Integrated sample preparation device for point-of-care processing of whole blood

Abstract

A biological sample processing device including a blood filtration module and an extraction module incorporated within a common housing. The blood filtration module has a dual filter structure. The extraction module includes a series of chambers that hold agents to effect any one or more of lysis, binding of target material to capture beads, washing, elution, and / or nucleic acid amplification. Moveable magnets are used to move the beads through the chambers to perform the extraction process. A second magnet-based device for preparing largely pure plasma samples from whole blood in the field.

Description

[0001] Atty. Dkt. No. 93597-7427

[0002] INTEGRATED SAMPLE PREPARATION DEVICE FOR POINT-OF-CARE PROCESSING OF WHOLE BLOOD

[0003] STATEMENT OF GOVERNMENT SUPPORT

[0004] [1] This invention was made with government support under EB034654 awarded by the National Institutes of Health and 2036197 awarded by the National Science Foundation. The government has certain rights in the invention.

[0005] RELATED APPLICATIONS

[0006] [2] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 704,356, entitled INTEGRATED SAMPLE PREPARATION DEVICE FOR POINT-OF- CARE PROCESSING OF WHOLE BLOOD and filed October 7, 2024, and U.S. Provisional Patent Application No. 63 / 770,069, entitled INTEGRATED SAMPLE PREPARATION DEVICE FOR POINT-OF-CARE PROCESSING OF WHOLE BLOOD and filed March 11, 2025, the entire contents of which are incorporated herein by reference.

[0007] FIELD

[0008] [3] This application relates to devices for sample preparation, and more specifically for preparation of blood plasma and nucleic acid samples extracted from whole blood.

[0009] BACKGROUND

[0010] [4] Blood is a critical sample matrix in clinical diagnostics, serving as a rich source of molecular biomarkers, such as proteins, circulating nucleic acids, and metabolites. These biomarkers enable a broad range of diagnostic applications, from detecting bloodborne pathogens and metabolic disorders to early screening and treatment monitoring through liquid biopsy approaches. Because cellular components such as red blood cells (RBCs), white blood cells (WBCs), and platelets can reduce the accuracy of many diagnostic assays, the separation of whole blood into plasma is a critical step for many blood-based diagnostic tests.

[0011] [5] Typically, plasma is separated from whole blood via centrifugation, which separates blood components by density. However, this process requires a bulky, expensive centrifuge and careful

[0012] 1

[0013] 4909-5947-0701V.14818-7263-4587V.2

[0014] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 micropipetting, raising the need for trained personnel and sophisticated laboratory infrastructure. This severely limits the accessibility of many blood-based diagnostic tests in decentralized settings. For example, although the Cepheid GeneXpert expands access to molecular diagnosis of bloodborne pathogens through user-friendly sample-to-answer test cartridges, these assays (for HIV, HBV, and HCV viral load) still require 1 mL of plasma or serum as the starting sample, necessitating centrifugation of at least 2 mL of whole blood. Many POC settings that would benefit from on-site viral load testing, including harm reduction centers, prisons, rural clinics, and sexual health facilities, lack the laboratory centrifuges and trained personnel needed to prepare these samples for testing.

[0015] [6] In addition, in remote or rural settings, transporting whole blood from the point of collection to centralized laboratories poses major logistical and biological challenges. Whole blood is highly perishable and must be kept at controlled temperatures — typically 2-8°C — to prevent hemolysis or cellular degradation. Hemolysis causes up to 40-70% of blood sample rejections, compromising test accuracy by contaminating plasma with intracellular contents such as heme, proteins, and nucleic acids, which can skew analyte levels, inhibit diagnostic assays like PCR, and distort instrumental measurements. General laboratory protocols recommend plasma separation within 4 hours of blood collection. It has been demonstrated that plasma separated at the point of collection results in significantly fewer hemolyzed samples and lower sample rejection rates compared with plasma isolated after transport. Further, analytes of interest may degrade or be consumed more rapidly in unprocessed whole blood, leading to inaccurate measurements. For example, it has been observed that glucose concentrations can be reduced by 5-7% per hour in unprocessed whole blood due to glycolysis by blood cells. Therefore, the ability to isolate plasma directly at the point of collection not only enables immediate on-site testing but also preserves sample integrity for accurate downstream analysis in centralized laboratories.

[0016] [7] Despite these clear advantages, most existing POC tools for plasma separation are limited to processing small blood volumes (typically under 100 pL), which is inadequate for molecular diagnostic applications requiring quantitation or high sensitivity, such as viral load testing or liquid biopsy. Most rely on microfluidic or filter-based systems that cannot scale to milliliter volumes without introducing significant clogging of RBCs, which results in hemolysis and / or low, inconsistent plasma yields. Many such methods require significant dilution of the blood sample to

[0017] 2

[0018] 4909-5947-0701V.14818-7263-4587V.2

[0019] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 mitigate clogging, reducing diagnostic sensitivity by decreasing analyte concentration and leading to large, impractical sample volumes for downstream assays.

[0020] [8] Although a few reported methods can accommodate slightly higher blood volumes, these methods are still limited to relatively small volumes (200 pL to 1.8 mL), demonstrate low plasma yields, and rely on manual operation and micropipetting, posing biosafety and reproducibility concerns, especially in resource-constrained settings with minimally trained personnel. Nevertheless, there are currently no centrifuge-free plasma separation systems that are capable of processing >2 mL of whole blood in POC settings.

[0021] [9] As discussed, sample preparation presents a major challenge in point-of-care (POC) diagnostic assays, including the detection of nucleic acids from bloodborne pathogens. In wholeblood specimens, blood cells can interfere with the accuracy of nucleic acid detection (1). As such, nucleic acid detection assays typically require plasma or serum, obtained via separation from whole blood using techniques such as centrifugation, which requires expensive equipment and is restricted to central testing laboratories. Aside from centrifugation, other plasma separation techniques (such as fdtration) have been explored for POC use (2-5), but it remains a challenge to integrate plasma separation with downstream sample preparation steps (such as nucleic acid extraction).

[0022]

[0010] Further, extraction of nucleic acids is often a critical step in sample preparation. Plasma contains substances (e.g. heme and immunoglobulins) that could inhibit amplification (1). Traditional RNA extraction uses silica columns or magnetic beads in a tube, and features a series of manual pipetting steps (6). A recent elegant strategy has been developed by several groups (7- 12) to perform nucleic-acid extraction in POC microfluidics, based on magnetofluidics and immiscible phases, sometimes referred to as IF AST (Immiscible Filtration Assisted by Surface Tension) (7,13-16); specifically, nucleic acid-bound magnetic particles are transported through aqueous phases separated by immiscible oil or liquid wax, accomplishing some of the individual steps of a RNA extraction workflow (i.e. lysis, washing, and elution) within a single connected system without pipetting steps.

[0023]

[0011] However, there still remain important challenges. In assays where a whole-blood specimen is obtained at the POC (including finger-pricked blood tests which involve 100 pL or less of whole blood), plasma separation and nucleic acid extraction are still done as separate steps and with

[0024] 3

[0025] 4909-5947-0701V.14818-7263-4587V.2

[0026] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 separate devices (8,17,18). For example, Ngo et al. 2023 (8) reports a POC HIV viral load quantification method from blood using a power-free plasma separation device and a separate magnetofluidic cartridge. Multiple liquid handling steps are required to transfer filtered plasma from device to a tube containing RNA extraction reagents to the magnetofluidic cartridge, adding complexity to the workflow. In addition, this method and others (11) still require additional equipment such as micropipettes for sample transfer before the magnetofluidic steps and a laboratory shaking mixer for bead mixing and RNA capture prior to sample loading (8).

[0027] SUMMARY

[0028]

[0012] To address these limitations, an object of the present invention is to provide a biological sample processing device that is a simple, automated platform for large-volume plasma separation at the POC. It combines immunodepletion of red blood cells using antibody-coated magnetic particles with dual membrane size-exclusion filtration to efficiently separate large volumes of whole blood into plasma without membrane clogging, hemolysis, or significant dilution. RBCs are depleted prior to filtration, which minimizes membrane clogging and significantly increases the blood volume capacity of the system. Highly efficient (>99.9%) separation of blood cells from plasma from up to 5 mL of whole blood is achieved in under 10 minutes without significant dilution or hemolysis, while preserving clinically relevant biomarkers. In exemplary embodiments, the invention thus addresses a critical gap in point-of-care diagnostics by enabling efficient, reproducible, high-volume plasma separation at the point of collection, supporting broader access to high-sensitivity molecular testing and improving sample transport and stability for decentralized healthcare.

[0029]

[0013] Another object of the present invention is to provide an integrated, field-deployable device for sample preparation starting from whole blood and including nucleic acid extraction to increase access to testing and treatment for a variety of infectious diseases. A prime example of one such disease is Hepatitis C virus (HCV), a leading cause of liver-related mortality worldwide and responsible for half of the annual liver cancer cases in the U.S. (19). Encouragingly, recently developed direct acting antivirals can cure HCV in over 95% of people infected (20), but -40% of infected people in the U.S. are unaware of their infection (21). A major barrier lies in the two-step diagnostic process, requiring an initial antibody screen followed by an expensive RNA test to

[0030] 4

[0031] 4909-5947-0701V.14818-7263-4587V.2

[0032] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 confirm active infection. Hence, availability of single-visit HCV tests could vastly improve detection and treatment. An ideal one-step diagnostic would be accurate, simple, rapid, affordable, and able to be performed by minimally trained users (22). RT-PCR assays are the gold standard test for HCV diagnosis, as they are sensitive, specific, and are robust across variants (23).

[0033]

[0014] To overcome these limitations, exemplary embodiments of the present invention provide a point-of-care RNA / DNA extraction cartridge using immiscible phase separation, which leverages integration of plasma separation from whole blood with magnetofluidic immiscible phase RNA extraction for simple, power-free sample preparation for blood-borne pathogen detection. The device features a dual-membrane size exclusion filter (containing glass fiber membranes for separating plasma from whole blood) integrated with a magnetofluidic nucleic acid extraction module, requiring only a syringe accessory for operation. An objective of the device is to simplify sample preparation for whole blood specimens, to improve feasibility for POC high- performing PCR or RT-PCR diagnostic testing, as demonstrated herein for detection of HCV.

[0034]

[0015] A biological sample processing device according to an exemplary embodiment of the present invention comprises: a first axis having disposed in order there along: a first samplereceiving chamber; one or more intermediate second chambers; a third processed-sample chamber; the first, second, and third chambers being sequentially in fluid communication with adjacent chamber(s), the chambers having a top surface and a bottom surface, wherein all chambers are configured to contain a fluid so as to form a continuous fluid pathway from the first chamber through the intermediate second chambers to the third chamber; a first magnet; a second magnet; the first magnet and the first, second and third chambers being configured for movement of the first magnet relative to the chambers, the second magnet and the first, second and third chamber being configured for movement of the second magnet relative the chambers, independent of the movement of the first magnet, wherein relative movement of either the first and / or second magnet along the first axis from the first chamber to the third chamber gives rise to transfer of magnetic material from the first chamber, when contained therein, through the second chambers and into the third chamber.

[0035]

[0016] In an exemplary embodiment, the device further comprises a blood filtration component (a) in fluid connection with said first chamber, or (b) not in fluid connection with said first

[0036] 5

[0037] 4909-5947-0701V.14818-7263-4587V.2

[0038] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 chamber, said blood filtration component configured to separate plasma from a blood sample moved therethrough.

[0039] [171 In an exemplary embodiment, the blood filtration component comprises a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough.

[0040]

[0018] In an exemplary embodiment, the blood filtration component comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

[0041]

[0019] In an exemplary embodiment, the first magnet is present in or on a first track or guide, via which track or guide the first magnet is slidable across the first, second and third chambers, and wherein the first track or guide is adjacent to a top surface of the first, second and third chambers.

[0042]

[0020] In an exemplary embodiment, the second magnet is present in or on a second track or guide, via which track or guide the second magnet it is slidable across the first, second and third chambers, and wherein the second track or guide is adjacent to a bottom surface of the first, second and third chambers.

