody. FIG. 4(b) shows an EV labeling workflow. Enriched EVs were transferred to the bead reservoir, wherein EVs were captured onto microbeads via physisorption and subsequently biotinylated. The EV-bead complexes were then dispensed into eight labeling compartments. In each compartment, EVs were stained with two fluorescent labels: one for the total EV content (StAv-dye) and the other for a target protein marker (1 ° Ab and 2° Ab-dye). FIG. 4(c) shows the metering design to distribute equal volumes of the EV-bead solution into the labeling compartments. Initially, the solution was loaded into metering wells, which were gated by capillary valves. A brief disc spinning (600 rpm, 30 sec) opened these valves, allowing the metered solution to be injected into the labeling compartments. After the transfer, the disc was placed in a rocking motion to disperse beads and enhance their mixing with reagents. FIG. 4(d) shows photographs of a disc section were taken during the EV-bead transfer process. To aid in visualization, beads were suspended in a blue-colored buffer, while the labeling compartments were filled with a green-colored buffer. FIG. 4(e) shows eight compartments in a SpinEx disc were filled with alternating red-colored and clear buffer solutions. After SpinEx operation (disc spinning, rocking), color intensities were compared with the original buffer intensities. No significant differences were observed among buffers of the same color type (one-way ANOVA). Data are displayed as mean ± s.d. from technical duplicates.

[0033] FIGs. 5(a) - 5(e) show validation of EV capture and labeling. FIG. 5(a) shows that scanning electron microscopy revealed that EVs were successfully captured on the surface of the microbeads via adsorption. FIG. 5(b) shows the EV-bead complexes were labeled for total EV loading (using StAv- AF647) and for the expression of the EpCAM biomarker (using an anti-EpCAM antibody and a secondary antibody with AF488). Confocal microscopy confirmed the dual staining of the captured EVs. FIG. 5(c) shows the stability of EV capturing. EVs were captured on microbeads via adsorption and fluorescently labeled with a CD63 antibody. The EV-bead complexes were then subjected to multiple centrifugal washing steps, followed by detection via flow cytometry. For a given EV concentration, no intensity ( / CD63) changes (P > 0.05, ANOVA) were observed between samples subjected to different wash steps. Data are displayed as mean ± s.d. (n = 4). FIG. 5(d) shows the comparison of EV capturing. (Left) The adsorption approach captured a higher number of EVs compared to using antibody-conjugated beads. (Right) This higher EV loading on the beads resulted in a stronger fluorescent signal for the target protein. Median fluorescence intensity ( / ) was determined from flow cytometry measurements of EV-bead complexes (n = 1000). Data are displayed as mean ± s.d. from technical triplicates. FIG. 5(e) shows fluorescent measurements of control and CD63-targeted EV samples. Controls included a bead-only sample and a control-IgG labeled sample. No intensity ( / CD63) differences were observed between an IgG control and a no-EV sample (P = 0.96, non-paired two-sided t-test). The dotted red line indicates mean + 3 x s.e.m. from the IgG control data. Data are displayed as mean ± s.e.m. from 5000 individual beads, a.u. arbitrary unit.

[0034] FIGs. 6(a) -6(d) show EV protein profiling. SpinEx was used to prepare EVs for protein detection via flow cytometry (FCM). FIG. 6(a) shows examples of scatter plots and histograms from FCM measurements. Each dot reports the EV loading (fetAv) and the marker expression ( / M) per bead. The fetAv histograms (top panel) indicated that overall EV capture was consistent across different samples. The marker histograms (right panel) revealed varying expressions of target protein markers, a.u., arbitrary unit. FIG. 6(b) shows EV detection sensitivity. Samples containing different amounts of EVs were processed by SpinEx, and fetAv values were measured via FCM. For comparison, conventional enzyme- linked immunosorbent assay (ELISA) was performed on biotinylated EV samples. The detection limit was 8.4 x 105 EVs / mL for SpinEx-FCM, which was over 100-fold lower than that of ELISA (1.5 x 108 EVs / mL). Data are displayed as mean ± s.d. from technical duplicates. FIG. 6(c) shows FCM measured two intensities, / StAv and / CD9, from individual beads (inset). From these data, two analytical metrics for marker (CD9) expression were compared: the intensity ratio of individual beads [fcD9 = median(fcD9 / fetAv)] and the bulk intensity ratio [= median(fcD9) / median(zstAv)]. The CD9 metric effectively normalized for variations in EV loading per bead, achieving higher precision than the bulk ratio. Data are displayed as mean ± s.d. from technical quintuplicates. FIG. 6(d) shows a SpinEx-FCM profiling results showed a strong linear correlation (Pearson coefficient, r= 0.93) with those from ELISA. The grey area denotes the 95% confidence band. For ELISA, the same amount of EVs (109 EVs / mL) was used for each marker. EVs were isolated from SW480 and CaOV3 cell lines. Both SpinEx-FCM and ELISA data are from technical duplicates and displayed as mean ± s.d.

[0035] FIGs. 7(a) - 7(h) show an application of SpinEx for cancer detection and classification. FIG. 7(a) shows an overall workflow. SpinEx was used to isolate EVs and label them for a panel of protein markers. The labeled EVs were subjected to high-throughput flow cytometry, and the results were utilized for cancer diagnostics and classification. Plasma samples were collected from patients with confirmed primary tumors in five cancer types: breast (n = 30), lung (n = 32), liver (n = 27), pancreas (n = 31 ), and colon (n = 37). Non-cancer control samples (n = 64) were obtained from healthy donors. FIG. 7(b) shows the marker selection. The algorithm identified tumor mRNA targets from the Human Protein Atlas and filtered them to select proteins likely present on the EV surface, using UniProt and Gene Ontology data. The list was further curated based on a literature review and the availability of antibodies. The final set had 30 protein markers. FIG. 7(c) shows a cancer diagnosis model. A least absolute shrinkage and selection operator (LASSO) regression identified the most relevant input markers, and a support vector machine (SVM) classifier was trained on these selected markers to generate a cancer risk score. FIG. 7(d) shows the diagnosis model was trained on a dataset of 154 patients (left), and its performance was evaluated on an independent test set of 67 patients (right). A threshold of 0.5 was used for classification (dotted line). FIG. 7(e) shows receiver operating characteristic curves were generated based on the cancer risk score. The area under the curve (AUC) values were 0.985 for the training set and 0.974 for the test set. These two values were statistically not different (P = 0.40, Delong's test). FIG. 7(f) shows the classification of tumor types using EV profiling data from cancer patients (n =157). The input markers were chosen by a multinomial logistic regression (with an L1 penalty). The classification SVM model employed a one-versus-one comparison strategy and generated probability scores for each of the five tumor types. FIG. 7(g) shows the multi-dimensional outputs (probability scores) that were visualized by applying t-distributed stochastic neighbor embedding (t-SNE). This dimensionality reduction revealed distinct clusters for different tumor types. FIG. 7(h) shows the model achieved a high overall classification accuracy of 0.96 and a macro F1 score of 0.96. The predicted tumor type corresponded to the class with the highest probability score.

[0036] FIG. 8 shows SpinEx operation steps.

[0037] (T) A whole blood (150 L) sample is loaded.

[0038] @ Plasma is separated from the whole blood.

[0039] @ Plasma is transferred to an on-disc chromatography column, and an elution buffer is introduced.

[0040] © A dense oil pushes the enriched EVs toward the inner side for further processing.

[0041] @ EVs are transferred to an assay chamber containing polystyrene beads. EVs bind to the bead surface via physisorption.

[0042] @ EV-bead complexes are washed by introducing a buffer.

[0043] @ TFP-biotin reagent is introduced to the assay chamber, and EVs are biotinylated. TFP, 2, 3, 5, 6- tetrafluorophenyl.

[0044] ® Excess biotinylation reagent is removed via washing.

[0045] © A block solution (10% Superblock) is introduced to the assay chamber.

[0046] ® The blocking solution is removed with another washing step.

[0047] @ EV-bead complexes are dispensed into eight labeling compartments, each containing a primary antibody (1 ° Ab) against a target protein.

[0048] © Dye-conjugated streptavidin and secondary antibody (2° Ab) are introduced to the compartments. The labeled EV-bead complexes are ready for fluorescent measurements (e.g., microscopy, flow cytometry).

[0049] FIG. 9 shows a SpinEx processing unit in detail. Each disc integrated two identical SpinEx processing units, allowing for parallel processing of samples. A processing unit consisted of 24 main chambers, connecting channels, and thirteen reversible valves. Key functional areas are the following: Q plasma separation unit; Q & 0 on-disc chromatography column; 0 oil column for EV transfer; 0 beadbased EV labeling section containing six reagent chambers, a waste chamber; and 0 eight labeling compartments pre-loaded with primary antibodies to protein targets. A single disc allows for the analysis of 16 markers. TFP, 2,3,5,6-tetrafluorophenyl.

[0050] FIGs. 10(a) -10(b) show spinning setup for SpinEx. FIG. 10(a) shows a photograph of the spinning hardware. The cradle and motor housing were fabricated using a 3D printer. The system incorporated a microcontroller that communicates with an external computer for user input. Based on these inputs, the microcontroller regulated the motor speed and spinning time. FIG. 10(b) shows the system acquired top-down images of the disc during a SpinEx operation, enabling real-time process monitoring.

[0051] FIG. 11 shows sample transfer kinetics. The simulation results from FIG. 11 were used to estimate the fraction of the aqueous solution transferred toward the disc center. The transfer was completed (100%) within 2 sec.

[0052] FIG. 12 shows plasma separation in straight and slanted channels. Time-lapse photographs of a SpinEx disc were taken during the plasma separation process, comparing the performance in straight (0°) and tilted (60°) channels. Rapid plasma separation was achieved in the slanted channel configuration. FIGs. 13(a) - 13(b) show elution patterns from a dual-mode chromatography column device. FIG. 13(a) shows that cell culture EV samples were subjected to dual-mode chromatography (size and charge). EV detection occurred across a broad range of elution fractions, including later fractions. Data are presented as mean ± s.d. from technical duplicates. FIG. 13(b) shows a plasma (0.5 mL) elution profile obtained through dual-mode chromatography. EV fractions exhibited overlap with later eluting LDL fractions. To mitigate LDL contamination, a restricted selection of elution fractions (shaded in yellow) is required. Mean values are displayed from technical replicates (n = 4). Data are adapted from our previous report.

[0053] FIG. 14 shows sequential filling of the elution chambers during an on-disc chromatography process. Three buffer solutions, each in a different color (red, blue, and clear), were sequentially introduced to the on-disc chromatography column: the red-colored buffer first, followed by the bluecolored buffer, and then the clear buffer lastly. The disc was spun at 600 rpm. (T) Red buffer was injected. @ The early elute (red buffer) filled the elution chambers (F3, F2) at the column outlet. @ Blue buffer was injected. © As the blue buffer exited the column, it filled the F3 chamber, while the previously eluted red buffer was transferred to the neighboring F2 and F1 chambers. @ Blue buffer was injected. @ As the blue buffer exited the column, it filled the F3 chamber, while the previously eluted red buffer was transferred to the neighboring F2 and F1 chambers. @ A clear buffer was injected. ® As the clear buffer exited the column, it started pushing the blue buffer present in the F3 chamber. The blue buffer was transferred to the neighboring F2 and F1 chambers. The previous red buffer was removed through a vent hole.

[0054] FIG. 15 shows characterization of SpinEx eluates. The three elution fractions (F1 , F2, F3) were analyzed. The top row shows images from transmission electron microscopy (TEM). The bottom rows show immunofluorescence images for specific markers: ApoAl (HDLs), CD63 (EVs), and ApoB100 (LDLs). The early elute (F1 fraction) contained few particles, as observed in the TEM image. In contrast, the late elute (F3 fraction) was predominantly composed of ApoB100-positive particles, indicating the presence of LDLs. Under TEM, the F2 fraction was observed to be enriched with EV-like particles, exhibiting EVs' characteristic morphology and size range. The immunofluorescence imaging revealed the enrichment of CD63-positive particles in the F2 fraction, corroborating the presence of EVs in this fraction.

[0055] FIGs. 16(a) - 16(b) show Western blotting analysis on the EV fraction. Human plasma from a colorectal cancer patient was processed to enrich EVs. FIG. 16(a) shows a high expression of established EV markers (CD63 and Alix) was observed, whereas the expression of a non-EV marker (histone H2B) was negligible. The whole blot image is shown. L, ladder; S, sample. FIG. 16(b) shows that protein signal intensity was normalized to that of Alix. ND, not detected.

[0056] FIG. 17 shows a transmission electron micrograph of a plasma sample processed by sizeexclusion chromatography (SEC). Human plasma was spiked with EVs isolated from a cancer cell line (SW620). The sample was then subjected to SEC processing to enrich EVs. A large amount of lipoproteins remained after the SEC processing step.

[0057] FIG. 18 shows unbiased-EV isolation. The amount of tetraspanins (CD63, CD9, CD81 ) was analyzed via ELISA before (purple-shaded) and after dual-mode chromatography (DMC). The input sample volume was varied to modulate the total EV load. Among pre- and post-processed samples, the relative composition of the tetraspannin amounts was maintained.

[0058] FIG. 19 shows the mixing mode operation. A rocking mode was implemented to enhance fluidic mixing. This mode involved the following sequence: ±120° oscillation / sec for 30 sec, followed by 50 rpm rotation for 5 min. (Left) The photograph was taken immediately after injecting microbead solutions (blue) into the eight compartments containing green-colored buffer. Initially, the beads appeared clumped within the compartments. (Right) After the rocking mode was executed twice, the beads were dispersed within the green buffer. The side view of the compartments further confirmed the effective mixing of the beads within the fluid.

[0059] FIG. 20 shows spontaneous protein adsorption to hydrophobic surfaces. (Left) Water molecules exhibit restricted configurations near a hydrophobic surface due to the absence of hydrogen bonding with the surface. Proteins may also possess hydrophobic pockets. (Right) Protein adsorption releases these restricted water molecules into the bulk aqueous solution, decreasing the system's overall free energy (G). This process is spontaneous and entropy-driven. The resulting state (protein adsorbed) is thermodynamically stable under the given buffer conditions.

[0060] FIG. 21 shows EV capture on microbeads using SpinEx. Scanning electron microscopy confirmed EV capture on the bead surface. The adsorption method captured more EVs than the CD9 antibody-based method. Microbeads coated with control IgG antibodies showed negligible EV binding.

[0061] FIG. 22 shows dual fluorescent staining of EVs. SpinEx was used to process a human plasma sample. EVs were captured on beads and biotinylated. The captured EVs were stained with two fluorescent probes: streptavidin-phycoerythrin (StAv-PE) and anti-CD63 antibody conjugated with fluorescein isothiocyanate (CD63-FITC). An imaging flow cytometer (ImageStreamX Mkll; Amnis Corporation) was used to acquire images of the same bead in brightfield, DAPI (4',6-diamidino-2- phenylindole), PE, and FITC channels.

[0062] FIGs. 23(a) - 23(b) show EV capture conditions. FIG. 23(a) shows that EVs were captured on carboxylated polystyrene microbeads under varying pH buffer conditions. Captured EVs were fluorescently labeled with a CD63 antibody for detection via flow cytometry. EV capture efficiency progressively increased under more acidic conditions. Data are displayed as mean ± s.d. from technical triplicates. FIG. 23(b) shows that EVs became less negatively charged at lower pH, which presumably enhanced their interaction with carboxyl (COO-)- functionalized microbeads. The green shade indicates the optimal buffer pH to maximize the charge difference between EVs and LDLs. Data are displayed as mean ± s.d. (n = 2). a.u. arbitrary unit. Data in panel (b) were adapted from our previous reportl .

[0063] FIG. 24 shows an example of optimizing bead size for EV detection in one embodiment. Biotinylated EVs were adsorbed onto polystyrene beads with different diameters (0.8, 2.5, and 5 pm). Captured EVs were labeled with fluorescent streptavidin (StAv) and detected by a flow cytometer. The median fluorescent intensity (fetAv) from 1000 beads was used as an analytical metric. Using the 5-pm beads led to the highest sensitivity with the detection limit of 8.4 x 105 EVs / mL. The detection limits with smaller beads were 4.8 x 107 EVs / mL (2.5-pm bead) and 2.4 x 108 EVs / mL (0.8-pm bead). Data from technical duplicates are displayed as mean ± s.d. FIG. 25 shows a comparison of analytical metrics. Two different metrics were defined: the intensity ratio of individual beads [£M= median( / M / stAv)] and the bulk intensity ratio [= median( / ivi) / median( / stAv)]. The coefficient of variation (CV) values for each metric were calculated from quintuplicate (n = 5) measurements. For a given bead count (Afe), the metric consistently had lower CV values than the bulk ratio. The dotted lines indicate the power law fit, CV ~ Ne~a. The obtained a values were 0.48 (green line) and 0.55 (blue line), which aligned with the theoretical expectation of a = 0.5.

[0064] FIGs. 26(a) - 26(c) shows cancer marker selection. FIG. 26(a) shows the marker selection pipeline. The algorithm accessed the Human Protein Atlas database and retrieved mRNA targets (n = 2311 ) that were differentially upregulated in different tumor types. This initial collection was then reduced to 233 proteins for their known presence in EVs based on information from the UniProt and the Gene Ontology databases. The list was further narrowed down by applying the following selection criteria: the marker's published use in cancer detection and the commercial availability of antibodies. The final set comprised 30 protein markers (Table 3). FIG. 26(b) shows a heatmap of mRNA targets (n = 2311 ) retrieved from the Human Protein Atlas. The data-driven approach selected 16 highly expressed markers (indicated on the left margin of the heatmap) with minimal or no overlap in expression across other cancer types. For each mRNA target, its expression across tumor types was converted into z-scores from FPKM (fragments per kilobase of transcript per million mapped reads) values. FIG. 26(c) shows the gene ontology analysis showing that i) the selected 30 markers were strongly associated with cancer pathways and ii) their presence was in the extracellular region.

[0065] FIGs. 27(a) - 27(b) show control samples in Spin Ex. FIG. 27(a) shows bead-antibody controls. Microbeads were treated with a blocking buffer (Superblock™) and subsequently incubated with a primary antibody (Ab) and a fluorescent secondary antibody (2° AbFL). These controls exhibited uniformly low baseline signals, with median intensities below 0.46 x 103(a.u.) across all samples. Data representing 5000 beads are displayed for each marker, a.u., arbitrary unit. FIG. 27(a) shows EV labeling controls. (Left) Preparation of a sample pair. For a given marker, EVs were captured and labeled with a target-specific antibody, followed by a fluorescent secondary antibody (targeted sample). A corresponding control sample used an isotype-matched control IgG instead of a primary antibody. In both samples, EVs were biotinylated and labeled with fluorescent streptavidin (StAvpi) to quantify EV loading per bead. (Right) The expression level (f) was determined for each sample type by calculating the median of the per-bead intensity ratio {IM / / stAv, M = IgG or on-target antibody) distribution. Then, the difference A£M = - ^igG was used as an analytical metric for the marker expression. Data from CD63 profiling were shown as an illustrative example (5000 beads measured per sample). Dashed vertical lines indicate median values.

[0066] FIGs. 28(a) - 28(c) show a comparison between whole blood and plasma samples. FIG. 28(a) shows that whole blood samples were spiked with EVs from EpCAM-positive SW620 cancer cells. The samples were divided and subjected to two different methods for plasma separation: SpinEx and standard centrifugation (2,000 x g, 20 min). Each plasma sample then underwent EV isolation, EV capture on microbeads, and fluorescent labeling for EV loading (streptavidin; StAv) and target markers (M). Signal intensities ( / stAv, / M) were measured via flow cytometry. FIG. 28(b) shows that no statistically significant differences {P > 0.05; all paired, two-sided t-tests) were observed between the two plasma types for overall EV capture ( / stAv), marker expressions ( / CD63, / EPCAM), and control signals ( / igc). Data are displayed as mean ± s.d. from technical quadruplicates. FIG. 28(c) shows that from the normalized marker expressions, [= median( / M / / stAv)] and ^IgG [= median(llgG I IStAv)], net marker levels were calculated as A^M = ^igc (M = CD63, EpCAM). These levels exhibited no significant differences (P> 0.05, paired, two-sided t-tests) between the two plasma types. Data are displayed as mean ± s.d. from technical quadruplicates.

[0067] FIG. 29 shows a quality check of clinical EV assays. A strong correlation (Pearson coefficient, r = 0.86) between SpinEx-FCM and ELISA results was observed, validating a SpinEx-FCM assay. Both SpinEx-FCM and ELISA data are displayed as mean ± s.d. from technical duplicates.

[0068] FIGs. 30(a) - 30(b) show a workflow for machine learning analyses. FIG. 30(a) shows constructing a model for cancer diagnosis. The LASSO regression determined the most informative marker sets from the training set (n = 154; left). The selected markers were used to train an SVM model for binary classification (cancer versus non-cancer). This trained model was evaluated using an independent test set (n = 67; right). FIG. 30(b) shows the model for five-cancer classification. Data from all cancer patients (n = 157) were used. The model performed a one-versus-one classification to differentiate between five tumor types.

[0069] FIG. 31 shows early-stage cancer detection. A diagnostic model for early cancer was constructed using EV profiling data from the following groups: early-stage (I & II) cancer patients and non-cancer controls. The model used a combination of LASSO (least absolute shrinkage and selection operator) and SVM (support vector machine), similar to the overall cancer detection model. The training set comprised 74 early-stage cancer patients and 45 non-cancer controls; the independent test set comprised 33 early- stage cancer patients and 19 non-cancer controls. From the receiver operating characteristic analysis, the area under the curve (AUC) values were 0.983 for the training set and 0.953 for the test set. These two AUC values were statistically non-different (P= 0.112, Delong’s test).