[0043]

[0021] In an exemplary embodiment, the blood filtration component is adapted for blood sample volumes of 100 uL or less.

[0044]

[0022] In an exemplary embodiment, the blood filtration component comprises a dual membrane filter comprising a first top membrane filter having a first and second face on opposite sides of the first filter, and a second bottom membrane filter having a first and second face on opposite sides of the second filter, the second filter adjacent to the first filter such that a fluid applied to a first face of the first top membrane filter under negative pressure will flow through the first face of the top membrane filter, out of the second face of said top filter, through a first face of the second bottom filter which faces the second face of the first filter, and through the adjacent second bottom filter membrane and exit through the second face thereof.

[0045]

[0023] In an exemplary embodiment, the pore size of the first filter is larger than the pore size of the second filter.

[0046]

[0024] In an exemplary embodiment, the pore size of the first filter is 2.0 microns to 5.0 microns, optionally about 2.7 microns.

[0047] 6

[0048] 4909-5947-0701V.14818-7263-4587V.2

[0049] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0050]

[0025] In an exemplary embodiment, the pore size of the second fdter is about 0.1 microns to 3.0 microns, optionally about 0.7 microns.

[0051]

[0026] In an exemplary embodiment, the filters of the dual membrane size-exclusion filter comprise glass fiber.

[0052]

[0027] In an exemplary embodiment, the device further comprises channels between adjacent chambers for fluid communication with the adjacent chamber(s), wherein said channels narrow triangularly from one chamber to an adjacent subsequent chamber in sequential order along the first axis of the device from the first sample-receiving chamber to the third processed-sample chamber.

[0053]

[0028] In an exemplary embodiment, the chambers are circular.

[0054]

[0029] In an exemplary embodiment, the first chamber contains a plurality of magnetic particles.

[0055]

[0030] In an exemplary embodiment, the plurality of magnetic particles is functionalized with a capture molecule.

[0056]

[0031] In an exemplary embodiment, the first chamber contains a liquid immiscible with a liquid in an adjacent intermediate second chamber.

[0057]

[0032] In an exemplary embodiment, the first chamber contains an aqueous solution and the adjacent intermediate second chamber contains an oil.

[0058]

[0033] In an exemplary embodiment, the intermediate second chambers are a plurality of chambers and are an odd number of chambers in total.

[0059]

[0034] In an exemplary embodiment, the intermediate second chambers are five chambers in total.

[0060]

[0035] In an exemplary embodiment, in sequential order between the first sample-receiving chamber and the third processed-sample chamber, a first intermediate second chamber contains an oil phase, a second intermediate second chamber contains an aqueous phase, a third intermediate second chamber contains an oil phase, a fourth intermediate second chamber contains an aqueous phase, and a fifth intermediate second chamber contains an oil phase.

[0061]

[0036] In an exemplary embodiment, the third chamber contains an aqueous solution.

[0062]

[0037] In an exemplary embodiment, the first magnet is moveable in an automated fashion.

[0063]

[0038] In an exemplary embodiment, the first magnet is moveable in a manual fashion.

[0064]

[0039] In an exemplary embodiment, the second magnet is moveable in an automated fashion.

[0065]

[0040] In an exemplary embodiment, the second magnet is moveable in a manual fashion.

[0066] 7

[0067] 4909-5947-0701V.14818-7263-4587V.2

[0068] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0069]

[0041] In an exemplary embodiment, wherein said first and / or second magnet comprises a handle attachment for manually moving the magnet along the track or guide, wherein the handle is substantially perpendicular to the first axis of the device.

[0070]

[0042] In an exemplary embodiment, the device further comprises a supply of nanoparticles disposed in a chamber of the device, the nanoparticles optionally comprising at least one of plasmonic outer shell and a magnetic core.

[0071]

[0043] In an exemplary embodiment, the sample processing device comprises agents in chambers thereof to effect any one or more of lysis, binding of target material to capture beads, washing, elution, and / or nucleic acid amplification.

[0072]

[0044] In an exemplary embodiment, the device further comprises a source of illumination configured to illuminate the contents of a chamber of the device.

[0073]

[0045] In an exemplary embodiment, the device further comprises in the first sample-receiving chamber magnetic material in the form of functionalized nanoparticles.

[0074]

[0046] In an exemplary embodiment, the functionalized nanoparticles are functionalized to bind one or more nucleic acids.

[0075]

[0047] In an exemplary embodiment, the functionalized nanoparticles are functionalized to bind a virus, a viral antigen, a bacteria, a bacterial antigen, a protein or a cell.

[0076]

[0048] A method of processing a sample in accordance with an exemplary embodiment of the present invention comprises operating the device so as to process a sample liquid supplied thereto.

[0077]

[0049] In an exemplary embodiment, the method comprises at least moving the first and second magnet in an alternate fashion relative to each other and the first chamber in order to effect mixing of the sample in the first chamber by movement of magnetic material therein.

[0078]

[0050] In an exemplary embodiment, the method comprises at least moving the first and second magnet relative to each other and the third chamber in order to effect mixing of the processed sample in the third chamber by movement of magnetic material therein.

[0079]

[0051] In an exemplary embodiment, the method comprises at least moving the first and / or second magnet along the track or guide from the first chamber through the intermediate second chambers to the third processed-sample chamber in order to effect the passing of the sample through the contents of the intermediate second chambers.

[0080] 8

[0081] 4909-5947-0701V.14818-7263-4587V.2

[0082] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0083]

[0052] A blood filtration device according to an exemplary embodiment of the present invention comprises a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough, the device configured for blood sample volumes of 100 uL or less, and wherein the dual membrane size-exclusion filter comprises two membranes, with a first membrane having a pore size larger than a second membrane.

[0084]

[0053] In an exemplary embodiment, the pore size of the first membrane is 2.5 to 5 microns, optionally about 2.7 microns.

[0085]

[0054] In an exemplary embodiment, the pore size of the second membrane is 0.1 to 3.0 microns, optionally about 0.7 microns.

[0086]

[0055] In an exemplary embodiment, the membranes comprise glass fiber.

[0087]

[0056] In an exemplary embodiment, the device comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

[0088]

[0057] In an exemplary embodiment, the device is configured for blood volumes of 50 uL or less.

[0089]

[0058] A biological sample processing device according to an exemplary embodiment of the present invention comprises: a container for placing in a chamber which is magnetic or through which a magnetic force can be applied, the container containing magnetic beads having thereon a molecule which attaches to human red blood cells; a chamber which is magnetic or through which a magnetic force can be applied for receiving the container; a blood filtration component (a) in fluid connection with said container, or (b) not in fluid connection with said container, said blood filtration component configured to separate plasma from a blood sample moved therethrough by suction from a syringe or pump component.

[0090]

[0059] In an exemplary embodiment, the blood filtration component comprises a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough.

[0091]

[0060] In an exemplary embodiment, the blood filtration component comprises a port for applying negative suction pressure and can be attached to the sample container, such that a blood sample in the sample container can be pulled therethrough.

[0092]

[0061] In an exemplary embodiment, the pore size of the first membrane is 2.5 to 5 microns, optionally about 2.7 microns.

[0093] 9

[0094] 4909-5947-0701V.14818-7263-4587V.2

[0095] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0096]

[0062] In an exemplary embodiment, the pore size of the second membrane is 0.1 to 3.0 microns, optionally about 0.7 microns.

[0097]

[0063] In an exemplary embodiment, the membranes comprise glass fiber.

[0098]

[0064] In an exemplary embodiment, the chamber which is magnetic or through which a magnetic force can be applied is a chamber comprising permanent magnets.

[0099]

[0065] A method of processing a blood sample in accordance with an exemplary embodiment of the present invention comprises: applying the blood sample into the chamber of the sample processing device and incubating the sample therein for a time sufficient for red blood cells to attach to the magnetic beads via the molecule which attaches to human red blood cells; and placing the container prior to, during, or after incubating, in the chamber which is magnetic or through which a magnetic force can be applied so as to thereby hold the red blood cells bound to the magnetic beads in the chamber and extracting via suction pressure, through the blood filtration component attached to the chamber of the sample processing device, a red blood cell-depleted supernatant, whereby passage of the supernatant through the blood filtration component and through the outlet removes a majority of any remaining cells and platelets in the red blood cell- depleted supernatant.

[0100]

[0066] In an exemplary embodiment, the sample is incubated for a time sufficient for red blood cells bound to a magnetic bead to settle at a bottom of the chamber, optionally for about 3 to about 5 minutes.

[0101]

[0067] In an exemplary embodiment, the blood sample is 0.5 to 7.5 ml.

[0102]

[0068] In an exemplary embodiment, the molecule is an antibody to a red blood cell surface antigen.

[0103]

[0069] In an exemplary embodiment, the chamber which is magnetic or through which a magnetic force can be applied surrounds a lower portion of the container when the container is placed therein and whereby red blood cells bound to the magnetic beads are held in the lower portion of the container when the supernatant is removed.

[0104]

[0070] In an exemplary embodiment, the method effects a processed blood sample having 102cells / ml or less once processed.

[0105]

[0071] In an exemplary embodiment, the method effects a processed blood sample having less than 102cells / ml once processed.

[0106] 10

[0107] 4909-5947-0701V.14818-7263-4587V.2

[0108] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0109] BRIEF DESCRIPTION OF THE DRAWINGS

[0110]

[0072] Exemplary embodiments of the present disclosure will be described with references to the accompanying figures, wherein:

[0111]

[0073] FIG. 1 is a perspective view of a biological sample processing device according to an exemplary embodiment of the present invention;

[0112]

[0074] FIG. 2A is a side view of a blood filtration module according to an exemplary embodiment of the present invention;

[0113]

[0075] FIG. 2B is an exploded view of the blood filtration module of FIG. 2A;

[0114]

[0076] FIG. 3A is a top view of a biological sample processing device according to an exemplary embodiment of the present invention;

[0115]

[0077] FIG. 3B is an exploded perspective view of a biological sample processing device according to an exemplary embodiment of the present invention;

[0116]

[0078] FIG. 3C are various views of an extraction module according to an exemplary embodiment of the present invention;

[0117]

[0079] FIG. 4 shows an extraction process using an extraction module according to an exemplary embodiment of the present invention;

[0118]

[0080] FIG. 5 illustrates a workflow for processing whole blood at the point of care using an integrated device in accordance with an exemplary embodiment of the present invention;

[0119]

[0081] FIG. 6 illustrates a workflow for separating plasma from a starting blood sample according to an exemplary embodiment of the present invention;

[0120]

[0082] FIG. 7 illustrates a workflow for processing whole blood at the point of care using an integrated device in accordance with an exemplary embodiment of the present invention;

[0121]

[0083] FIG. 8 is a chart of cell counts of whole blood and as processed by centrifugation and by PRECISE filter;

[0122]

[0084] FIG. 9 shows absorbance measure of lysed whole blood and as processed by centrifugation and by PRECISE filter;

[0123]

[0085] FIG. 10 shows blood input and plasma output resulting from use of a blood filtration module according to an exemplary embodiment of the present invention;

[0124] 11

[0125] 4909-5947-0701V.14818-7263-4587V.2

[0126] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0127]

[0086] FIG. 11 is a chart showing comparison of volume recovery of filter and centrifugation methods of plasma separation;

[0128]

[0087] FIG. 12 is a chart showing comparison of virus recovery of an exemplary device disclosed herein (“PRECISE filter”) and centrifugation methods of plasma separation;

[0129]

[0088] FIG. 13 is a chart of blood input volume versus recovered volume at different hematocrit levels (see Fig. 14);

[0130]

[0089] FIG. 14 is a chart of blood input volume versus normalized absorbance at different hematocrit levels;

[0131]

[0090] FIG. 15 is a chart of cycle versus normalized fluorescence with and without mixing magnet cycles;

[0132]

[0091] FIG. 16 is a chart of cycle versus normalized fluorescence measurements for the device / protocol disclosed herein (e.g., “PRECISE cartridge”) and benchtop magnetic bead-based protocol;

[0133]

[0092] FIG. 17 is a chart of whole virus concentration in plasma for RT-PCR amplification showing performance of an exemplary device herein;

[0134]

[0093] FIG. 18 is a chart of whole virus concentration in blood versus RT-PCR amplification;

[0135]

[0094] FIG. 19 is a chart of cycle versus normalized fluorescence for exemplary device and labbased workflow;

[0136]

[0095] FIG. 20A is a chart showing plasma separation performance (by cell count) for filters with various combinations of single or dual membranes with differing pore sizes;

[0137]

[0096] FIG. 20B is a chart showing evaluation of hemolysis in plasma filtered by membranes with various combinations of single or dual membranes;

[0138]

[0097] FIG. 20C is a chart showing superior recovered volume of plasma filtered by dual glass fiber (2.7 pm and 0.7 pm pore dual membrane filter) versus polycarbonate membranes (3.0 pm and 0.4 pm pores);

[0139]

[0098] FIG. 20D show images of filter input, output, and filtrate quality for various combinations of dual and single membranes, with dual glass fiber (2.7 pm and 0.7 pm pore dual membrane filter) showing superior performance;

[0140]

[0099] FIG. 21 shows a process of using a device / extraction module according to an exemplary embodiment of the present invention;

[0141] 12

[0142] 4909-5947-0701V.14818-7263-4587V.2

[0143] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0144]

[0100] FIG. 22A is a chart showing evaluation of the effect of various bottom surfaces on cartridge RNA extraction performance by PCR Ct;

[0145]

[0101] FIG. 22B is a chart showing PCR amplification curves of cartridge-extracted RNA using various magnet sizes;

[0146]

[0102] FIG. 22C is a chart showing evaluation of the effect of number of wash chambers on RNA extraction performance;

[0147]

[0103] FIG. 22D is a chart showing PCR amplification curves of cartridge-extracted RNA with and without on-cartridge mixing in the wash chambers;

[0148]

[0104] FIG. 23 is a perspective view of a a biological sample processing device according to an exemplary embodiment of the present invention;

[0149]

[0105] FIG. 24 is a illustrates a workflow for processing whole blood using the device of FIG. 23 in accordance with an exemplary embodiment of the present invention;

[0150]

[0106] FIG. 25 is a chart of magnetic bead volume versus supernatant volume recovered;

[0151]

[0107] FIG. 26 is a chart of mixing time versus supernatant volume recovered;

[0152]

[0108] FIG. 27 is a chart of magnetic separation time versus supernatant volume recovered;

[0153]

[0109] FIG. 28 is a chart of flow rate versus plasma volume recovered;

[0154] [HO] FIG. 29 is a chart of filter diameter versus plasma volume recovered;

[0155] [HI] FIG. 30 is a chart of filter diameter versus absorbance at 414 nm;

[0156]

[0112] FIG. 31 is a chart showing comparison of plasma volume recovered by centrifugation and a device according to an exemplary embodiment of the present invention;

[0157]

[0113] FIG. 32 is a chart showing comparison of cell count between whole blood, and samples obtained using centrifugation and a device according to an exemplary embodiment of the present invention;

[0158]

[0114] FIG. 33 is a chart showing comparison of absorbance at 414 nm between lysed whole blood, and samples obtained using centrifugation and a device according to an exemplary embodiment of the present invention;

[0159]

[0115] FIG. 34 is a chart of hematocrit versus plasma volume recovered;

[0160]

[0116] FIG. 35 is a chart of hematocrit versus plasma recovery;

[0161]

[0117] FIG. 36 is a chart of hematocrit versus normalized absorbance at 414 nm;

[0162] 13

[0163] 4909-5947-0701V.14818-7263-4587V.2

[0164] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0165]

[0118] FIG. 37 is a chart showing comparison of total protein concentration in samples obtained using centrifugation and a device according to an exemplary embodiment of the present invention;

[0166]

[0119] FIG. 38 is a chart showing comparison of glucose concentration in samples obtained used centrifugation and a device according to an exemplary embodiment of the present invention;

[0167]

[0120] FIG. 39 is a chart showing comparison of total cholesterol in samples obtained used centrifugation and a device according to an exemplary embodiment of the present invention;

[0168]

[0121] FIG. 40 is a chart showing comparison of total DNA concentration in samples obtained used centrifugation and a device according to an exemplary embodiment of the present invention;

[0169]

[0122] FIGS. 41 A-41B show proof-of-concept, (A) volume of plasma recovered and (B) cell count of recovered plasma, for combining immunomagnetic depletion with filtration;

[0170]

[0123] FIGS. 42A-42B shows comparison of three magnetic separator and chamber designs for efficient immunomagnetic RBC separation, with square prototype no. 3 showing the best results.

[0171]

[0124] FIG. 43: Different prototype arrangements for RBC separation.

[0172] DETAILED DESCRIPTION

[0173]

[0125] The present disclosure is generally related to an integrated, field-deployable device for sample preparation starting from whole blood and including nucleic acid extraction.

[0174]

[0126] A biological sample processing device, comprising: a first axis having disposed in order there along: a first sample-receiving chamber; one or more intermediate second chambers; a third processed-sample chamber; the first, second, and third chambers being sequentially in fluid communication with adjacent chamber(s), the chambers having a top surface and a bottom surface, wherein all chambers are configured to contain a fluid so as to form a continuous fluid pathway from the first chamber through the intermediate second chambers to the third chamber; a first magnet; a second magnet; the first magnet and the first, second and third chambers being configured for movement of the first magnet relative to the chambers,

[0175] 14

[0176] 4909-5947-0701V.14818-7263-4587V.2

[0177] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 the second magnet and the first, second and third chamber being configured for movement of the second magnet relative the chambers, independent of the movement of the first magnet, wherein relative movement of either the first and / or second magnet along the first axis from the first chamber to the third chamber gives rise to transfer of magnetic material from the first chamber, when contained therein, through the second chambers and into the third chamber.

[0178]

[0127] In some embodiments, the sample processing device further comprises a blood filtration component (a) in fluid connection with said first chamber, or (b) not in fluid connection with said first chamber, said blood filtration component configured to separate plasma from a blood sample moved therethrough.

[0179]

[0128] In some embodiments, the blood filtration component comprises a dual membrane sizeexclusion filter which separates plasma from a blood sample moved therethrough.

[0180]

[0129] In some embodiments, the blood filtration component comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

[0181]

[0130] In some embodiments, the first magnet is present in or on a first track or guide, via which track or guide the first magnet is slidable across the first, second and third chambers, and wherein the first track or guide is adjacent to a top surface of the first, second and third chambers.

[0182]

[0131] In some embodiments, the second magnet is present in or on a second track or guide, via which track or guide the second magnet it is slidable across the first, second and third chambers, and wherein the second track or guide is adjacent to a bottom surface of the first, second and third chambers.

[0183]

[0132] In some embodiments, the blood filtration component is adapted for blood sample volumes of 100 uL or less.

[0184]

[0133] In some embodiments, the blood filtration component comprises a dual membrane filter comprising a first top membrane filter having a first and second face on opposite sides of the first filter, and a second bottom membrane filter having a first and second face on opposite sides of the second filter, the second filter adjacent to the first filter such that a fluid applied to a first face of the first top membrane filter under negative pressure will flow through the first face of the top

[0185] 15

[0186] 4909-5947-0701v. l4818-7263-4587v.2

[0187] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 membrane filter, out of the second face of said top filter, through a first face of the second bottom filter which faces the second face of the first filter, and through the adjacent second bottom filter membrane and exit through the second face thereof.

[0188]

[0134] In some embodiments, the pore size of the first filter is larger than the pore size of the second filter.

[0189]

[0135] In some embodiments, the pore size of the first filter is 2.0 microns to 5.0 microns, optionally about 2.7 microns.

[0190]

[0136] In some embodiments, the pore size of the second filter is about 0.1 microns to 3.0 microns, optionally about 0.7 microns.

[0191]

[0137] In some embodiments, the filters of the dual membrane size-exclusion filter comprise glass fiber.

[0192]

[0138] In some embodiments, the sample processing device comprises channels between adjacent chambers for fluid communication with the adjacent chamber(s), wherein said channels narrow triangularly from one chamber to an adjacent subsequent chamber in sequential order along the first axis of the device from the first sample-receiving chamber to the third processed-sample chamber.

[0193]

[0139] In some embodiments, the chambers are circular.

[0194]

[0140] In some embodiments, the first chamber contains a plurality of magnetic particles.

[0195]

[0141] In some embodiments, the plurality of magnetic particles is functionalized with a capture molecule.

[0196]

[0142] In some embodiments, the first chamber

[0197]

[0143] contains a liquid immiscible with a liquid in an adjacent intermediate second chamber.

[0198]

[0144] In some embodiments, the first chamber

[0199]

[0145] contains an aqueous solution and the adjacent intermediate second chamber contains an oil.

[0200]

[0146] In some embodiments, the intermediate second chambers are a plurality of chambers and are an odd number of chambers in total.

[0201]

[0147] In some embodiments, the intermediate second chambers are five chambers in total.

[0202]

[0148] In some embodiments, in sequential order between the first sample-receiving chamber and the third processed-sample chamber, a first intermediate second chamber contains an oil phase, a

[0203] 16

[0204] 4909-5947-0701V. 14818-7263-4587V.2

[0205] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 second intermediate second chamber contains an aqueous phase, a third intermediate second chamber contains an oil phase, a fourth intermediate second chamber contains an aqueous phase, and a fifth intermediate second chamber contains an oil phase.

[0206]

[0149] In some embodiments, the third chamber contains an aqueous solution.

[0207]

[0150] In some embodiments, the first magnet is moveable in an automated fashion.

[0208]

[0151] In some embodiments, the first magnet is moveable in an manual fashion.

[0209]

[0152] In some embodiments, the second magnet is moveable in an automated fashion.

[0210]

[0153] In some embodiments, the second magnet is moveable in an manual fashion.

[0211]

[0154] In some embodiments, said first and / or second magnet comprises a handle attachment for manually moving the magnet along the track or guide, wherein the handle is substantially perpendicular to the first axis of the device.

[0212]

[0155] In some embodiments, the sample processing device further comprises a supply of nanoparticles disposed in a chamber of the device, the nanoparticles optionally comprising at least one of plasmonic outer shell and a magnetic core.

[0213]

[0156] In some embodiments, the sample processing device comprises agents in chambers thereof to effect any one or more of lysis, binding of target material to capture beads, washing, elution, and / or nucleic acid amplification.

[0214]

[0157] In some embodiments, the sample processing device further comprises a source of illumination configured to illuminate the contents of a chamber of the device.

[0215]

[0158] In some embodiments, the sample processing device further comprises in the first samplereceiving chamber magnetic material in the form of functionalized nanoparticles.

[0216]

[0159] In some embodiments, the functionalized nanoparticles are functionalized to bind one or more nucleic acids.

[0217]

[0160] In some embodiments, the functionalized nanoparticles are functionalized to bind a virus, a viral antigen, a bacteria, a bacterial antigen, a protein or a cell.

[0218]

[0161] A method of processing a sample, comprising operating a device according to claim 1 so as to process a sample liquid supplied thereto.

[0219]

[0162] In some embodiments, the method comprises at least moving the first and second magnet in an alternate fashion relative to each other and the first chamber in order to effect mixing of the sample in the first chamber by movement of magnetic material therein.

[0220] 17

[0221] 4909-5947-0701v. l4818-7263-4587v.2

[0222] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0223]

[0163] In some embodiments, the method comprises at least moving the first and second magnet relative to each other and the third chamber in order to effect mixing of the processed sample in the third chamber by movement of magnetic material therein.

[0224]

[0164] In some embodiments, the method comprises at least moving the first and / or second magnet along the track or guide from the first chamber through the intermediate second chambers to the third processed-sample chamber in order to effect the passing of the sample through the contents of the intermediate second chambers.