[0070] FIG. 32 shows intravesicular marker detection. Biotinylated EVs, captured on polystyrene beads, were fixed and then permeabilized to allow antibodies to access the interior of the EVs. Subsequently, EVs were immunofluorescently labeled for an intravesicular protein marker, Alix. The fluorescent intensity (lAlix) of the beads was measured by flow cytometry. The signal increased in an EV-dose-dependent manner. Control samples that were not permeabilized displayed negligible signals. Data are shown as mean ± s.d. from technical duplicates.

[0071] FIG. 33 shows flow cytometry gating strategy. Gates for EV-bound beads were determined using no-EV bead controls. Microbeads were incubated with fluorescent streptavidin (StAv-PE) and markerspecific antibodies, followed by fluorescent secondary antibodies (AF488). Thresholds were set at the 95th percentile of the StAv-PE (EVs) and AF488 (marker) signal distributions.

[0072] FIG. 34 shows calibration curves for human serum albumin, ApoAl, ApoB100, and CD63. These curves were generated using purified protein standards as target analytes for ELISA.

[0073] FIG. 35 shows a schematic of a device of the invention.

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention relates to an integrated centrifugal device and method for analyte processing, particularly for the separation, enrichment, labeling, and analysis of nanovesicles such as extracellular vesicles (EVs). The device leverages a compact, rotatable substrate to execute multi-step assay protocols that may include chromatographic separation, centripetal fluid transport, immunocapture, and protein labeling, all under low-speed centrifugal conditions. The device architecture enables automated, hands-free operation without requiring ultracentrifugation or specialized personnel. Fluidic modules including sedimentation, chromatography, displacement, and multiple assay chambers may be functionally integrated through a series of valves, channels, and immiscible liquid-based displacement mechanisms, facilitating precise sample handling, minimal loss, and multiplexed analysis.

[0076] Devices

[0077] Devices of the invention include a rotatable substrate. The substrate may be planar and circular in shape. Substrates may also include nonplanar portions and / or other shapes, so long as the substate can be rotated at sufficient speed. Any suitable material may be employed including glass, quartz, silicon, metals, e.g., aluminum, brass, or stainless steel, ceramics, and polymers, e.g., polycarbonate, poly(methyl methacrylate), polystyrene, cyclic olefin copolymer, cyclic olefin polymer, and combinations thereof. In some embodiments, the substrate is at least partially transparent to visible light. Manufacturing techniques are well known in the art, such as CNC milling, molding, e.g., injection molding, photolithography, etching, etc. The substrate may include a hole, mating plate, or other element for connection to a spindle or other rotating part of a device that rotates the substrate.

[0078] The substrate includes various chambers and channels. Chambers are generally wider than channels and have sufficient volume to contain a desired volume of liquid or suspension for one or more operations. Chambers may generally have dimensions of widths: 4-50 mm, lengths: 2-40 mm, and heights: 1 -4 mm. Channels may generally have dimensions of widths: 0.1 -1 mm, e.g., about 1 mm, lengths: 2-55 mm, and heights: 0.01 to 1 mm, e.g., about 0.035 mm.

[0079] As described herein, the substrate may be modular, where two or more segments are joined to create a device. Each segment may include channels and / or chambers to carry out various tasks as described herein. Alternatively, two segments may include channels and / or chambers to carry out the same task on different samples or sample aliquots.

[0080] In one embodiment, the substrate includes an inlet, in fluid communication with a first channel via a first valve, an eluant chamber in fluid communication with the chromatography chamber via a second valve, first and second eluate chambers in fluid communication with the chromatography chamber, a displacement chamber in fluid communication with the second eluate chamber via a third valve, wherein the displacement chamber is located centrally relative to the second eluate chamber. Such a device may be employed to enrich desired components, e.g., EVs, relative to undesired components, e.g., LDLs and HDLs, and to move an enriched aliquot centrally, e.g., for removal or further manipulation or analysis on the device. Additional, e.g., third, fourth, etc., eluate chambers may be arranged azimuthally. The azimuthal position of additional eluate chamber(s) may be arranged so that the desired aliquot is delivered to the second eluate chamber. Additional eluant chambers, e.g., second, third, etc., may also be in fluid communication with the chromatography chamber. In certain embodiments, the substrate further includes a sedimentation channel in fluid communication with the inlet and having a proximal end located centrally relative to a distal end, wherein the first channel intersects the sedimentation channel between the proximal and distal ends. In some embodiments, the substrate further includes a sample chamber between the inlet and the sedimentation channel. The elements in this paragraph may be disposed in a single segment of the device.

[0081] In one embodiment, the device includes a second channel in fluid communication with the displacement chamber via a sixth valve; an immobilization chamber containing a solid support in fluid communication with the second channel; a first assay chamber in fluid communication with the immobilization chamber via a seventh valve; and a waste chamber in fluid communication with the immobilization chamber via a liquid permeable barrier. The device may include one or more additional assay chambers, e.g., second, third, fourth, fifth, sixth, etc. Assays chambers may be arranged such that a more centrally located assay chamber dispenses liquid into a more radially located assay chamber. The elements in this paragraph may be disposed in a single segment of the device or in a segment including the elements of the preceding paragraph.

[0082] In one embodiment, the device includes a third channel in fluid communication with the immobilization chamber via a thirteenth valve; and first and second labeling chambers in fluid communication with the third channel via fourteenth and fifteenth valves. The device may include additional labeling chamber, e.g., third, fourth, fifth, sixth, seventh, eighth, etc., e.g., in fluid communication with the third channel by a corresponding valve. Valves leading to the labeling chambers may be capillary valves that allow passage of liquids or suspensions when the device is rotated above of threshold speed. The elements in this paragraph may be disposed in a single segment of the device or in a segment including the elements of the preceding paragraph or with the elements of the preceding two paragraphs.

[0083] An exemplary device having a sedimentation channel, first through sixth assay chambers, an immobilization chamber, and first through eighth labeling chambers is shown in Fig. 35. The locations of valves are indicated by number in triangles. Valves for the labeling chambers are capillary valves.

[0084] Devices may also include additional inlets, e.g., for loading fluids into assay or labeling chambers or for loading solid materials in the chromatography or immobilization chambers. The device may also include outlets, e.g., for removal of liquids or suspensions after one or more operations, e.g., from a labeling chamber. Devices may also include vents as necessary.

[0085] The chromatography chamber may include one or more chromatography separation media. Exemplary media include that for affinity, adsorption, size exclusion (SEC), ion exchange, and reversed-phase chromatography. In some embodiments, the chromatography chamber includes two types of media, e.g., SEC medium and ion exchange medium, e.g., cation exchange medium. The cation exchange medium may be located radially relative to the SEC medium. The SEC may be selected to enrich particles below a selected size, e.g., about 40 nm. Such media are known in the art and may include beads or particles. Exemplary ion-exchange resins include, but are not limited to, strong cation exchange resins with sulfonate or sulfoisobutyl functional groups on a polymethacrylate matrix, agarose-based strong cation exchange resins with sulfopropyl groups, and methacrylate-based strong cation exchange resins, e.g., Fractogel®. Exemplary size-exclusion resins include, but are not limited to, porous polymer beads such as cross-linked agarose resins, cross-linked dextran, crosslinked polyacrylamide; and hydrophilic surface-modified silica. Representative volumes of media, e.g., ion-exchange resin or size-exclusion resin, within the chromatography chamber include, but are not limited to, about 0.05 mL to 0.5 mL, or about 0.1 mL to 0.5 mL, or about 0.25 mL to 0.5 mL.

[0086] Suitable eluants are known in the art and are selected based on the type of chromatography and the materials being separated. Exemplary eluants include, but are not limited to, physiological buffers such as phosphate-buffered saline (PBS) and HEPES-buffered saline. Other eluants include Tris-HCI, sodium phosphate, citrate buffers, and acetate buffers.

[0087] The displacement chamber may include a displacement liquid, e.g., one immiscible with water and having a density greater than water. An exemplary density is greater than or equal to 1 .3 g / cm3, e.g., about 1 .6 g / cm3. Exemplary liquids immiscible with water include hydrocarbons and halogenated solvents. In some embodiments the immiscible liquid is an oil (e.g., fluorinated hydrocarbon), dichloromethane, methylene chloride, chloroform, carbon tetrachloride. In some embodiments the immiscible liquid is bromoform or phenylamine. The volume of displacement liquid will typically exceed the volume of the second eluate chamber, e.g., by a factor of 1 .25, 1 .5, 2, 3, or more. An excess volume of displacement fluid can be used to transport the eluate further centrally.

[0088] A sedimentation channel may intersect the first channel at a point where undesired particulate matter has sedimented radially to the intersection, e.g., to allow for transport of the desired liquid phase to be transported into the first channel. The location of the intersection may therefore be selected based on the nature and volume of the sample, e.g., whole blood. In some embodiments, the sedimentation channel is oriented at an angle with respect to a centripetal force generated by rotating the device. The angle is selected to enhance sedimentation efficiency, and the device may further include one or more structural features configured to enhance sedimentation efficiency within the sedimentation channel. Exemplary angles are greater than or equal to 10°, e.g., about 10°, or about 20°, or about 30°, or about 40°, or about 50°, about 60°, or about 70°.

[0089] The immobilization chamber may include a solid support for capturing a molecule or particle of interest and / or for capturing an undesired molecular or particle. Exemplary solid supports include beads, membranes, and fibers. Molecules or particles may be captured by the solid support by any mechanism, e.g., affinity, charge, or physical adsorption. Suitable solid support materials include, but are not limited to, polystyrene, polymethacrylate, agarose, dextran, silica, magnetic materials, and affinity agents, such as biotin, avidin / streptavidin, and antibodies. Solid supports conjugated with antibodies to target tetraspanins (CD9, CD63, or CD81 ) may be employed in affinity capture. Other exemplary tetraspanins to target include, but are not limited to, CD37, CD53, CD82, CD151 , or any one of TSPANs 1 -23, e.g., TSPAN7, TSPAN8, TSPAN10, TSPAN20, TSPAN21 , TSPAN22, TSPAN23, TSPAN24, TSPAN25, TSPAN26, TSPAN27, TSPAN28, TSPAN29, TSPAN30, TSPAN31 , TSPAN32, and TSPAN33.

[0090] Assay chambers may include any liquids for use in an operation. Suitable liquids may include labeling reagents, blocking agents, wash liquids, buffers, eluants, etc. Exemplary assay labeling reagents include, but are not limited to, enzyme labels that conjugate to antibodies, fluorescent labels, chemiluminescent labels, and affinity labels such as biotin and / or avidin / streptavidin. Exemplary wash liquids include, but are not limited to, PBS, TBS, and imidazole-buffered saline. Wash liquids may also include detergents, e.g., non-ionic detergents such as polysorbate 20, and salts, such as sodium chloride and potassium chloride. Blocking agents may be employed to reduce non-specific binding, examples of which include Superblock®, non-fat dry milk in PBS or TBS, bovine serum albumin in PBS or TBS, casein, and blocking detergents (e.g., polysorbate 20).

[0091] The physical barrier in the immobilization chamber may be any barrier sufficient to retain a solid support in the immobilization chamber. Examples include a liquid permeable membrane or posts or other physical objects disposed with a spacing to retain the solid support.

[0092] Labeling chambers may include a labeling reagent. Exemplary labeling reagents include, but are not limited to, enzyme labels, fluorescent labels, chemiluminescent labels, and affinity labels such as antibodies and biotin and / or avidin / streptavidin. Primary and secondary antibodies may be employed as labels. The present disclosure may employ biotin-streptavidin interactions for extracellular vesicle labelling. In some embodiments, other high-affinity ligand-binding systems may be utilized, including, but not limited to, using a short peptide tag (Strep-tag II) with a high affinity for strep-tactin (Strep-tag@ / Strep-Tactin®); a protein peptide tag (FLAG) that is recognized by a highly-specific anti- FLAG monoclonal antibody (FLAG-tag / anti-FLAG); HA-tag / anti-HA; antibody; GST-tag / glutathione.

[0093] The device may include various valves. Suitable valves for controlling liquid flow are known in the art. Valves may be mechanical, e.g., electrically or manually controlled. Capillary valves may also be employed. Electrically controlled mechanical vales, or active valves requiring external actuation, may include, but are not limited to, electromagnetic, piezoelectric, electrostatic, and thermopneumatic components that activate upon an electrical signal activating an actuator. Manually controlled vales requiring physical manipulation by a user include, but are not limited to, push-pin valves, screw valves, and clamp valves. Capillary valves, or passive valves, do not require any moving mechanical parts or external actuation. Examples of capillary valves that may be employed include, but are not limited to, hydrophobic barriers, expansion / contraction valves, and a siphon inverted “U” shaped valve.

[0094] Systems of the invention may include a device of the invention and liquid reagents, e.g., as discussed herein.

[0095] Methods

[0096] Devices of the invention may be employed to enrich a component of a sample, such as particles or molecules in the sample, e.g., EVs. Exemplary samples include biological fluids (such as whole blood, plasma, serum, lymphatic fluid, saliva, cerebrospinal fluid (CSF), ascites, amniotic fluid, sputum, urine, semen, vaginal fluid, perspiration, tears, mucus, bile, breast milk, intracellular fluid, synovial fluid, amniotic fluid, pleural fluid, interstitial fluid, pericardial fluid, exudates, pus and fluid secretions from wounds, and vomit), liquids cultures, and environmental samples, e.g., originating from surface water, ground water, drinking water, waste water, storm runoff, rainwater samples, and leachate. Devices may also be employed to perform operations on the enriched components, e.g., labeling, further purification, aliquoting, or quantifying. The devices transport liquids by rotating the device. The device may also be oscillated back and forth for mixing or incubation within a chamber. The speed and duration of rotation or oscillation can be determined by one skilled in the art according to the sample and liquids involved. Exemplary protocols are provided herein.

[0097] The device of the invention may enrich a component of a sample by loading a sample, such as whole blood, into the device, e.g., in a sample chamber. The sample is transported to the chromatography chamber by rotating the device. In some embodiments, the device includes a sedimentation channel, and rotating the device transports a sample into the sedimentation channel. Additional rotation may be used to sediment particulate components of the sample towards the distal end leaving liquid at the proximal end. For example, cells and larger particles in whole blood can be sedimented from plasma. Further rotation may then transport the liquid from the sedimentation channel into the chromatography channel. Once loaded onto chromatography separation media, the device may be rotated to transport eluant from the eluant chamber to the chromatography chamber. Eluate is then transported to one or more eluate chambers. Further rotation transports a displacement fluid into the eluate chamber with the desired eluate. The eluate is displaced by the displacement fluid and transported to the displacement chamber, located centrally on the device.

[0098] In some embodiments, the device further includes an immobilization chamber and one or more assay chambers. The eluate can be moved to the immobilization chamber by further rotation. Components of the eluate bind to solid support in the immobilization chamber. One or more liquids may be transported from one or more assay chambers to the immobilization chamber. Such liquids may be transported sequentially, or two liquids may be combined in an assay chamber prior to being transported to the assay chamber. In some embodiments, an assay chamber contains a labeling reagent that is transported to the immobilization chamber to bind to a component, e.g., particle or molecule, in the sample. Once operations in the immobilization chamber are complete, the component can be released from the solid support, and the device may be rotated to transport liquid to a labeling chamber. Multiple aliquots from the immobilization chamber may be transported to multiple labeling chambers. Additional reagents may be in the labeling chamber or added from as an assay chamber.

[0099] For any of these steps, chambers may be separated from each other by valves. Thus, after a particular operation, a valve may be opened or closed to transport liquid to a desired location. Exemplary valving schemes are provided herein.

[0100] In one embodiment, the methods are employed to enrich EVs, e.g., relative to LDLs and / or HDLs. This method may include separating plasma from cells in a sedimentation channel, performing SEC and cation exchange chromatography in a chromatography chamber and separating eluates into eluate chambers, displacing the desired eluate centrally into a displacement chamber, and binding EVs to a solid support in an immobilization chamber. The EVs can then be biotinylated, and the solid support cab be blocked with a blocking buffer. Aliquots can then be transported to labeling chambers that contain primary antibodies, e.g., a different antibody or combination of antibodies in each chamber. Labeled streptavidin and secondary antibodies can then be delivered to each labeling chamber.

[0101] EXAMPLES

[0102] Below we described a centrifugal disc platform, termed SpinEx (Separation-processing / ntegration for Extracellular vesicles), for comprehensive EV-sample processing. SpinEx incorporates several innovative designs: i) on-disc chromatography for EV purification, ii) liquid transfer toward a disc center (i.e., centripetal transfer) to maximize system integration inside a centrifugal device, and iii) unbiased, label-free EV capture on microspheres. Integrating these features empowered SpinEx to seamlessly perform a series of preparatory processes: separating plasma from whole blood, enriching EVs, and immunolabeling them for downstream assays. Our SpinEx prototype proved the concept by enriching EVs from whole blood (150 pL) within eight minutes while removing >96% of the LPP population. SpinEx further processed EVs to be ready for protein assays. It allocated enriched EVs and fluorescently labeled them for target proteins, with a single disc enabling the detection of sixteen protein markers. Notably, all operations were carried out within a single disc, offering a consistent and robust sample preparation. In a pilot clinical study, we applied SpinEx to process clinical plasma samples (n = 221 ) for multi-marker profiling, aimed at detecting and differentiating five common cancers: breast, lung, liver, pancreas, and colon. SpinEx significantly accelerated EV characterization, isolating and labeling EVs from these samples for the analysis of 30 protein markers. The profiling data led to high accuracies in cancer diagnostics (90% with an independent test set of 67 samples) and cancer-type classification (96%, n = 157).

[0103] Results

[0104] SpinEx design

[0105] FIG. 1(a) - FIG. 1(d) shows an overview of a SpinEx system. We designed a disc to execute the entire EV preparation within a single device (FIG. 1(a)): i) separating plasma from the whole blood, ii) enriching EVs by removing lipoprotein particles and soluble proteins, iii) capturing EVs on pre-loaded microbeads and subsequently biotinylating the captured EVs, and iv) labeling the EVs with detection probes (fluorescent streptavidin for EV quantification and fluorescent antibodies for the target protein). In a prototype disc, we laid out two assay units (FIG. 1(b)), each integrating functional blocks corresponding to the assay steps. These units took the whole blood sample (150 L per unit), isolated EVs, and distributed them into eight separate chambers, with each chamber's EVs labeled for a distinct protein marker (see FIG. 8(f)).

[0106] We fabricated a SpinEx disc in plastics (FIG. 1(c); see Methods for details). We first defined a fluidic structure on polycarbonate sheets via computer-numerical-control (CNC) milling, making plates for a top cover (thickness, 1 .6 mm) and a bottom chamber (thickness, 5 mm). It is worth noting that this patterning process can also be achieved by injection moldings for large-scale production. After patterning, we incorporated other components (e.g., membrane filters and chromatography resins) and bonded the top and bottom plates using a thin (35 pm), pressure-sensitive adhesive sheet. Torque-activated valves were then installed on the top plate. The completed disc had a diameter of 11 .4 cm. Individual assay units had 24 chambers and 13 valves (FIG. 9).

[0107] To operate a SpinEx disc, we custom-designed a desktop spinning system (FIGs. 1(d) and FIG. 10(a)). Inside a safety housing, the system contained a cradle and a magnetic retainer to mount a disc, a direct-current motor for disc spinning, and a digital camera for process monitoring. A microcontroller within the system i) regulated the motor speed using feedback from a rotary encoder, controlled a light source and the camera to capture disc images during SpinEx operation (FIG. 10(b), and iii) communicated with an external computer to receive user inputs (e.g., spinning schedules).

[0108] On-disc EV enrichment from the whole blood A SpinEx assay started with isolating EVs from the whole blood, which entailed the separation of plasma through centrifugation, on-disc chromatography, and the preparation of EVs for protein labeling. FIG. 2(a) illustrates the operational schematic (top row) and the snapshot (bottom row) of a SpinEx disc during this process (see Table 1 for detailed operation parameters). For plasma separation, we designed an input chamber (150 L) linked to a narrow channel (FIG. 2(a), i). Spinning the disc would sediment cellular components, leaving plasma in the channel nearer to the disc center (FIG. 2a, ii). Notably, we laid out the channel inclined from the sedimentation force direction; this arrangement is known to generate a convective flow that enhances cell settling. Subsequently, the isolated plasma (50 L) was introduced to an on-disc chromatography column (see Methods for material details) along with an elution buffer (300 pL; FIG. 2(a), iii), which concentrated EVs in a defined elution fraction by removing abundant LPPs.

[0109] The collected EVs were then moved from the disc periphery to its central position (FIG. 2(a), iv). This centripetal transfer was crucial to further manipulating EVs within the centrifugal fluidics; its implementation, however, was difficult as the EV fraction had to move against the centrifugal force. We solved this challenge by exploiting a density-based phase separation between immiscible fluids. Specifically, we preloaded a dense oil (1 .6 g / mL) in a reservoir located radially above the EV elution chamber (FIG. 2(b)). Upon opening the reservoir and spinning the disc, the oil flowed radially to displace the less than EV fraction towards the disc center. EVs were then transferred to an adjacent chamber at the same radial position for subsequent assay steps. This compact fluidic design facilitated integration with other functional modules on the disc.

[0110] We have further characterized the density-based transport mechanism. Computational fluidic simulations demonstrated an efficient fluidic exchange (FIG. 2(c)), with >99% of the EV solution transferred to the upper chamber within two seconds of rotation (FIG. 11). These simulation results corroborated experimental observations, where fluidic transport was completed within 5 sec (1800 rpm). Using a colored aqueous buffer, we also measured its optical signal before and after the centripetal transfer. No significant signal changes were observed, indicating no concentration loss during the sample transfer (FIG. 2(d)).