[0225]

[0165] A blood filtration device, the device comprising a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough, the device configured for blood sample volumes of 100 uL or less, and wherein the dual membrane size-exclusion filter comprises two membranes, with a first membrane having a pore size larger than a second membrane.

[0226]

[0166] In some embodiments, the pore size of the first membrane is 2.5 to 5 microns, optionally about 2.7 microns.

[0227]

[0167] In some embodiments, the pore size of the second membrane is 0.1 to 3.0 microns, optionally about 0.7 microns.

[0228]

[0168] In some embodiments, the membranes comprise glass fiber.

[0229]

[0169] In some embodiments, the device comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

[0230]

[0170] In some embodiments, the device is configured for blood volumes of 50 uL or less.

[0231]

[0171] A biological sample processing device, comprising: a container for placing in a chamber which chamber is magnetic or through which a magnetic force can be applied, the container containing magnetic beads having thereon a molecule which attaches to human red blood cells; a chamber which is magnetic or through which a magnetic force can be applied for receiving the container; a blood filtration component (a) in fluid connection with said container, or (b) not in fluid connection with said container, said blood filtration component configured to separate plasma from a blood sample moved therethrough by suction from a syringe or pump component.

[0232] 18

[0233] 4909-5947-0701V.14818-7263-4587V.2

[0234] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0235]

[0172] In some embodiments, the device further comprises a lid sealed or sealable on a top of the container and having a port therethrough for an attachable blood filtration component, or wherein the device further comprises a lid sealed or sealable on a top of the container and having the blood filtration component attached thereto or configured as part of said lid. In some embodiments, the container is substantially square in cross-section. For example, for a non-limiting exemplary 5ml sample size, container dimensions can be, for example, 3cm to 4cm in height (e.g., 3.4 cm). For example, for a non-limiting exemplary 5ml sample size, container dimensions can be, for example, 2 to 3 cm in width (e.g., 2.4 cm). For example, for a non-limiting exemplary 5 mL sample size, container dimensions can be, for example, 0.5 to 1.5cm in depth (front to back) (e.g., 1.0 cm). In embodiments, the container volume can be made for any size of blood sample volume desired, including volumes of 0.01 mL to 10 mL and any size in between. For example, container volumes can be of from 3 mL to 7 mL. For example, container volumes can be of from 3 mL to 6 mL. For example, container volumes can be 5 mL, or thereabouts, e.g., 4.8 mL to 5.2 mL. Larger volume containers can also be employed.

[0236]

[0173] In some embodiments, the blood filtration component comprises a dual membrane sizeexclusion filter which separates plasma from a blood sample moved therethrough.

[0237]

[0174] In some embodiments, the blood filtration component comprises a outlet port on an external face thereof for applying negative suction pressure and can be attached to the sample container, and on an opposite face thereof an inlet port such that when the blood filtration component is placed on the container, a blood sample in the sample container can be pulled therethrough.

[0238]

[0175] In some embodiments, suction pressure can applied to the container via a peristaltic pump on the outset side. In some embodiments, the peristaltic pump is operatively attached to a microprocessor controller. In some embodiments the outlet port is positioned on an external face such that it can collect fluid that has passed through an exit face of the filter of the smaller pore size. In some embodiments the inlet port is positioned on an external face of the blood filtration component, opposite the external face on which the outlet port is positioned, such that the inlet port can collect fluid and channels it towards an external face of the filter of the larger pore size. In some embodiments, the inlet port comprises a tubular component which extends into the fluid when placed on the container containing a fluid. In some embodiments, the outlet port comprises

[0239] 19

[0240] 4909-5947-0701V.14818-7263-4587V.2

[0241] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 a tubular component which extends into a receptacle for containing filtered plasma. In some embodiments, the tubular components comprise a stainless steel. In embodiments, the inlet port comprises, e.g,, a tube that extends, e.g., from the top of the container down into the container, so as to thereby be immersed in the liquid sample once the sample is placed in the container. For example, a lower end of the tube may extend 75%, 70%, 65%, 60% or 55% into the depth of the container from the lid or the top of the container. For example, a lower end of the tube may extend from 50% to 80% of the chamber height into the depth of the container from the lid or the top of the container. For example, in an embodiment the container is 3.4cm tall and the inlet tube extends down 2cm from the top of the container.

[0242]

[0176] In some embodiments, the pore size of the first membrane is 2.5 to 5 microns, optionally about 2.7 microns. In some embodiments, the pore size of the first membrane is 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 microns. In some embodiments, the pore size of the second membrane is 0.1 to 3.0 microns, optionally about 0.7 microns. In some embodiments, the pore size of the second membrane is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 microns.

[0243]

[0177] In some embodiments, the magnetic beads comprise attached thereto one or more antibodies which bind red blood cell surface markers.

[0244]

[0178] In some embodiments, the one or more antibodies which bind red blood cell surface markers comprise anti-Glycophorin A antibodies. In some embodiments, the magnetic bead concentration is 50 to 150 uL / ml per milliliter of blood sample. In some embodiments, the magnetic bead concentration 100 uL / ml per milliliter of blood sample. In some embodiments, the magnetic bead concentration is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 uL / ml per milliliter of blood sample

[0245]

[0179] In some embodiments, the membranes comprise glass fiber.

[0246]

[0180] In some embodiments, the chamber which is magnetic or through which a magnetic force can be applied is a chamber comprising permanent magnets.

[0247]

[0181] A centrifuge-free plasma separation device is provided which can process more than 2mL of blood, e.g., up to 5 mL or lOmL, at point-of care. In some embodiments, the device separates whole blood into plasma without filter membrane clogging or hemolysis. In some embodiments,

[0248] 20

[0249] 4909-5947-0701V.14818-7263-4587V.2

[0250] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 the device separates whole blood into plasma with 10% or less dilution. In some embodiments, the device separates 99% or greater, or 99.9% or greater separation of blood cells from plasma from up to 5ml blood. In some embodiments, the device or method achieves 90.0, 90.1, 90.2, 90.3,

[0251] 90.4, 90.5, 90.6, 90.7, 90.8, 90.9, 91.0, 91.1, 91.2, 91.3, 91.4, 91.5, 91.6, 91.7, 91.8, 91.9, 92.0,

[0252] 92.1, 92.2, 92.3, 92.4, 92.5, 92.6, 92.7, 92.8, 92.9, 93.0, 93.1, 93.2, 93.3, 93.4, 93.5, 93.6, 93.7,

[0253] 93.8, 93.9, 94.0, 94.1, 94.2, 94.3, 94.4, 94.5, 94.6, 94.7, 94.8, 94.9, 95.0, 95.1, 95.2, 95.3, 95.4,

[0254] 95.5, 95.6, 95.7, 95.8, 95.9, 96.0, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97.0, 97.1,

[0255] 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8,

[0256] 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% separation of blood cells from plasma. In some embodiments this is achieved in 10 minutes or less. In some embodiments, the separated plasma retains biomarkers from the subject, such as nucleic acids, proteins or lipids.

[0257]

[0182] A method of processing a blood sample, comprising: applying the blood sample into the chamber of the sample processing device described herein and incubating the sample therein for a time sufficient for red blood cells to attach to the magnetic beads via the molecule which attaches to human red blood cells and sealing the container with a lid having the blood filtration component attached thereto or configured as part of said lid; and placing the container prior to, during, or after incubating, in the chamber which is magnetic or through which a magnetic force can be applied so as to thereby hold the red blood cells bound to the magnetic beads in the chamber and extracting via suction pressure, through the blood filtration component, a red blood cell-depleted supernatant, whereby passage of the supernatant through the blood filtration component and through the outlet removes a majority of any remaining cells and platelets in the red blood cell-depleted supernatant.

[0258]

[0183] In some embodiments, the method further comprises inverting the chamber, when it contains the blood sample, at least once, so as to aid mixing, prior to incubating.

[0259]

[0184] In some embodiments the sample is incubated for a time sufficient for red blood cells bound to a magnetic bead to settle at a bottom of the chamber, optionally for about 3 to about 6 minutes.

[0260]

[0185] In some embodiments, the time sufficient is 60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115

[0261] 21

[0262] 4909-5947-0701V.14818-7263-4587V.2

[0263] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0264] ,1 16,117,118,1 19,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137, 138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159, 160, 161,162,163,164,165,166,167,168,169,170, 171,172,173,174,175,176,177,178,179,180, 181, 182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203, 204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225, 226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247, 248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269, 270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291, 292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313, 314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335, 336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357, 358,359, or 360 seconds.

[0265]

[0186] In some embodiments, time sufficient for red blood cells bound to a magnetic bead to settle at a bottom of the chamber is 6 minutes.

[0266]

[0187] In some embodiments the blood sample is 0.5 to 7.5 ml. In some embodiments, the blood sample is 5 mL. In some embodiments the blood sample is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mL.

[0267]

[0188] In some embodiments, the molecule is an antibody to a red blood cell surface antigen.

[0268]

[0189] In some embodiments, the chamber which is magnetic or through which a magnetic force can be applied surrounds a lower portion of the container when the container is placed therein and whereby red blood cells bound to the magnetic beads are held in the lower portion of the container when the supernatant is removed.

[0269]

[0190] In some embodiments, the method effects a processed blood sample having 102cells / ml or less once processed.

[0270]

[0191] In some embodiments, the method effects a processed blood sample having less than 102cells / ml once processed.

[0271]

[0192] FIG. 1 shows a biological sample processing device, generally designated by reference number 1, according to an exemplar embodiment of the present invention. The device 1 includes a housing 5 that contains or otherwise supports the various components of the device 1, which include a blood filtration module 10 and an extraction module 50.

[0272] 22

[0273] 4909-5947-0701V.14818-7263-4587V.2

[0274] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0275]

[0193] FIGS. 2A and 2B show a blood filtration module 10 of the device 1 according to an exemplary embodiment of the present invention. The blood filtration module 10 includes an input section 12, a first filter 14, a second filter 16 and an output section 18. The first and second filters 14, 16 are disposed between the input and output sections 12 and 18. O-rings 13 and 17 are disposed between the filters 14, 16 and the input and output sections 12, 18. The blood filtration module 10 is configured to filtrate plasma from blood. In exemplary embodiments, the first filter 14, which is disposed above the second filter 16, has a larger pore size as compared to that of the second filter 16. The larger pore membrane functions to block larger blood components (such as white blood cells and some red blood cells), while the smaller pore membrane blocks the remaining cells and allows plasma and any present virus particles to pass through. Healthy red blood cells range from about 7-9 pm in diameter, so filtration using subsequent membranes of smaller pore sizes down to <1 pm effectively filters red blood cells (RBCs), white blood cells (up to 20 pm) and platelets (3-4 pm), while retaining the constituents of plasma (i.e. water, coagulants, proteins, viruses, and nucleic acids) (24). In exemplary embodiments, the first filter 14 may have a pore size of 2.7 pm and the second filter 16 may have a pore size of 0.7 pm.

[0276]

[0194] The first and second filters 14, 16 are preferably made of glass fiber, although other materials may be used, including, for example, polycarbonate or asymmetric polysulfone membrane (commercially available from Cytiva US LLC, Marlborough, Massachusetts, USA).

[0277]

[0195] The input section 12 includes an input port 13 and the output section 18 includes an output port 19. As explained in more detail below, the input section 12 receives a blood sample through the input port 13 and filtrate is removed from the output port 19 of the output section 18.

[0278]

[0196] In exemplary embodiments, the blood filtration module 10 is generally disk-shaped, with the ports 13, 19 surrounded by walls that protrude upwards from the top and bottom surfaces of the module 10.