[0111] Validation of EV isolation. We confirmed the operation of each EV isolation component. Firstly, the inclined channel indeed separated plasma faster than a straight channel (FIG. 3a). The PNK theory could explain this enhancement. In the inclined channel, the height / ? of the plasma fraction would increase over time (t) as h = c-[1 - exp(-u-t / w-sin0)], where c is the final plasma height, w is the channel width, u is the sedimentation speed in a straight channel, and 9 is the tilt angle from the radial direction (W.D. Hill, 1977). Conversely, plasma separation in a straight channel would progress linearly with spinning time (h = u-t). To test these models, we prepared a disc containing both inclined (0 = 60°) and straight (0 = 0°) channels and compared their performance (FIG. 3(a) and FIG. 12). Within the straight channel, the plasma height exhibited linear growth over time (FIG. 3(a), green dotted line), with the measured sedimentation speed u = 0.014 mm / sec (at 1800 rpm). Using this value and c = 10.5 mm from experiments, we estimated h in the inclined channel. The expected h closely matched the experimental observation (FIG. 3a, red dotted line). Importantly, the inclined channel supported about 5.6 times higher sedimentation rate (d / ? / dt|r = o = 0.078 mm / sec) than the straight channel, completing plasma separation within 4 minutes (at 1800 rpm).

[0112] For EV isolation, we adopted our dual-mode chromatography (DMC) approach, which uses two types of resins (FIG. 3(b)). The upper section of the column housed a size-exclusion resin, which delayed the flow of particles below a specified size cutoff (~ 40 nm). The lower section contained an ion-exchange resin with an affinity to positively charged particles. As plasma traversed the column, soluble proteins and HDLs were captured in the size exclusion segment; (V)LDLs, possessing lower negative charges than EVs, were retained in the ion exchange region. Consequently, EVs emerged from the column earlier than other particles (FIG. 13(a) - 13(b)).

[0113] In contrast to the column device, the on-disc DMC exhibited a distinct elution profile. The early chromatography eluate was recovered in the chamber (F1 ), farthest from the chromatography outlet, whereas the latest eluate was collected in the chamber (F3), positioned next to the outlet. Fluidic 6 simulations revealed a cascading liquid transfer during disc rotation, explaining this phenomenon (FIG. 3c). The eluate initially filled chamber F3 at the column exit and then sequentially transferred toward the next chambers (F2 and F1 ). To validate this simulated flow pattern, we conducted experiments using colored buffer solutions (FIG. 14). A red-colored buffer solution was injected into the column, and the disc was spun. The process was then repeated using a blue-colored buffer. As predicted, the red-colored buffer initially filled chamber F3 and then sequentially moved to chambers F2 and F1 . These findings guided us to fine-tune the disc operation to maximize EV isolation. Specifically, we designated the second chamber (F2) for collecting EVs eluted from the column. The farthest chamber (F1 ) was used to collect the initial elution buffer, while the closest chamber (F3) was designated for collecting late elution fractions potentially containing (V)LDLs.

[0114] The optimized protocol effectively enriched EVs in the F2 fraction, as qualitatively confirmed by electron microscopy (FIG. 3(d) and FIG. 15). We further analyzed elution fractions through immunofluorescent microscopy (FIG. 3(e) and FIG. 15), staining them for representative markers for EVs (CD63), HDLs (ApoAl), and LDLs (ApoBl OO). The F2 fraction exhibited a high abundance of CD63- positive particles, corroborating EV enrichment in this fraction (FIG. 3(f)). Western blot analyses further revealed a high expression of established EV markers (CD63, Alix) and the absence of a non-EV marker (histone H2B; FIG. 16(a) - 16(b)).

[0115] Overall, SpinEx outperformed conventional and disc-based size-exclusion chromatography (SEC) in EV enrichment (FIG. 3(g) and FIG. 17). We processed EV-spiked plasma samples using different filtering methods and quantified the amounts of serum albumin, ApoAl, ApoBl OO, and CD63 via ELISA (see Methods). While albumin removal efficiency (98%) remained comparable among the tested methods, LDL removal was superior with SpinEx (96%), followed by disc-SEC (87%) and conventional SEC (81%). The EV recovery was about 29% for SpinEx, which was comparable to that of the conventional SEC column (40%). Consequently, EV enrichment was higher with SpinEx, as evidenced by the CD63 / ApoB100 expression ratio: this value was 0.48 for SpinEx, representing a 3.6- fold increase over conventional SEC (0.13). Furthermore, SpinEx demonstrated unbiased EV isolation. For instance, the expression profiles of tetraspanins (CD63, CD9, CD81 ) remained consistent between samples before and after SpinEx-based EV isolation (FIG. 18).

[0116] Design of EV-labeling module

[0117] Following EV isolation, a SpinEx assay advanced to EV labeling. The specific disc section (FIG. 4(a)) had chambers filled with reagents (e.g., fluorescent labels, biotin, buffers), a reservoir containing microbeads, and an array of compartments designed for protein labeling. FIG. 4(b) shows the labeling steps. Inside the reservoir, EVs were captured on the microbeads and biotinylated; the beads served as a solid substrate for the assay, and the biotinylation was employed to enable universal EV labeling. Note that the reservoir bottom was sealed with a membrane filter to retain the microbeads throughout this process. After being treated with a blocking buffer (SuperBlock), the EV-bead conjugates were distributed into eight separate compartments (FIG. 4(b)), wherein EVs were labeled with a primary antibody specific to a target protein. Finally, fluorescent reagents were introduced to generate analytical signals for total EV amount (via fluorescent streptavidin) and protein expression (via fluorescent secondary antibody).

[0118] To ensure consistent EV handling across all compartments, we controlled their fluid intake (FIGs. 4(c), (d)). Each labeling compartment was connected to the main distribution channel through a capillary valve, which dispensed a fixed volume (10 pL) of fluid. Employing capillary valves enabled this dispensing to occur all at once. This valve exerts a barrier pressure (Pc) that blocks the fluidic flow until the centrifugal pressure (Pm) exceeds Pc. The barrier pressure is given as Pc= 4-y-sin0c-c / / )-n, where y is the surface tension at the water-air interface (72.8 mN / m), 9c is the contact angle (20s), dh is the hydraulic diameter (0.067 mm), and n = 1 .14 for a rectangular cross-section; the centrifugal pressure is Pm= p-uj2-r-Lr, where p is the liquid density (1 g / mL), a> is the angular velocity, r is the average distance of the liquid from the disc center (51 .5 mm), and Ar is the radial length of the liquid (8 mm). The estimated UJ required for Pm> Pcwas 71 rad / sec (680 rpm), which closely matched the experimental value of 63 rad / sec (600 rpm). Once EVs were transferred to the designated compartments, the disc was operated in a rocking mode to promote sample dispersion and mixing (FIG. 19). No spillover contamination occurred between compartments during disc operations (FIG. 4(e)), ensuring negligible crosstalk in EV labeling.

[0119] EV capture protocol

[0120] We compared two methods of capturing EVs on microbeads inside a SpinEx disc: i) adsorption, which used carboxylated microbeads directly to capture EVs through hydrophobic interactions, and ii) immunocapture, wherein microbeads were conjugated with antibodies to target tetraspanins (CD9, CD63, or CD81 ), canonical EV markers (see Methods for details). The adsorption method is commonly used to immobilize proteins on plastic surfaces (e.g., ELISA plates). The governing mechanism is entropy-driven thermodynamics: protein adsorption releases ordered water molecules from the vicinity of hydrophobic surfaces and minimizes the hydrophobic surface area exposed to water molecules (FIG. 20). This hydrophobic interaction is stronger than the van der Waals interactions, leading to stable and irreversible protein adsorption.

[0121] We performed both adsorption and immunocapture, using test samples containing known amounts of EVs (from SW620 cells) in a phosphate buffer solution. Both methods were compatible with SpinEx. Electron microscopy confirmed effective EV capture on microbeads (FIG. 5(a) and FIG. 21); fluorescent imaging verified dual EV staining (FIG. 5(b) and FIG. 22), which facilitated the assessment of both EV quantity and the expression of a target protein. For adsorption-based EV capture, we confirmed robust and stable EV adherence to microbeads. Following EV capture and fluorescent labeling, EV-bead complexes underwent repeated centrifugal washes (1200 rpm, 30 sec) and subsequent signal detection by flow cytometry (FCM). The observed signal levels showed no statistically significant differences (P> 0.05, ANOVA) across multiple washing rounds (FIG. 5(c)).

[0122] Comparative analysis revealed that the adsorption approach yielded a higher EV-capture yield than immunocapture (FIG. 5(d), left). This is likely because adsorption is less affected by the variable expression of tetraspanins (CD9, CD63, CD81 ) in EVs. Prior single-EV imaging studies have indicated that tetraspanins (CD9, CD63, CD81 ) are not ubiquitously present on EVs, but rather occur at ratios below 50%. Consequently, relying on a single tetraspanin for EV capture may restrict downstream analysis to specific EV subpopulations.

[0123] Conversely, EV capture via adsorption enables unbiased sampling of the entire EV population, resulting in improved capture efficiency and a stronger fluorescent signal for the target protein (FIG. 5(d), right).

[0124] Optimization of EV labeling

[0125] We further optimized a SpinEx protocol to maximize analytical signals and minimize non-specific EV labeling. Lowering the buffer pH during the adsorption step improved the EV capture yield (FIG. 23(a) - 23(b)). EVs are less negatively charged in acidic buffers, which may have reduced electrostatic repulsion between EVs and microbeads. We specifically selected a buffer pH of 6.4, which was previously determined to maximize the surface charge difference between EVs and LDL particles.

[0126] To mitigate non-specific EV labeling, we incorporated a blocking step. After EV capture, the beads were incubated with a blocking buffer (Superblock™) to prevent fluorescent reporters (e.g., fluorescent antibodies) from adsorbing onto beads. A SpinEx assay also included control samples, prepared by incubating EV-bead complexes with control IgG antibodies. Signal levels from these control samples established the threshold for marker positivity. FIG. 5(e) compares signals between various controls and targeted samples. No significant differences were observed between the IgG control and no- EV samples (P = 0.96, non-paired two-sided t-test), indicating minimal non-specific binding between antibodies and microbeads. The cutoff value for CD63 positivity was set at mean + 3 x standard error (red dotted line), based on the IgG control results.

[0127] EV protein profiling

[0128] To quantify EV protein expression, we detected EV-bound microbeads via FCM (see Methods for details). Following the EV labeling with SpinEx, samples were collected from the eight labeling compartments and dispensed into a 96-well plate for automated FCM measurements. This approach facilitated high-throughput, simultaneous measurement of dual fluorescent signals from individual beads (FIG. 6(a)): fetAv from fluorescent StAv and I from the secondary antibody. We used these signals to assess EV loading (fetAv) and marker expression ( / M). EV loading was consistent across samples, as indicated by the similar intensity distributions of fluorescent StAv (FIG. 6(a), top panel). Conversely, the intensity distributions varied for different markers (FIG. 6(a), right panel), indicating differential protein expression patterns in EVs.

[0129] We first evaluated EV detection sensitivity. SpinEx processed samples containing varying amounts of EVs, and FCM measured fetAv from labeled beads (5 m in diameter). For comparison, we analyzed the same EV samples via a conventional enzyme-linked immunosorbent assay (ELISA; see Methods for details). The experiment showed dose-dependent signal increases, establishing titration curves for EV concentration (FIG. 6(b)). From these curves, the estimated detection limit was 8.4 x 105 EVs / mL for SpinEx-FCM, which was >100-fold lower compared to ELISA (1.5 x 108 EVs / mL). We further changed the bead size (0.8 and 2.5 pm) and tested its impact on sensitivity (FIG. 24). The FCM detection sensitivity correlated with the bead size: 5-pm beads achieved the lowest detection limit followed by 2.5 pm beads (4.8 x 107 EVs / mL) and 0.8 pm beads (2.4 x 108EVs / mL). Although even larger beads (>5 pm) could further enhance the sensitivity, they were excluded due to their tendency to sediment during FCM analysis. We therefore selected 5-pm beads for this study. However, we note that SpinEx can accommodate microbeads of different types and sizes to suit specific detection needs, for 9 example, larger beads (~10 pm) for microscopy or smaller beads (~2.5 pm) for electrochemical EV assays (Park et al., 2021 , 34183802) (Jeong et al., 2016, 26808216).

[0130] For a quantitative marker analysis, we adopted the following ratiometric approach. For each bead, we calculated the intensity ratio, W / stAv, effectively mitigating variations arising from differential EV loading across individual beads. From the ratio data set, we subsequently obtained its median [= median( / M I fetAv)] as a metric for the marker (M) expression normalized to the EV loading. This method produced highly consistent marker expression results (FIG. 6(c)). For instance, the coefficient of variation remained below 2%, even at a low bead count of 100 (FIG. 25). In contrast, calculating a bulklevel ratio [= median( / M) / median( / stAv)] resulted in about 10-fold increase in CV values compared to M.

[0131] Applying SpinEx-FCM, we measured various protein markers in EVs isolated from different cell lines (see Methods for details). ELISA was also performed as a gold standard. A SpinEx-FCM results exhibited a strong linear correlation (Pearson coefficient r= 0.93) with ELISA (FIG. 6(d)), demonstrating the quantitative marker profiling capabilities of a SpinEx approach. However, due to its superior sensitivity, SpinEx-FCM required substantially lower amounts of EVs (105 EVs per marker) compared to ELISA (107 EVs per marker).

[0132] Cancer detection and classification through EV profiling.

[0133] To evaluate SpinEx’s practical utility, we applied the technique to process clinical plasma samples, facilitating high-throughput protein profiling for cancer diagnostics and subtype classification (FIG. 7(a)). We focused on five prevalent tumor types, breast, lung, liver, pancreas, and colon, as potential detection targets. Plasma samples were collected from patients with a confirmed primary tumor in one of these organs (Table 2): breast (n = 30), lung (n = 32), liver (n = 27), pancreas (n = 31 ), and colon (n = 37). In addition, non-cancer control samples (n = 64) were obtained from non-cancer donors.

[0134] We determined protein marker candidates through our customized algorithm (FIG. 7(b) and FIG. 26(a)). From a public database (the Human Protein Atlas), we retrieved a set of mRNAs (n = 2311 ) differentially upregulated in our tumor types (FIG. 26(b)). Each selected mRNA for a given tumor type had >50% higher expression than the average expression in the other four tumor types. This initial collection was refined to exclude cytosolic proteins based on the UniProt annotation (#Cytoplasm) and further filtered to include proteins (n = 233) known to be present in EVs (Gene Ontology terms: extracellular exosome, extracellular vesicle, extracellular region). We included the term “extracellular region” to account for secreted proteins known to be present on EV surfaces (Hallal et al., 2022, 36239734). Subsequently, we manually curated the list through a literature review, ensuring i) markers’ relevance in cancer detection, ii) experimental evidence of its presence on the cell membrane and / or in the extracellular space, and Hi) the availability of suitable antibodies. The final set comprised 30 protein markers (Table 3) with functionalities strongly associated with cancer pathways and cancer proteoglycans (FIG. 26(c)).

[0135] We further evaluated the compatibility of selected antibodies (Table 4) with a SpinEx process. Following a SpinEx protocol, microbeads were subjected to treatment with a blocking buffer, incubation with primary antibodies, and labeling with fluorescent secondary antibodies. Subsequently, 10 FCM was performed to quantify fluorescent signals. These controls exhibited consistent, low baseline signals (FIG.- 27(a) - 27(b)), confirming the efficacy of the blocking and bead-washing procedures. We proceeded to process clinical samples. SpinEx process is compatible with both whole blood and plasma samples. Furthermore, plasma prepared from whole blood by SpinEx was confirmed to be equivalent to plasma prepared through conventional centrifugation (FIG. 28(a) - 28(c)). As our clinical samples were already available as plasma, we bypassed the whole-blood separation step and directly injected 50 L samples into the chromatography region. The subsequent SpinEx process performed EV isolation, biotinylation, capture on microbeads, and labeling with i) fluorescent StAv to estimate EV loading per bead (fetAv) and ii) primary and fluorescent secondary antibodies to assess marker expression ( / M). Corresponding control samples were prepared using isotype-matched control IgG antibodies in place of marker-specific primary antibodies. Note that permeabilization was not used for clinical EV samples. Both targeted and control sample pairs were analyzed via FCM, yielding normalized metrics [= median( / M I fetAv)] and lgG [= median( / igG I fetAv)], respectively. We then used the difference M = igG as an analytical metric for statistical analysis (see FIG. 27(b) for an illustrative example). This metric showed good concordance with ELSI A (r= 0.86) when a subset of clinical samples was spot-checked for quality control (FIG. 29).

[0136] We used the obtained data for cancer diagnostics and classification (see FIG. 30(a) - 30(b) for the workflow). We first established a cancer diagnostic model (FIG. 7(c)). EV-profiling data were differentially split into training (70% of cohorts, n = 154) and test (30%, n = 67) sets. To the training set, we applied the Lasso regression; this process identified a subset of informative markers (n = 21 ), which were used as input features for training a support vector machine (SVM) model to generate a cancer risk score (FIG. 7(d)). The trained model demonstrated a high detection sensitivity (95%), specificity (97%), and accuracy (96%) for cancer diagnostics. Importantly, the model remained effective when applied to the independent test set, achieving a detection sensitivity of 80%, specificity of 97%, and accuracy of 90%. In the receiver operating characteristic analysis, the area under the curve (AUC) values were 0.985 (95% confidence interval: 0.973 - 0.998) for the training set and 0.974 (0.952 - 0.996) for the testing set (FIG. 7(e)). Using a similar machine-learning approach, we developed a model to differentiate early-stage cancer (stages I and II) from non-cancer controls (FIG. 31). The training set consisted of 74 early-stage cancer patients and 45 non-cancer controls, and the independent test set included 33 early-stage cancer patients and 19 non-cancer controls. This diagnostic model achieved area under the curve (AUC) values of 0.983 (training set) and 0.953 (test set).

[0137] Next, we developed a classification model to differentiate between five distinct tumor types (FIG. 7(f)). We leveraged the EV profiling data from cancer patients (n = 157), considering the limited number of samples for each cancer type. Utilizing an approach similar to that used for cancer detection, we employed a logistic regression (multi-nominal with L1 penalty) to identify informative markers. These markers were then used to train an SVM classification model with a one-versus-one algorithm (see Methods). We mapped the prediction probabilities from five to two dimensions (FIG. 7(g)). This visualization revealed distinct clusters of EV profiles corresponding to different tumor types. The trained model achieved high accuracy in predicting tumor types (95.5%, FIG. 7(h)).

[0138] SpinEx enriched EVs from whole blood and processed them for multi-panel protein detection. Importantly, these capabilities were accomplished within a disc-fluidic cartridge, a format that is amenable to i) the design of a compact system (i.e., no external fluidic components), ii) versatile and programmable fluidic controls, and Hi) parallel and automated sample processing. It isolated EVs from blood samples while removing >96% of lipoprotein particles, captured EVs on microbeads, and fluorescently labeled EVs for sixteen different protein markers. SpinEx completed these operations inside a single disc within 75 minutes.

[0139] SpinEx processed over 200 plasma samples (n = 221 ), targeting 30 proteins per sample. The EV profiling data effectively differentiated cancer from non-cancer cases and classified cancer samples according to their primary tumor types. SpinEx features innovative fluidic modules (e.g., on-disc chromatography, centripetal fluidic transport, metering capillary) that bring new functionalities to the lab- on-a-disc platform. Implementing dual-mode chromatography (DMC) as an on-disc column automated the operation, eliminating user intervention and reducing processing time to <5 minutes. This adaptation effectively enhanced the throughput and reproducibility of EV isolation. Moreover, the compact fluidic design allows for the assembly of multiple DMC sections in a single disc, which would facilitate the development of dedicated high-throughput EV isolation platforms (see Table 5 for comparison).

[0140] Table 1. SpinEx operation parameters. tRocking mode: ±!20* osdllatfofosec for 30 sec, followed by 50 rpm rotation for 300 sec. Table 2. Information on clinical samples.

[0141] Table 3. EV protein targets in this study.

[0142] Table 4. List of antibodies.

[0143] Table 5. Comparison of SpinEx’s isolation system with existing platforms.

[0144] The above-described results were obtained using the following material and methods.

[0145] Materials and Methods

[0146] Methods

[0147] SpinEx disc fabrication. We designed the disc's top and bottom layers using computer-aided design software and then transferred the design onto polycarbonate (PC) sheets (8574K24, 8574K809; McMaster Carr) via computer numerical control (CNC) milling (MDX-50 SRP, Roland). At the valve locations on the top layer, we formed diaphragms by applying encapsulant material (4019862; Ellsworth adhesives) and cured it for 3 h (20 °C). Both top and bottom PC layers were then treated with a 1% Pluronic F-127 solution (0.2-pm filtered, Thermo Fisher Scientific) for 1 hour, followed by washing with distilled water and air-drying. We separately patterned a pressure-sensitive adhesive film (SELECT DF132311 ; FLEXcon) using a plotter (CE7000-60; Graphtec). We bonded the top and bottom layers using the patterned film as a double-sided adhesive. Next, we integrated other components into the disc. The torque-activated valve adaptor units (93800A350; McMaster Carr) were assembled by aligning onto the diaphragm and bonded with epoxy glue (CA-11 ; 3M). Afterward, the valve actuators (91067A114; McMaster Carr) were assembled on the adaptors (Kim et al., Lab Chip, Vol. 16, 2016, 3741 -3749; Woo et al., ACS Nano, Vol. 11 , 2017, 1360-1370).