[0279]

[0197] In exemplary embodiments, for compatibility with point-of-care diagnostic use cases, using small volumes of fingerstick capillary blood, the blood filtration module 10 is configured to accommodate up to 50 pL of blood. It should be appreciated that the blood filtration module 10 may be configured to accommodate less or more volumes of blood. A volume of 50 pL may be chosen to maximize assay sensitivity and ensure practicality with fmgerstick blood collection, as collection of larger volumes (>100 pL) is difficult with a single fmgerstick (25). Further, the blood

[0280] 23

[0281] 4909-5947-0701V.14818-7263-4587V.2

[0282] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 filtration module 10 is preferably configured to be compatible with capillary commercially available blood collection and dispensing tools (including ones sized to collect 50 pL, such as the Minivette POCT (Sarstedt)) to simplify the workflow and improve reproducibility. For example, the user may collect a capillary blood sample using the Minivette tool and dispense the sample into the inlet of the blood filtration module 10.

[0283]

[0198] The primary advantage of the dual-layer membrane approach is stringent filtration of blood components while limiting membrane clogging, maximizing the blood volume that is able to be filtered. It was found that filtration of 50 pF of blood with only a 2.7 pm glass fiber membrane resulted in inefficient filtration and red blood cell breakthrough, likely due to red blood cells’ ability to deform to minimize resistance to flow (26). Using only the 0.7 pm glass fiber membrane led to improved filtration but resulted in membrane clogging, leading to significant hemolysis. Hemolysis is undesirable, as the release of heme from lysed RBCs is known to cause PCR inhibition (27). Overall, it was found that glass fiber membranes with a pore size of 2.7 pm followed by 0.7 pm enabled efficient filtration while minimizing clogging and hemolysis.

[0284]

[0199] Glass fiber exhibits high liquid absorption, enabling high recovery of plasma when applying negative pressure. Polycarbonate membranes (of 3 pm and 0.4 pm pore sizes) have more consistent pore sizes and densities, but present a higher degree of hemolysis compared to the dual glass fiber design. Decreased volume recovery were also observed when using two polycarbonate membranes, likely due to low liquid absorption properties of polycarbonate. The asymmetric Vivid plasma separation membrane, used by several other membrane-based blood filtration approaches, exhibits inefficient filtration and red blood cell breakthrough compared to dual glass fiber membranes.

[0285]

[0200] FIGS. 3A, 3B and 3C show the extraction module 50 of the device 1 according to an exemplary embodiment of the present invention. The extraction module 50 includes a filter holder 52, an extraction cartridge 54, an upper track 56, and upper arm (or upper handle attachment) 58, a lower track 60 and a lower arm (or lower handle attachment) 62. The extraction cartridge 54 include the following components disposed in order along a common axis: first sample-receiving chamber 70, one or more intermediate second chambers 72, and a third processed-sample chamber 74. The first, second, and third chambers 70, 72, 74 are sequentially in fluid communication with adjacent chamber(s). The chambers 70, 72, 74 have a top surface and a bottom surface, and all

[0286] 24

[0287] 4909-5947-0701V.14818-7263-4587V.2

[0288] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 chambers 70, 72, 74 are configured to contain a fluid so as to form a continuous fluid pathway from the first chamber(s) 70 through the intermediate second chambers 72 to the third chamber 74.

[0289]

[0201] The first chamber 70 is generally cylindrical shaped and contains a liquid immiscible with a liquid in an adjacent intermediate second chamber 72. In exemplary embodiments, the first chamber 70 contains an aqueous solution containing agents to effect lysis and / or binding of target material. For example, the first chamber 70 may contain a lysis buffer, a purification buffer, proteinase K, and magnetic material functionalized for binding nucleic acids. The magnetic material may be in the form of beads, and the beads may be nanoparticles that are functionalized to bind one or more nucleic acids. In exemplary embodiments, the nanoparticles are functionalized to bind a virus, a viral antigen, a bacteria, a bacterial antigen, a protein or a cell. The nanoparticles may include a plasmonic outer shell and / or a magnetic core.

[0290]

[0202] The intermediate second chamber 72 immediately adjacent to the first chamber contains an oil. In exemplary embodiments, in sequential order between the first sample-receiving chamber 70 and the third processed-sample chamber 74, a first intermediate second chamber 72 contains an oil phase, a second intermediate second chamber 72 contains an aqueous phase, a third intermediate second chamber 72 contains an oil phase, a fourth intermediate second chamber 72 contains an aqueous phase, and a fifth intermediate second chamber 72 contains an oil phase. In exemplary embodiments, the aqueous phase includes a wash buffer. In exemplary embodiments, the oil phase may include mineral oil. In exemplary embodiments, the intermediate second chambers 72 are a plurality of chambers and are an odd number of chambers in total (e.g., three, five or seven chambers in total).

[0291]

[0203] The third chamber 74 contains an aqueous solution that includes an elution buffer.

[0292]

[0204] A first magnet is disposed on or otherwise held by the upper arm 58 and a second magnet is disposed on or otherwise held by the lower arm 62. The first and second magnets and the first, second and third chambers 70, 72, 74 are configured so that the first and second magnets can be moved relative to the chambers 70, 72, 74, where movement of the first and second magnets can be achieved independent of one another. Relative movement of either the first and / or second magnet along the common axis from the first chamber to the third chamber gives rise to transfer

[0293] 25

[0294] 4909-5947-0701V.14818-7263-4587V.2

[0295] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 of magnetic material from the first chamber 70, when contained therein, through the second chambers 72 and into the third chamber 74.

[0296] [2051 The upper arm 58 that holds the first magnet is present in or on the upper track 56. The upper track 56 is adjacent to and disposed over the top surfaces of the first, second and third chambers 70, 72, 74. The upper track 56 guides movement of the upper arm 58 so that the first magnet is slidable across the top of the first, second and third chambers 70, 72, 74.

[0297]

[0206] The lower arm 62 that holds the second magnet is present in or on the lower track 60. The lower track 60 is adjacent to and disposed below the bottom surfaces of the first, second and third chambers 70, 72, 74. The lower track 60 guides movement of the lower arm 62 so that the second magnet is slidable along the bottom of the first, second and third chambers 70, 72, 74.

[0298]

[0207] In exemplary embodiments, channels 80 extend between adjacent chambers 70, 72, 74 for fluid communication therebetween. The channels 80 preferably have a circular cross section with triangular narrowing between chambers 70, 72, 74 (FIG. 3C). It has been found that the triangular narrowing aids in guiding magnetic particles towards the centerline to cross into subsequent chambers 70, 72, 74.

[0299]

[0208] It has been found that increasing volumes of liquid in the lysis / binding chamber result in leakage of reagents into the oil phase, owing to the significantly lower surface tension of lysis buffers containing detergents compared to water (44). As discussed by previous studies utilizing immiscible phase separation (9), the separation of the aqueous and oil phases during device loading relies upon the dominance of surface tension over gravity. This relationship can be quantified using Bond number (Bo = pgL2 / y), and Bo«l indicates that surface tension forces dominate the effects of gravity; the L2term (characteristic length, or phase interface area at the barrier between oil and reagent in this case) dominates this relationship, thus the aim was to minimize L in order to minimize Bo. Experimentally, observations demonstrated phase interface influenced how well the barrier was maintained without leaking for the lysis chamber. Decreasing sizes of phase interfaces were first test, and found that smaller interfaces better maintained the barrier (module design with larger interface 2 x 0.5mm leaked more severely than 1 x 0.5mm design, when adding 200pL sample volume). Additionally, oil volume held in the oil chamber appeared to be important in maintaining the interface. Square oil chambers holding lOOpL oil, compared to 20-30pL oil in previous, with 1 x 0.5mm interface did not leak at 200pL sample volume, yet began to show signs

[0300] 26

[0301] 4909-5947-0701V.14818-7263-4587V.2

[0302] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 of encroachment of the oil barrier at this sample volume, likely indicating that 200 pL approaches the lysis chamber’s volume capacity for that geometry with a larger phase interface. Thus, combining the concepts of minimizing phase interface and increasing oil volume, a circular oil chamber design was created to hold lOOpL oil with a 0.5 x 0.25mm phase interface that could accommodate 200 pL without leaking or signs of encroachment into the oil. The lysis / binding chamber can accommodate a sample volume of up to 300 pL without leaking into the first oil chamber, which is well within the expected yield of the filter (200pL average yield). Sample volumes up to 450 pL can be added without leaking into subsequent reagent chambers.

[0303]

[0209] In exemplary embodiments, the first and second magnets may be moveable in an automated or manual fashion. For example, the upper and lower arms 58, 62 may be operatively connected to one or more actuators (e.g., one or more motors) that are controllable to cause automated movement of the arms 58, 62. Alternatively, one or both of the upper and lower arms 58, 62 are manually slidable relative to the housing 5 so that a user can manually adjust the position of the first and second magnets using the upper and lower arms 58, 62. As discussed in further detail below, movement of the first and second magnets in an alternate fashion relative to each other and to the first chamber 70 results in mixing of the sample in the first chamber 70 by movement of the magnetic material disposed in the first chamber 70, movement of the first and second magnets relative to each other and to the third chamber 74 results in mixing of the processed sample in the third chamber 74 by movement of the magnetic material contained in the third chamber 74, and movement of the first and / or second magnet along the tracks 56, 60 from the first chamber 70 through the intermediate second chambers 72 to the third processed-sample chamber 74 results in the passing of the sample through the contents of the intermediate second chambers 72.

[0304]

[0210] FIG. 4 shows an extraction process using the extraction module 50 according to an exemplary embodiment of the present invention. The chambers of the module 50 are pre-loaded with reagents, and the extraction process functions as follows: 1) plasma sample is added (via dualmembrane filtration as in FIG. 2B, or conventionally separated plasma) to the first chamber containing lysis buffer, purification buffer, proteinase K, and magnetic beads functionalized for binding nucleic acids. Magnet-assisted mixing is performed by alternating presence of upper and lower magnets above and below the chamber, respectively, to draw beads through solution. Beads are then collected with the lower magnet and transferred through the oil chambers into the

[0305] 27

[0306] 4909-5947-0701V.14818-7263-4587V.2

[0307] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 subsequent washes and finally into the elution chamber, where the magnet mixing is performed again before removal of eluate from the final chamber.

[0308] [2111 FIG. 5 illustrates a workflow for processing whole blood at the point of care using the device 1 in accordance with an exemplary embodiment of the present invention. Specifically, the device 1 integrates plasma separation and RNA extraction for processing of whole blood at the point of care. The device 1 operates with as little as 50 pL of whole blood sample (which is compatible with fingerstick blood collection methods), drawn up by user with a tool that is then used to dispense the blood into the plasma separation component of the device. The dualmembrane filter separates out plasma from the sample, which is then withdrawn by user with a syringe from the filter to be dispensed into the extraction component of the device. Extraction is accomplished by using a magnetofluidic immiscible phase reagent separation approach to purify the RNA for PCR performed off-device. Overall, for the user, no pipetting steps are needed upstream of the cartridge (commonly used blood collection tools such as Minivette POCT (Sarstedt) or MICROSAFE tube (Drummond Scientific) fit right into the filter), the plasma separation step involves fluid handling with similar complexity to running LFA rapid tests, and the nucleic acid extraction requires the user to move two magnets either around a chamber to mix or along tracks to move the beads.

[0309]

[0212] The following Example illustrates further exemplary details and advantages of the present invention.

[0310]

[0213] EXAMPLE 1

[0311]

[0214] A device was fabricated that integrated both a blood filtration module and an extraction module as described in accordance with various exemplary embodiments of the present invention. The following provides the fabrication details of the device used in this Example.

[0312]

[0215] DEVICE FABRICATION

[0313]

[0216] Fabrication of plasma filter for dual-membrane size-exclusion filtration:

[0314]

[0217] Filters were designed using AutoCAD Fusion360 and 3D printed using VeroWhite resin on a Stratasys Objet 30 Pro. Filters were composed of two identical printed pieces: a syringe inlet port piece and an outlet piece. Each piece included a ringed spoke design printed on the surface in contact with the membranes to help liquid distribute evenly across the membranes. Squareprofile 20mm ID O-rings (1171N113, McMaster-Carr) were inserted into the syringe inlet port

[0315] 28

[0316] 4909-5947-0701V.14818-7263-4587V.2

[0317] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 and outlet layers. 2.7 pm and 0.7 pm pore size glass fiber membranes (APFD02500 and APFF04700, Millipore) were cut to a diameter of 25mm using a leather hole puncher. The membranes were stacked with the 2.7 pm pore size on top facing the syringe port and the 0.7 pm membrane pore size at the outlet piece. The pieces were assembled with edges sealed using lightcure adhesive (Loctite 3525) and cured with 3000 flashes in a UV curing unit (Otoflash G171).