[0148] A track-etched PC membrane (13 mm diameter, 0.2 m pore size; Millipore Sigma) was placed in the designated bead chamber through the backside of the disc (Woo et al., ACS Nano, Vol. 11 , 2017, 1360-0370; Kim et al., Anal. Chem. Vol. 89, 2017, 1155-1162. The column chamber was sequentially filled with 0.1 mL of Fractogel EMD SO3- (M) resin (Millipore Sigma) and 0.5 mL of Sepharose CL-4B resin (GE Healthcare). The disc was then spun at 1200 rpm (<70 x g) to stack the resins within the column.

[0149] Spinning system. We custom-built a spinner system controlled by a computer. The key components of the system were a direct-current (DC) motor (RM-ESMO-06N; E-S Motor), a white LED (5050 9SMD LED Panel; YM E-Bright), a CMOS camera (C920; Logitech), and a microcontroller (Arduino Uno). A custom Python program running on the computer served as the central hub for user interaction and system control. It received the users' commands (e.g., motor speed, running time) and sent them to the microcontroller for the motor control. The program also coordinated image capture with motor movement to ensure clear visualization of the liquid movements within the disc (see FIG. 10(a) - 10(b) for an example).

[0150] SpinEx assay. Table 1 shows the assay process in detail. Plasma separation. A 150 pL whole blood sample was loaded into a plasma separation chamber, and the disc was spun at 1800 rpm (4 min). The separated plasma (50 pL) was then transferred to the on-disc chromatography column. This step involved opening a connecting valve within the disc and spinning the disc at 1800 rpm (20 sec). Once the transfer was complete, the valve was closed. EV isolation. The transferred plasma was injected into the column section along with an elution buffer (300 pL). To achieve this, a valve connecting the column to the elution buffer reservoir was opened, and the disc was at 600 rpm (4 min). The isolated EVs were collected at the F2 fraction chamber (100 pL). EV relocation. Two valves around the F2 chamber were closed, and the valve to the oil chamber was opened. With the disc spinning at 1800 rpm (20 sec), a dense oil was introduced, pushing the isolated EVs from the F2 chamber towards the inner portion of the disc. We then opened the value to the bead chamber and briefly (1 sec) spun the disc at 1200 rpm, allowing the EVs to flow into the bead chamber. Bead assay. The assay chamber was initially filled with carboxylated PS beads (0.5 mg beads / mL; 50 pL). EVs (100 pL) were transferred from the isolation section and allowed to attach to beads via physisorption (20 min). To promote the sample mixing, we operated the disc in one cycle of oscillatory mixing mode (±120eoscillation / sec for 30 sec, followed by 5- min rotation at 600 rpm). After incubation, we washed 14 the sample with a buffer (PBS, 120 pL) using a spinning step (1200 rpm, 0.5 min). Subsequently, TFP-biotin solution (0.1 mM, 100 pL) was introduced (1200 rpm, 0.5 min) to the bead sample, and the mixture was placed in the mixing mode (2 cycles, 10 min). After the biotinylation reaction, we washed the samples as described above, introduced a blocking buffer (50 pL, superblock; 1200 rpm, 0.5 min), and placed the disc in the mixing mode (2 cycles, 10 min). Following this blocking step, we washed the sample (90 pL wash buffer; 1200 rpm, 0.5 min). The bead solution (100 pL) was then distributed (600 rpm, 0.5 min) into the eight labeling chambers, each preloaded with a primary antibody solution (15 pL, 10 pg / mL). The disc was placed in a mixing mode (2 cycles, 10 min) for target protein labeling. Following the incubation, a solution (100 pL) of fluorescent reagents was introduced (600 rpm, 0.5 min) to the labeling chambers, and the disc was placed under the mixing mode (2 cycles, 10 min). The fluorescent solution consisted of a cocktail of fluorescent secondary antibodies (20 pg / mL) and streptavidin (20 pg / mL). All reactions were performed at room temperature (20 °C). See Table 4 for detailed information on antibodies and reagents used in this study.

[0151] High-throughput bead analysis. SpinEx-processed bead samples were dispensed into a 96- well U-bottom plate (Corning, cat#CLS3788, Sigma Aldrich), washed twice with PBS (50 pL), and resuspended in PBS (50 pL). For high-throughput signal readout, we used a flow cytometer (CytoFlex B2- R0-V2; Beckman Coulter) that accommodated a 96-well plate as a sample input. Dual fluorescent signals were read from individual beads (488 nm excitations; 525 / 40 nm and 585 / 42 nm bandpass filters; see FIG. 33 for gating examples). A total of 5000 events were recorded for each sample. We used FlowJo (v10; BD) for data export. Exported data were post-processed to compensate for signal bleed-through between FITC and PE channels (Roederer, Curr. Protoc. Cytom., Ch. 1 , 2002) and remove outliers using the median absolute deviation (MAD) algorithm (Leys et al., J. Exp. Soc. Psychol., Vol. 49, 764-766, 2013) with a threshold of 2.5.

[0152] Cell lines and culture for EV production. Cell lines used in this study (SW480, SW620, and CaOV3) were purchased from the American Type Culture Collection. Cells were seeded in a T175 flask and cultured in Dulbecco's modified Eagle medium (Thermo Fisher Scientific) supplemented with 1% exofree fetal bovine serum (Thermo Fisher Scientific). For EV collection, cell-culture supernatants (100 mL) were collected and centrifuged at 300 x g for 10 min. After centrifugation, supernatants were collected and centrifuged again at 2,000 x g for 10 min. Finally, clear supernatants were filtered through 0.22-pm membrane filters (cat#430767, Corning), and the filtrates were concentrated using centrifugal filter units (10 kDa cutoff; Centricon-70, Millipore Sigma).

[0153] Nanoparticle tracking analysis (NTA). NanoSight LM10 (Malvern), equipped with a 405 nm laser, was used. Samples were diluted in filtered (0.22 pm) phosphate-buffered saline (fPBS) to obtain the recommended particle concentration (25-100 particles / frame). For each test sample, three 30-sec videos were recorded (camera level, 14). Recorded videos were analyzed by NTA software (version 3.2) at a detection threshold of 3.

[0154] Enzyme-linked immunosorbent assay (ELISA). EV protein markers. Streptavidin (4 pg / mL) was loaded (50 pL / well) in a 96 well-plate (Nunc MaxiSorp flat-bottom, Thermo Fisher Scientific) and incubated overnight at 4 °C. After washing twice with a 200-pL wash buffer (PBS containing 0.1 % BSA), the plate was blocked with 1% BSA (200 pL / well) for 2 h (20 °C). The plate was then washed and loaded with biotinylated EV samples (50 pL / well). After 2-h incubation at 20 °C, we washed the plate 15 twice and loaded HRP-conjugated StAv (1 :20,000 diluted in 0.1% BSA; #405210, BioLegend) or HRP- conjugated antibodies (50 pL / well). After 20-min incubation (20 °C), the sample was washed thrice. Subsequently, 3,3’,5,5’-tetramethylbenzidine (TMB, BioLegend) was loaded (100 pL / well) and incubated for 30 min (20 °C). We stopped the reaction by adding 50 pL of stop solution and measured the absorbance (450 nm) on a plate reader (Tecan). Albumin quantification. We used the human albumin ELISA kit (EHALB; Invitrogen) according to the manufacturer’s protocol. Briefly, we loaded 100 pL of samples into capture antibody-coated 96-well plates and incubated them (2.5 h, 20 °C). After washing 4 times with a 1 x wash buffer (300 pL), we added biotinylated antibody (100 pL / well) for labeling (1 h, 20 °C). After another wash step, we introduced StAv-HRP solution (100 pL / well). Following 45-min incubation (20 °C) and subsequent washing, TMB substrate was added (100 pL / well) and allowed to react (30 min, 20 °C). Finally, we stopped the reaction by adding 50 pL of stop solution and measured the absorbance (450 nm) on a plate reader (Tecan). All buffers and reagents were provided within the kit. HDL and LDL quantification. We loaded samples (50 pL / well) in a 96 well-plate (Nunc MaxiSorp flatbottom) for adsorption (2 h, 20 °C). After washing twice with a wash buffer (200 pL), we blocked the plates with 1% BSA (200 pL / well) for 1 h (37 °C). We then washed the plate twice and added anti-ApoAl (MAB36641 -SP; R&D systems) or anti-ApoB100 (MAB4124; R&D systems) antibodies (500 ng / mL; 50 pL / well). Following 1 -h incubation (20 °C), we washed the plate twice and loaded HRP-conjugated antirabbit (1 :5000 dilution in 0.1% BSA; cat#7074S; Cell signaling) or anti-mouse (1 :2500 dilution in 0.1% BSA; cat#7076S; Cell signaling) antibodies (50 pL / well). After another 1 -h incubation (20 °C), analytical signals were generated using the TMB substrate as described above. CD63 quantification. We coated a 96 well-plate (Nunc MaxiSorp flat-bottom) with anti-CD63 antibody (cat#215-820; Ancell). The antibody solution (4 pg / mL) was loaded (50 pL / well) and kept overnight at 4 °C. Subsequently, the plate was washed twice as described above and blocked with 1% BSA (200 pL / well) for 2 h at 20 °C. The plate was washed, and samples were loaded (50 pL / well) for 2-h incubation (20 °C). We then washed the plate twice and added (50 pL / well) biotinylated anti-CD63 antibody (cat#215-030; Ancell) into the wells (500 ng / mL, 50 pL / well). After 1 -h incubation (20 °C), the plate was washed twice, followed by the loading (50 pL / mL) of HRP-conjugated streptavidin (1 :20000 dilution in 0.1% BSA). The mixture was incubated for 20 min (20 °C), and the analytical signals were generated using the TMB substrate (described above). We generated calibration curves using purified protein standards as targets (see FIG. 34 for albumin, ApoAl, ApoB100, and CD63).

[0155] Western blot. We processed a plasma sample from a colorectal cancer patient to enrich EVs. The isolated EVs underwent lysis in 10x RIPA buffer (Abeam) for 30 minutes in an ice bath. Following centrifugation at 14,000 x g (10 min, 4 °C), the supernatant was collected. Protein concentration was determined using a BCA assay (Thermofisher Scientific), and the sample was subsequently analyzed via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary antibodies employed in the Western blot analysis included Alix (1 :1000, JM85-31 , Invitrogen), biotinylated CD63 (1 :1000, AH N16.1 / 46-4-5, Ancell), and histone H2B (1 :1000, D2H6, Cell Signaling Technology). To mitigate potential signal interference from human immunoglobulin G present in the plasma, Alix and Histone H2B antibodies were modified with sulfo-NHS-Biotin (Thermo Fisher Scientific) according to the manufacturer's protocol. Singal detection was performed using HRP-conjugated streptavidin (1 :2000, R&D Systems). 16 Single particle imaging. We used samples eluted from the chromatography column in a SpinEx disc. Fifteen microliters of the processed samples were loaded on a glass slide and allowed to settle (2 h, 20 °C). The slide was then washed with fPBS and incubated (15 min, 20 °C) with 10 pL of fixation buffer (4% paraformaldehyde). After washing the slide with fPBS, we treated the slide with a blocking buffer (15 pL, Superblock) for 1 h (20 °C). For CD63 detection, we labeled samples (90 min, 20 °C) with 15 pL of anti-CD63 antibody (5 pg / mL, cat#215-020; Ancell) conjugated with AF555 fluorophore. For ApoAl detection, we first added 15 pL of biotinylated anti-ApoAl antibody (STA-361 , 1 / 200 dilution; Cell Biolabs). Following 90-min incubation (20 °C), the sample was washed with fPBS and labeled (30 min, 20 °C) with 15 pL of StAv (5 pg / mL) conjugated with FITC. For ApoB detection, samples were labeled (90 min, 20 °C) with 15 pL of anti-ApoB100 antibody (5 pg / mL, MAB4124; R&D systems) conjugated with AF647 fluorophore. All samples were washed with fPBS before imaging. We used an inverted microscope (Nikon, Eclipse TE2000S) equipped with an sCMOS camera (Andor, Zyla) for fluorescent microscopy. Images were analyzed with Imaged.

[0156] Preparation of microbeads for EV capture. We used carboxylated PS beads (CP-50-10; Spherotech) to capture EVs via physisorption or immunocapture. For immunocapture, we prepared PS beads coated with antibodies against CD9, CD63, or CD81 . For the antibody-coating, 10 pg of antibodies were incubated with the washed carboxylated PS beads (0.5 mg beads / mL, 100 pL) for 1 h at 20 °C under rotation. After washing twice with fPBS, we suspended beads in 100 pL of Superblock buffer (#37580, Thermo Fisher Scientific), incubated the mixture at 4 °C overnight, and washed beads with fPBS. Control beads were prepared using the same protocol as above but with isotype IgG antibodies instead of EV-specific antibodies. For EV capture, we mixed biotinylated EVs (80 pL) with the bead solution (0.7 pL). After 30-min incubation (20 °C), beads were washed with PBS containing 0.05% (v / v) Tween 20 (PBST). Then, beads were mixed with 30 L of anti-EpCAM (4 g / mL in 0.1% BSA) antibody conjugated with AF488 fluorophore. After 10-min incubation (37 °C), beads were washed with PBST and mixed with 30 pL of StAv (4 pg / mL in 0.1% BSA) conjugated with -AF647 fluorophore. After incubation (10 min, 37 °C), beads were washed twice in PBST and suspended in PBS (15 pL). We imaged labeled beads using a confocal microscope (Nikon, Eclipse Ti2E).

[0157] Fluidic simulation. We used COMSOL Multiphysics 5.6 (COMSOL, Sweden) equipped with a microfluidics module to model the centrifugal force-driven flow within a SpinEx disc. The disc geometry, designed using AutoCAD 2019 (Autodesk, USA), was imported into COMSOL for simulation. We used the laminar flow model, with the fluid assumed to be water at 20 °C. At the fluid inlet, the entering flow velocity was set to 0.4 mm / s based on the disc spinning speed. The fluid outlet was set as an open boundary. Laminar flow models were generated under centrifugal forces ranging from 0 to 200 times gravity. This corresponded to spinning speeds ranging from 0 to 1800 rotation per minute (rpm) within a 55 mm disc radius. The simulations were run for 200 seconds with a 0.5-second time step. We exported the flow velocity fields and streamlines at each time step for further analysis. We exported the flow velocity fields and streamlines at each time step for further analysis. Additionally, for centripetal transport, the fluidic system consisted of two immiscible fluids with different densities: the lighter fluid was an aqueous solution, and the heavier fluid was a mineral oil. The aqueous solution was assumed to have the same physical properties as water, whereas the mineral oil properties were defined as follows: density =

[0158] I .61 mg / mL, interfacial tension = 16.2 mN / m, and dynamic viscosity = 1 .24 mPa-s. External forces at

[0159] 1800 rpm introduced both centrifugal and Coriolis effects. To track the interface 17 dynamics and shape evolution during this centripetal transport process, we used a level set method (Osher and Fedkiw J. Comput. Phys. 169, 463-502 (2001 )).

[0160] Particle tracking simulation. We simulated particle movement in the disc's flow field generated by COMSOL. Three key assumptions were made for particle behavior, i) Particles were massless and solid, enabling them to instantly adapt to the fluid flow and follow the streamlines within the channels (Han

[0161] J. Appl. Meeh. 27, 403-409 (1960); Robert A. Granger, Fluid Mechanics (Dover publications, 1995)). ii) Particles were assumed to be uniformly distributed across the inlet of the microfluidic channel. Hi) Particles in contact with the walls are reflected back into the channel. Based on these assumptions and the velocity data obtained from COMSOL, we developed MATLAB codes to numerically calculate and visualize particle trajectories within the microfluidic disc.

[0162] Machine learning analysis. We imported the profiling data that listed the expression of 30 markers per patient. For the cancer diagnosis model, the Least Absolute Shrinkage and Selection Operator (LASSO) method was applied to select the most informative markers for distinguishing between cancer and non-cancer samples. The penalty parameter ( ) of LASSO was obtained from cross- validation, and the best performance was obtained when = 0.005. The importance of each marker was ranked by the summation of the LASSO coefficients. The selected markers were then used as input for a Support Vector Machine (SVM) classifier (Cortes and Vapnik Mach. Learn. 20, 273-297 (1995)) to predict sample categories (cancer vs. non-cancer). For cancer typing (multi-class classification), we implemented the logistic regression for marker selection and the one-versus-one (OVO) SVM approach for classification. This OVO strategy decomposed the multi-class problem into a series of binary classification tasks, where each SVM classifier differentiates between one pair of classes. The final prediction for a sample was assigned to the class with the most positive outcome from the individual binary SVMs. Detailed workflow is shown in FIG. 30(a) - 30(b).

[0163] Clinical samples. We drew peripheral blood samples (~10 mL) from participants. The samples were then centrifuged (2,000 *g, 20 min) to separate plasma from red blood cells and buffy coat. The plasma samples were stored at -80 °C until use. For EV isolation, stored plasma samples (50 L) were thawed and processed with Spin Ex.

[0164] OTHER EMBODIMENTS

[0165] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0166] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0167] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0168] Other embodiments are within the following claims.

Claims

CLAIMS1 . A device comprising a rotatable substrate comprising: a) an inlet in fluid communication with a first channel via a first valve; b) a chromatography chamber comprising chromatography separation media that is in fluid communication with the first channel; c) an eluant chamber in fluid communication with the chromatography chamber via a second valve; d) first and second eluate chambers in fluid communication with the chromatography chamber; and e) a displacement chamber in fluid communication with the second eluate chamber via a third valve, wherein the displacement chamber is located centrally relative to the second eluate chamber.

2. The device of claim 1 , further comprising a sedimentation channel in fluid communication with the inlet and having a proximal end located centrally relative to a distal end, wherein the first channel intersects the sedimentation channel between the proximal and distal ends.

3. The device of claim 2, wherein the sedimentation channel is slanted at an angle of at least 10°relative to a centripetal force generated by rotating the device.

4. The device of claim 2, further comprising a sample chamber between the inlet and the sedimentation channel.

5. The device of claim 1 , wherein the chromatography separation media comprises a size-exclusion stationary phase and / or an ion-exchange stationary phase.

6. The device of claim 5, wherein the size-exclusion stationary phase is disclosed centrally relative to the ion-exchange stationary phase.

7. The device of claim 1 , wherein the displacement chamber comprises a liquid immiscible with water that has a density greater than water.

8. The device of claim 1 , further comprising a third eluate chamber in fluid communication with the chromatography chamber, wherein a fourth valve is disposed between the second and third eluate chambers and a fifth valve is disposed between the second and first eluate chamber, wherein the first eluate chamber is disposed azimuthally further from the chromatography chamber, the third eluate chamber is disposed azimuthally closest to the chromatography chamber, and the second eluate chamber is disposed azimuthally between the first and third eluate chambers.

9. The device of claim 1 or 2, further comprising a second channel in fluid communication with the displacement chamber via a sixth valve; an immobilization chamber comprising a solid support in fluid communication with the second channel; a first assay chamber in fluid communication with the immobilization chamber via a seventh valve; and a waste chamber in fluid communication with the immobilization chamber via a liquid permeable barrier.

10. The device of claim 9, further comprising second, third, fourth, fifth, and sixth assay chambers in fluid communication with the immobilization chamber via eighth, ninth, tenth, eleventh, and twelfth valves.11 . The device of claim 9, further comprising a third channel in fluid communication with the immobilization chamber via a thirteenth valve; and first and second labeling chambers in fluid communication with the third channel via fourteenth and fifteenth valves.

12. The device of claim 11 , further comprising third, fourth, fifth, sixth, seventh, and eighth labeling chambers in fluid communication with the third channel via sixteenth, seventeenth, eighteenth, nineteenth, and twentieth valves.

13. The device of claim 11 , wherein the fourteenth and fifteenth valves are capillary valves.

14. A method for sample processing comprising: a) providing a device of claim 1 ; b) rotating the device to transport a portion of a sample into the chromatography chamber; c) opening the second valve and rotating the device to transport an eluant in the eluant chamber into the chromatography chamber, wherein eluate flows into the first and second eluate chambers to provide first and second eluates; and d) opening the third valve and rotating the device to transport a liquid immiscible with water that has a density greater than water from the displacement chamber into the second eluate chamber thereby displacing the second eluate into the displacement chamber.

15. A method for sample processing comprising: a) providing a device of claim 11 ; b) rotating the device to transport a sample comprising particulate components and liquid into the sedimentation channel, wherein particulate components of the sample sediment towards the distal end leaving liquid at the proximal end; c) opening the first valve and rotating the device to transport the liquid into the chromatography chamber; d) opening the second valve and rotating the device to transport an eluant in the eluant chamber into the chromatography chamber, wherein eluate flows into the first and second eluate chambers to provide first and second eluates;e) opening the third valve and rotating the device to transport a liquid immiscible with water that has a density greater than water from the displacement chamber into the second eluate chamber thereby displacing the second eluate into the displacement chamber; f) opening the sixth valve and rotating the device to transport the second eluate into the immobilization chamber, wherein particulate components in the second eluate bind to the solid support; g) opening the seventh valve and rotating the device to transport a labeling reagent from the first assay chamber to the immobilization chamber, wherein the labeling reagent binds to the particulate components; and h) opening the thirteenth valve and rotating the device to transport first and second aliquots of the solid support into the first and second labeling chambers.

16. The method of claim 15, wherein the device is oscillated between steps (f) and (g) and between steps (g) and (h).

17. The method of claim 16, wherein the first and second labeling chambers contain first and second antibodies.

18. The method of claim 17, wherein the device is oscillated after step (h).

19. The method of claim 15, further comprising quantifying the number of particulate components in the first or second aliquot.

20. The method of claim 15, further comprising analyzing protein expression in the first or second aliquot.

Images