[0318]

[0218] Fabrication of magnetofluidic nucleic acid extraction module:

[0319]

[0219] The module was 3D-printed with open chambers using PlasClear resin (Asiga) in an Asiga Max X 3D printer, cleaned with 100% isopropyl alcohol, and cured with 2000 flashes in the UV curing unit. The chambers were sealed to a sheet of acrylic (PMMA) measuring 60 x 15 x 0.25 mm using light-cure adhesive and cured using 2000 flashes in the UV curing unit. The overall module was 65 mm x 18 mm x 3.25 mm, with raised edges on both ends of the long side to hold the module in the designated space above the magnetic track. The chambers were designed as cylindrical for easy reagent filling; the lysis chamber had a diameter of 15 mm and height of 3.25 mm to hold a maximum of approximately 575 pL of liquid. The wash chambers had diameters of 6 mm and heights of 3.25 mm to hold a maximum of approximately 90 pL of liquid. The elution chamber had a diameter of 4 mm and height of 3.25 mm to hold a maximum of approximately 40 pL of liquid. Chambers for oil connected the chambers (sized 7 mm diameter for the oil after the larger lysis chamber, and 5 mm for the other two oil chambers), with triangular transitions between all chambers to aid in guiding beads for transfer (length of 3 mm between lysis chamber and first oil chamber, and 1 mm between all other chambers). All dimensions are shown in FIG. 3C. Reagents and oil could be pre-loaded directly into chambers from above via pipette.

[0320]

[0220] Fabrication of magnetic track for holding extraction module:

[0321]

[0221] The magnetic track was 3D-printed using VeroWhite Plus resin (Stratasys) in a Stratasys Objet 30 Pro 3D printer. Printed components were post-processed with an alkaline cleaning bath and dried in an oven at 40°C. The lower track on which the extraction module sits was designed to guide a cylindrical 12.7 x 12.7 mm neodymium magnet (D88-N52, K&J Magnetics) in four cardinal directions on one plane, held by a 3D-printed arm and manually controlled by the user. The extraction module was held in a slot on the lid of the magnetic track to position it above the lower track. The upper track to hold a magnet for mixing the magnetic beads in the module chambers was designed to guide a cylindrical 6.4 mm x 3 mm neodymium magnet (D42-N52, K&J

[0322] 29

[0323] 4909-5947-0701V.14818-7263-4587V.2

[0324] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0325] Magnetics) from left to right on one axis above the module chambers; it could also be flipped backward away from the module by rotating in place on the axis. A fdter holder was designed into which the filter could be slotted and removed as needed, for use with experiments involving whole blood starting samples. The holder positioned the filter’s outlet above the first chamber of the extraction module for initial blood sample deposit and PBS flush into inlet facing upward, to direct any early filtrate droplets that may come through the outlet before withdrawal via syringe. The holder could be flipped 180°, as needed for filtrate withdrawal, while still holding the filter securely and positioning the filter, now facing with outlet port upward, outside of the track away from the extraction module to allow for filtrate withdrawal via syringe.

[0326]

[0222] The following test methods were used to determine performance of the fabricated integrated device:

[0327]

[0223] TEST METHODS

[0328]

[0224] Separation efficiency of plasma from blood:

[0329]

[0225] Healthy control human whole blood was purchased from HumanCells Biosciences; all specimens were de-identified and tested negative for HIV1 / 2, HBV, HCV and syphilis prior to receipt. Whole blood was stored at 4°C and handled using BSL-2 facilities and protocols. For plasma separation using the filter, 50 pL of whole blood was inserted into the inlet port of the filter directly on top of the membrane using a pipette. A 1 mL Luer slip tip syringe pre-loaded with 450 pL of PBS was inserted into the inlet port, and the plunger was slowly pushed to push plasma through the membrane. The syringe was removed and the inlet was then sealed using a 3D-printed plug (PlasClear resin, Asiga). The filter was flipped over and the syringe was then placed into the outlet port and drawn upwards, drawing filtered plasma into the syringe using negative pressure. Once the plunger reached the end of the syringe, the filtered plasma was expelled either directly onto the first chamber of the extraction module or into a clean 1.5 mL tube.

[0330]

[0226] Gold-standard controls were centrifuged at 1,500g for 10 minutes, and plasma supernatant was removed carefully using a pipette into a clean 1.5 mL tube. 10 pL of each filter and centrifugation control sample were then diluted 1 : 1 with Trypan Blue (final volume 20 pL) and added to an Invitrogen Countess cell counting slide (Cl 0228, ThermoFisher Scientific). Input whole blood comparison samples were diluted 1000-fold prior to addition of Trypan Blue. Cell

[0331] 30

[0332] 4909-5947-0701V.14818-7263-4587V.2

[0333] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 counts were obtained using an automated cell counter (Invitrogen Countess 3, ThermoFisher Scientific).

[0334] [2271 Determination of hemolysis from plasma separation:

[0335]

[0228] The level of hemolysis was determined by measuring absorbance of each sample separated using the filter and gold-standard centrifugation at 414 nm, which is a known absorbance peak of hemoglobin (40) using a NanoDrop One spectrophotometer (ThermoFisher Scientific). 2 pL of each sample was measured using the spectrophotometer, using PBS as a blank. Gold-standard centrifugation and fully lysed whole blood samples were diluted to the same volume as the filtered plasma samples using PBS and used as controls. The obtained absorbance values were baseline- corrected.

[0336]

[0229] Viral recovery with plasma filter:

[0337]

[0230] 5 pL of inactivated HCV (NATHCV-0005, ZeptoMetrix) was spiked into 45 pL whole blood for a final concentration of 6.77 x 103 lU / mL. Spiked 50 pL whole blood samples were separated into plasma using the filter and gold-standard centrifugation. Resulting volumes of separated plasma were measured using a micropipette. HCV viral RNA was extracted from resulting plasma samples using a modified, optimized benchtop extraction protocol using the Invitrogen ChargeSwitch gDNA Mini Tissue Kit (ThermoFisher Scientific). RNA was quantified via RT-qPCR through generation of a standard curve using a range of concentrations of HCV quantitative synthetic RNA (VR-3233SD, ATCC). Virus recovery in separated plasma was back- calculated using the quantified concentration of eluted RNA with the assumption of 50% RNA extraction efficiency.

[0338]

[0231] Evaluation of plasma separation across range of hematocrit levels and blood sample input volumes:

[0339]

[0232] To evaluate the filter at a range of blood volumes and various hematocrit levels, blood samples at artificial blood hematocrit levels (50%, 55%, and 60%) were prepared by centrifuging whole blood at 1,500g for 10 minutes, removing the separated plasma to calculate the leftover volume of packed blood cells (while hematocrit refers to red blood cells percentage only, this is a close estimate, as the packed blood cells are >99% RBC (41,42). Plasma was then added back at appropriate volumes to generate the correct total % RBC in each sample. Different volumes of

[0340] 31

[0341] 4909-5947-0701V.14818-7263-4587V.2

[0342] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 the artificially-created hematocrit blood were added (25, 50, and 75pL) to filters and filtrate was collected for analysis. Hemolysis and cell count of filtrate were tested as described above.

[0343] [2331 HCV RNA extraction with benchtop protocol:

[0344]

[0234] The ChargeSwitch gDNA Mini Tissue kit (ThermoFisher Scientific) was selected for extraction, as it utilizes pH-induced nucleic acid binding and does not require alcohol-based washing. The ChargeSwitch protocol was modified and optimized for reduced reaction volumes. First, a lysis and binding mix was prepared, consisting of 140 pL of lysis buffer, 20 pL of proteinase K (20 pg / mL), 50 pL N5 purification buffer, and 8 pL of ChargeSwitch magnetic beads per reaction for a 200 pL filtered plasma input. These volumes were modified slightly to scale with different sample volumes, as needed (i.e. for undiluted centrifuged plasma samples with an expected volume of 25 pL, the reaction consists of 17.5 pL of lysis buffer and 6.25 pL of purification buffer, with the same volumes of proteinase K and magnetic beads). The mixture was added to each plasma sample in a 1.5 mb tube, gently pipette mixed ten times, and allowed to incubate at room temperature for 5 minutes. The samples were placed on a 1.5 mb magnetic rack (New England Biolabs) until the beads were separated out of solution, and the supernatant was removed using a pipette. The beads were washed twice with 200 pL of wash buffer (40pL for undiluted centrifuged plasma samples). 30 pL of elution buffer was added and the beads were pipette mixed ten times to resuspend. After a 5-minute incubation at room temperature, the beads were once again separated using the magnetic rack, and the eluted RNA was transferred to a clean PCR tube for further processing. The same reagent volumes were used in the magnetofluidic extraction module for a 200 pL plasma input, with the exception of 90 pL of wash buffer used in the wash chambers instead of 200 pL.

[0345]

[0235] Immiscible-phase magnetofluidic RNA extraction with whole Hepatitis-C virus in human plasma:

[0346]

[0236] Initial characterization of the RNA extraction module (optimizing magnet size, mixing, and washing steps) was performed by spiking inactivated HCV into DNA-cleared plasma for a final concentration of 6.77 x 103 lU / mL. The spiked plasma sample was added to the lysis / binding chamber and incubated for 3 minutes. The beads were then mixed by alternating the mixing magnet on top of the chamber with the extraction magnet underneath the chamber 10 times. Beads were then collected at the bottom of the chamber and transferred horizontally through the oil phase

[0347] 32

[0348] 4909-5947-0701V.14818-7263-4587V.2

[0349] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 into each wash chamber before transfer to the elution chamber. The beads incubated in the elution chamber for 3 minutes. The beads were mixed ten times and resuspended using the mixing magnet for an additional 2-minute incubation. The extraction magnet was used to separate the beads from solution, and the 30 pL eluate was transferred to a clean PCR tube using a pipette.

[0350]

[0237] To evaluate the limit of detection of magnetofluidic RNA extraction, serial dilutions of inactivated HCV in PBS were spiked into DNA-cleared plasma (System Biosciences) to obtain concentrations from 1.3 x 104 lU / mL to 85.5 lU / mL, with three replicates per concentration. Performance at each concentration was compared to the optimized benchtop extraction protocol.

[0351]

[0238] RT-qPCR quantification of extracted RNA:

[0352]

[0239] Previously published primers and probes designed from the conserved 5’ untranslated region (UTR) of the HCV genome were used to amplify HCV RNA (MAD-1 primer 5'- TGCTAGCCGAGTAGYGTTGG-3', MAD-2 primer 5'-ACTCGCAAGCACCCTATCAG-3', and MAD-3 probe 5'-ACCACAAGGCCTTTCGCGAC-3') (43). The RT-qPCR reaction mix consisted of PrimeScript One-Step RT-PCR master mix (lx final concentration), forward and reverse primers (500 nm), and probes (125 nm) with a final volume of 20 pL. RT-qPCR was performed using an Applied Biosystems QuantStudio 3 thermocycler (ThermoFisher Scientific) using the following cycling conditions: 52°C for 5 minutes, 95°C for 10 seconds, and 50 cycles of 95°C for 5 seconds and 60°C for 30 seconds. For quantification of extracted RNA concentration, a standard curve was generated using ten-fold serial dilutions of HCV quantitative synthetic RNA (ATCC, VR-3233SD) from 8.3 x 107copies / mL to 830 copies / mL, with three technical replicates per concentration. A best fit equation generated from the plot of Ct vs. log concentration was used to calculate PCR efficiency and estimate the concentration of extracted RNA based on the average Ct of three technical replicates.

[0353]

[0240] The integrated device fabricated as described above was used in the following workflow for RNA extraction from whole Hepatitis-C virus in human whole blood:

[0354]

[0241] WORKFLOW

[0355]

[0242] To investigate limit of detection of the integrated system, five serial dilutions of inactivated HCV spiked into whole human blood were prepared as samples: 1.35 x 104lU / mL, 6.77 x 103lU / mL, 3.39 x 103lU / mL, 1.71 x 103lU / mL, and 8.55 x 102lU / mL, as well as an no-template

[0356] 33

[0357] 4909-5947-0701V.14818-7263-4587V.2

[0358] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 control with TE buffer spiked into whole human blood. Three experimental replicates of each concentration were performed (three cartridge extractions per concentration).

[0359] [2431 FIG. 6 shows the workflow for separating plasma from the starting blood sample. The filter was placed in the holder of the integrated device to position the outlet directly above the first chamber of the extraction module. A pipette was used to generate a blood bead of 50 p , simulating a fingerstick blood bead, then a Minivette POCT (Sarstedt) capillary collection tube was used to draw up the blood. The collection tool was then used to add the blood directly to the top of the filter by pressing down on the plunger. 450 pF of PBS was next flushed into the inlet port of the filter via ImL slip tip syringe. The inlet was plugged with a 3D printed plug, then the filter was flipped 180° with the filter holder to position it away from the extraction module with outlet port facing upward. The syringe, in a starting position with the plunger pressed all the way down, was then inserted into the outlet port and used to draw up filtrate from the filter. The syringe was then used to deposit the filtrate into the first chamber of the extraction module. The extraction workflow described previously was then performed using the magnets. The entire workflow is shown in FIG. 7.

[0360]

[0244] A lab-based workflow was performed as a standard comparison. Inactivated HCV was spiked into whole human blood, and the blood samples were centrifuged at 1,500g for 10 minutes to separate the plasma. Separated plasma was pipetted off the top of the separated samples and used in the benchtop extraction protocol described previously.

[0361]

[0245] RT-PCR was performed using eluted samples; each experimental replicate was run in triplicate in the PCR plate on the QuantStudio 3 system, according to RT-PCR protocol described previously.

[0362]

[0246] All statistical analysis was performed with GraphPad Prism 10. All data were collected in triplicate (n=3) unless otherwise specified, t-tests were used for comparisons between two groups. Post-hoc multiple comparisons were performed when necessary using one-way ANOVA Tukey’s multiple comparisons test, or two-way ANOVA Sidak’s multiple comparisons test when multiple conditions were being compared at once.

[0363]

[0247] The following discussion provides results of testing performed using the integrated device fabricated as described above (the term “PRECISE filter” in the charts refer to the integrated device fabricated as described above).

[0364] 34

[0365] 4909-5947-0701V.14818-7263-4587V.2

[0366] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0367]

[0248] TEST RESULTS - I

[0368]

[0249] As shown in FIG. 8, plasma separation performance of the filter demonstrates its ability to separate the majority of cells from the blood sample with no significant difference from lab-based centrifugation.

[0369]

[0250] As shown in FIG. 9 filtration causes minimal hemolysis of red blood cells, with no significant difference from centrifugation control. Hemolysis measured via hemoglobin absorbance peak at 414 nm peak and compared to fully lysed whole blood.

[0370]

[0251] FIG. 10 show images of filter after filtering blood. The red color at input (left) from the blood sample on the membrane contrasts with the yellow color at output (middle) where only PBS- diluted plasma filtrate exits the filter, indicating successful retention of blood cells in the membranes. Images of the filtrate (right) are included, for three replicates after processing of 50 pL of whole blood.

[0371]

[0252] FIG. 11 shows comparison of volume recovery of filter and centrifugation methods of plasma separation. The filter produced 201.8 ± 18.75 pL filtrate after input of 50pL blood and 450pL PBS, and centrifugation of 50pL blood produced 24.6 ± 3.35 pL plasma.

[0372]

[0253] FIG. 12 shows virus recovery from plasma separated from blood, spiked with whole inactivated HCV virus at 6.77 x 103lU / mL final concentration in blood, using filter and traditional centrifugation methods, extracted via benchtop RNA extraction protocol and amplified via RT- PCR. Recovery was calculated by comparing Ct values to RNA standard curve and demonstrated no significant difference between filter and centrifugation separation methods.

[0373]

[0254] FIG. 13 shows recovered volumes of filtrate across hematocrit levels (50%, 55%, and 60%) and blood sample input volumes (25, 50, and 75 pL). There was no significant difference in recovered volumes across hematocrit levels. There was an observed trend in higher recovered volume with higher blood input, with significantly higher filtrate volume for 75 pL blood vs. 25 pL irrespective of hematocrit (p = 0.0012).

[0374]

[0255] FIG. 14 shows hemolysis level in filtrate across hematocrit levels and blood sample input volumes, measured at absorbance peak 414 nm for hemoglobin. Within blood sample input volumes, there was no significant difference in hemolysis between hematocrit levels. There was a significant difference between 25 pL and 75 pL (p = 0.0219) and 50 pL and 75 pL (p = 0.0237)

[0375] 35

[0376] 4909-5947-0701V.14818-7263-4587V.2

[0377] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 irrespective of hematocrit level, but there was no significant difference in hemolysis between 25 pL and 50 pL groups.

[0378] [2561 FIG. 15 shows that use of the upper magnet for magnet-assisted mixing improves RNA extraction performance on the module, as demonstrated by the differences in RT-PCR amplification between no magnet-assisted mixing, mixing 3 times, and mixing 10 times in the lysis / binding and elution steps.

[0379]

[0257] FIG. 16 shows representative RT-PCR curves for HCV RNA extraction using the extraction module and with benchtop magnetic bead extraction kit, from whole, inactivated HCV spiked into 25 pL of DNA-cleared plasma (estimated volume of plasma from 50pL blood sample) and diluted by PBS to 200pL to represent the estimated sample volumes determined from Fig. 2f, shown at 6,770 lU / mL starting concentration in plasma sample.

[0380]

[0258] FIG. 17 shows RT-PCR amplification at varying concentrations of whole, inactivated HCV spiked into 25 pL of DNA-cleared plasma and diluted by PBS to 200pL. All replicates amplified for both cartridge and benchtop comparison at 855 lU / mL, with no significant differences between extraction methods for any concentration.

[0381]

[0259] FIG. 18 shows detection of varying concentrations of whole, inactivated HCV spiked into 50pL blood when processed with lab-based workflow (centrifugation for plasma separation, benchtop magnetic bead protocol for RNA extraction) and the fabricated integrated device (integrated blood filter for plasma separation and magnetofluidic immiscible-phase cartridge for RNA extraction). Extracted samples from both approaches were amplified for detection via RT- PCR on QuantStudio 3 system. All replicates (n=3, each technical replicate run in triplicate on PCR plate) for the integrated device and lab-based workflow amplified at 6,770 lU / mL with no significant difference between workflows.

[0382]

[0260] FIG. 19 shows real-time fluorescence curves of RT-PCR comparing lab-based workflow and the integrated device for sample preparation from whole blood at 13,540 lU / mL.

[0383]

[0261] Error bars in all figures indicate mean ± standard deviation. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0384]

[0262] FIGS. 20A- 20D illustrate the advantages of the dual membrane structure according to exemplary embodiments of the present invention.

[0385] 36

[0386] 4909-5947-0701V.14818-7263-4587V.2

[0387] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0388]

[0263] FIG. 20A shows plasma separation performance for filters with various combinations of single or dual membranes. Dual membrane filtration with 2.7 pm and 0.7 pm pore size glass fiber membranes demonstrated the highest filtration efficiency indicated by low cell counts remaining in filtered plasma, compared to single glass fiber membranes, dual polycarbonate membranes (3.0 pm and 0.4 pm), and the Vivid plasma separation membrane.

[0389]

[0264] FIG. 20B shows evaluation of hemolysis in plasma filtered by membranes with various combinations of single or dual membranes. Dual membrane filtration with 2.7 pm and 0.7 pm pore size glass fiber membranes demonstrated the lowest degree of hemolysis, indicated by low absorbance at 414 nm, compared to dual polycarbonate membranes and a single 0.7 pm glass fiber membrane.

[0390]

[0265] FIG. 20C shows recovered volume of plasma filtered by dual glass fiber (2.7 pm and 0.7 pm) and polycarbonate membranes (3.0 pm and 0.4 pm). The dual glass fiber design yielded a higher average volume of 201.8 pL ± 18.75 pL, while filtration with dual polycarbonate membranes resulted in 137.9 ± 24.13 pL of filtrate.

[0391]

[0266] FIG. 20D show images of filter input, output, and filtrate for various combinations of dual and single membranes. Dual glass fiber membranes demonstrated high red blood cell filtration and low hemolysis, indicated by the red color at the filter input showing successful retention of red blood cells in the membranes with a clear yellow color at the output and filtrate. Single 2.7 pm and Vivid membranes showed limited filtration, shown by the appearance of red blood cells in the filtrate. Dual polycarbonate membranes and single 0.7 pm membranes resulted in higher hemolysis, indicated by the red tint in the filtrate and at the filter outputs.

[0392]

[0267] FIG. 21 shows a process of using the extraction module according to an exemplary embodiment of the present invention. The upper magnet moves vertically with respect to the extraction module to resuspend magnetic beads in the chamber, while the upper magnetic track moves horizontally to move the mixing magnet between the lysis / binding and elution chambers. The lower magnet moves vertically to draw and release magnetic beads and horizontally to transfer magnetic beads between chambers.

[0393]

[0268] FIGS. 22A-22D illustrate the advantages of the extraction module according to exemplary embodiments of the present invention.

[0394] 37

[0395] 4909-5947-0701V.14818-7263-4587V.2

[0396] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0397]

[0269] FIG. 22A shows evaluation of the effect of various bottom surfaces on cartridge RNA extraction performance by PCR Ct. There is higher variability in RNA extraction performance on cartridges with 3D-printed resin bases, likely due to variable surface roughness during DLP 3D- printing and post-processing. Cartridges with acrylic (PMMA) bases show similar performance to benchtop RNA extraction (p=0.2677) while there is a significant difference in Ct between benchtop extraction and cartridge extraction with a resin base (p=0.0139).

[0398]

[0270] FIG. 22B shows PCR amplification curves of cartridge-extracted RNA using various magnet sizes. The 12.7 x 12.7 mm cylindrical magnet demonstrated slightly earlier amplification than the smaller 6.35 x 3.18 mm cylindrical magnet, potentially due to decreased magnetic bead loss due to stronger magnetic field.

[0399]

[0271] FIG. 22C shows evaluation of the effect of number of wash chambers on RNA extraction performance. There was no significant difference in cartridges with one wash chamber vs. two wash chambers (p = 0.4999), but there was more variability in Ct in cartridges with only one wash chamber.

[0400]

[0272] FIG. 22D shows PCR amplification curves of cartridge-extracted RNA with and without on-cartridge mixing in the wash chambers. Mixing in the wash chambers resulted in delayed amplification compared to cartridge extraction runs where there was no mixing, which was similar in performance to benchtop RNA extraction.

[0401]

[0273] REFERENCES

[0402] 1. Al-Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001 Feb;39(2):485-93.

[0403] 2. Wang S, Sarenac D, Chen MH, Huang S-H, Giguel FF, Kuritzkes DR, et al. Simple filter microchip for rapid separation of plasma and viruses from whole blood. Int J Nanomedicine. 2012 Sep 17;7:5019-28.

[0404] 3. Su X, Zhang J, Zhang D, Wang Y, Chen M, Weng Z, et al. High-Efficiency Plasma Separator Based on Immunocapture and Filtration. Micromachines (Basel). 2020 Mar 28; 11(4).

[0405] 4. Liu C, Liao S-C, Song J, Mauk MG, Li X, Wu G, et al. A high-efficiency superhydrophobic plasma separator. Lab Chip. 2016 Feb 7;16(3):553-60.

[0406] 38

[0407] 4909-5947-0701V.14818-7263-4587V.2

[0408] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0409] 5. Gong MM, Macdonald BD, Vu Nguyen T, Van Nguyen K, Sinton D. Field tested milliliter-scale blood fdtration device for point-of-care applications. Biomicrofluidics. 2013 Aug 5;7(4):44111.

[0410] 6. Ali N, Rampazzo R de CP, Costa ADT, Krieger MA. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. Biomed Res Int. 2017 Jul 12;2017:9306564.

[0411] 7. Sur K, McFall SM, Yeh ET, Jangam SR, Hayden MA, Stroupe SD, et al. Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid. J Mol Diagn. 2010 Sep;12(5):620-8.

[0412] 8. Ngo HT, Jin M, Trick AY, Chen F-E, Chen L, Hsieh K, et al. Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument. Anal Chem. 2023 Jan 17;95(2): 1159-68.

[0413] 9. Berry SM, Alarid ET, Beebe DJ. One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST). Lab Chip. 2011 May 21;11(10): 1747-53.

[0414] 10. Ngamsom B, Truyts A, Fourie L, Kumar S, Tarn MD, Iles A, et al. A microfluidic device for rapid screening of e. coli O157:H7 based on IFAST and ATP bioluminescence assay for water analysis. Chem Eur J. 2017 Sep 18;23(52): 12754-7.

[0415] 11. Rodriguez-Mateos P, Ngamsom B, Walter C, Dyer CE, Gitaka J, Iles A, et al. A lab-on-a- chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resourcelimited settings. Anal Chim Acta. 2021 Sep 8; 1177:338758.

[0416] 12. Changtor P, Rodriguez-Mateos P, Buddhachat K, Wattanachaiyingcharoen W, Iles A, Kerdphon S, et al. Integration of IFAST-based nucleic acid extraction and LAMP for on- chip rapid detection of Agroathelia rolfsii in soil. Biosens Bioelectron. 2024 Apr 15;250: l 16051.

[0417] 13. Berry SM, LaVanway AJ, Pezzi HM, Guckenberger DJ, Anderson MA, Loeb JM, et al. HIV viral RNA extraction in wax immiscible filtration assisted by surface tension (IFAST)

[0418] 39

[0419] 4909-5947-0701V.14818-7263-4587V.2

[0420] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 devices. J Mol Diagn. 2014 May; 16(3):297-304.

[0421] 14. Berry SM, Strotman LN, Kueck JD, Alarid ET, Beebe DJ. Purification of cell subpopulations via immiscible filtration assisted by surface tension (IF AST). Biomed Microdevices. 2011 Dec; 13(6): 1033-42.

[0422] 15. Strotman LN, Lin G, Berry SM, Johnson EA, Beebe DJ. Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension). Analyst. 2012 Sep 7;137(17):4023-8.

[0423] 16. Moussavi-Harami SF, Annis DS, Ma W, Berry SM, Coughlin EE, Strotman LN, et al. Characterization of molecules binding to the 70K N-terminal region of fibronectin by IFAST purification coupled with mass spectrometry. J Proteome Res. 2013 Jul 5;12(7):3393-404.

[0424] 17. Shin DJ, Trick AY, Hsieh Y-H, Thomas DL, Wang T-H. Sample-to-Answer Droplet Magnetofluidic Platform for Point-of-Care Hepatitis C Viral Load Quantitation. Sci Rep. 2018 Jun 28;8(1):9793.

[0425] 18. Neto MF, Butzler MA, Reed JL, Rui X, Fisher MJ, Kelso DM, et al. Immiscible phase filter extraction and equivalent amplification of genotypes 1-6 of hepatitis C RNA: The building blocks for point-of-care diagnosis. J Virol Methods. 2017 Oct;248: 107-15.

[0426] 19. Lazarus JV, Picchio CA, Colombo M. Hepatocellular carcinoma prevention in the era of hepatitis C elimination. Int J Mol Sci. 2023 Sep 21;24(18).

[0427] 20. Brzdqk M, Zarqb ska-Mi chaluk D, Invernizzi F, Cilla M, Dobrowolska K, Flisiak R. Decade of optimizing therapy with direct-acting antiviral drugs and the changing profile of patients with chronic hepatitis C. World J Gastroenterol. 2023 Feb 14;29(6):949-66.

[0428] 21. Gnanapandithan K, Ghali MP. Self-awareness of hepatitis C infection in the United States: A cross-sectional study based on the National Health Nutrition and Examination Survey. PLoS ONE. 2023 Oct 24;18(10):e0293315.

[0429] 22. Ivanova Reipold E, Easterbrook P, Trianni A, Panneer N, Krakower D, Ongarello S, et al. Optimising diagnosis of viraemic hepatitis C infection: the development of a target product

[0430] 40

[0431] 4909-5947-0701V.14818-7263-4587V.2

[0432] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 profile. BMC Infect Dis. 2017 Nov l ;17(Suppl l):707.

[0433] 23. Feld JJ. What Is Needed to Move Toward Single-Step Diagnosis of Current HCV Infection? J Infect Dis. 2024 May 8;229(Supplement_3): S316-21.

[0434] 24. Diez-Silva M, Dao M, Han J, Lim C-T, Suresh S. Shape and Biomechanical Characteristics of Human Red Blood Cells in Health and Disease. MRS Bull. 2010 May;35(5):382-8.

[0435] 25. Jankowski CA, Casapao AM, Siller S, Isache C, Cani KV, Claudio AM, et al. Preanalytical challenges during capillary fingerstick sampling preclude its widespread use in adult hospitalized patients. Am J Clin Pathol. 2021 Feb 11 ; 155(3):412— 7.

[0436] 26. Barshtein G, Pajic-Lijakovic I, Gural A. Deformability of stored red blood cells. Front Physiol. 2021 Sep 22; 12:722896.

[0437] 27. Newman H, Hardie D. HIV-1 viral load testing in resource-limited settings: Challenges and solutions for specimen integrity. Rev Med Virol. 2021 Mar;31(2):e2165.

[0438] 28. Nabatiyan A, Parpia ZA, Elghanian R, Kelso DM. Membrane-based plasma collection device for point-of-care diagnosis of HIV. J Virol Methods. 2011 Apr;173(l):37-42.

[0439] 29. Dure P, Foret F, Kuban P. Fast blood plasma separation device for point-of-care applications. Taianta. 2018 Jun 1 ; 183 :55- 60.

[0440] 30. Liu C, Mauk M, Gross R, Bushman FD, Edelstein PH, Collman RG, et al. Membranebased, sedimentation-assisted plasma separator for point-of-care applications. Anal Chem. 2013 Nov 5;85(21): 10463-70.

[0441] 31. Baillargeon KR, Murray LP, Deraney RN, Mace CR. High-Yielding Separation and Collection of Plasma from Whole Blood Using Passive Filtration. Anal Chem. 2020 Dec 15;92(24): 16245-52.

[0442] 32. Daae LN, Halvorsen S, Mathisen PM, Mironska K. A comparison between haematological parameters in “capillary” and venous blood from healthy adults. Scand J Clin Lab Invest. 1988 Nov;48(7):723-6.

[0443] 33. Pereiro I, Fomitcheva Khartchenko A, Petrini L, Kaigala GV. Nip the bubble in the bud: a

[0444] 41

[0445] 4909-5947-0701V.14818-7263-4587V.2

[0446] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 guide to avoid gas nucleation in microfluidics. Lab Chip. 2019 Jul 9;19(14):2296-314.

[0447] 34. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012 Nov; 113(5): 1014-26.

[0448] 35. Thomas PC, Strotman LN, Theberge AB, Berthier E, O’Connell R, Loeb JM, et al. Nucleic acid sample preparation using spontaneous biphasic plug flow. Anal Chem. 2013 Sep 17;85(18):8641-6.

[0449] 36. El-Sabawi B, Huang S, Tanriverdi K, Perry AS, Amancherla K, Jackson N, et al. Capillary blood self-collection for high-throughput proteomics. Proteomics. 2024 May 24;e2300607.

[0450] 37. Poland DCW, Cobbaert CM. Blood self-sampling devices: innovation, interpretation and implementation in total lab automation. Clin Chem Lab Med. 2024 Jun 25;

[0451] 38. Blumenfeld NR, Bolene MAE, Jaspan M, Ayers AG, Zarrandikoetxea S, Freudman J, et al. Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis. Nat Nanotechnol. 2022 Sep;17(9):984-92.

[0452] 39. FDA Permits Marketing of First Point-of-Care Hepatitis C RNA Test | FDA [Internet], [cited 2024 Jul 3], Available from: https: / / www.fda.gov / news-events / press- announcements / fda-permits-marketing-first-point-care-hepatitis-c-rna-test

[0453] 40. Zijlstra WG, Buursma A, Zwart A. Molar absorptivities of human hemoglobin in the visible spectral range. J Appl Physiol. 1983 May;54(5): 1287-91.

[0454] 41. Dean L. Chapter 1, Blood and the cells it contains. Blood Groups and Red Cell Antigens [Internet], Bethesda, MD: National Center for Biotechnology Information (US); 2005 [cited 2024 Jul 3], Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK2263 /

[0455] 42. Cologne, Germany: Institute for Quality and Efficiency in Health Care (IQWiG). In brief: What does blood do? [Internet], InformedHealth.org. 2023 [cited 2024 Jul 3], Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK279392 /

[0456] 43. Candotti D, Temple J, Sarkodie F, Allain J-P. Frequent recovery and broad genotype 2 diversity characterize hepatitis C virus infection in Ghana, West Africa. J Virol. 2003

[0457] 42

[0458] 4909-5947-0701V.14818-7263-4587V.2

[0459] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0460] Jul;77(14):7914-23.

[0461] 44. Snyder SA, Boban M, Li C, VanEpps JS, Mehta G, Tuteja A. Lysis and direct detection of coliforms on printed paper-based microfluidic devices. Lab Chip. 2020 Nov 24;20(23):4413-9.

[0462]

[0274] EXAMPLE 2

[0463]

[0275] FIG. 23 shows a biological sample processing device, generally designated by reference number 100, according to an exemplary embodiment of the present invention. The device 100 includes three main device components: (1) a single-use, disposable sample chamber 110 and dualmembrane fdter cap 120, (2) a reusable magnetic separator 130 containing an array of permanent magnets 132, and (3) a miniature peristaltic pump 140 connected to a laptop or Arduino microcontroller for automated operation. The sample chamber 110 is pre-loaded with commercially-available magnetic particles functionalized with antibody complexes recognizing Glycophorin A, a major sialoglycoprotein found in the membrane of human RBCs, designed to enable immunomagnetic depletion of RBCs using the magnetic separator 130. The fdter cap 120 is made up of parallel glass fiber membranes of subsequently smaller pore sizes (e.g., 2.7 pm and 0.7 pm from bottom to top), adapted from the dual membrane syringe filter approach previously described. The filter cap 120 is designed to seal the sample chamber and, through attached stainless steel tubing at the inlet and outlet, enable integrated, in-line filtration of RBC -depl eted supernatant into cell-free plasma (removing residual RBCs, WBCs, platelets, and magnetic beads) using a peristaltic pump without necessary sample transfer steps.

[0464]

[0276] The device workflow is shown in FIG 24. Blood is first added by the user via syringe to the sample chamber 110 containing anti -RBC magnetic beads, sealed using the filter cap 120, and gently mixed by the user (via brief, gentle inversion of the sample chamber). The user then attaches the pump tubing to the filter cap 120, places the sample chamber into the magnetic separator 130, and activates the device (e.g., by pressing a “start” button). Once placed into the magnetic separator 130, the magnetic-particle-bound RBCs are drawn to the permanent magnets on the bottom and back, left, and right sides of the sample chamber 110 (“lifting” RBC-depleted plasma to the top and front of the chamber). After a short incubation period, the peristaltic pump activates or may be activated and the RBC-depleted plasma is drawn through the dual-membrane

[0465] 43

[0466] 4909-5947-0701V.14818-7263-4587V.2

[0467] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 filter cap 120 into an empty sample tube, yielding cell-free plasma for immediate on-site testing or sample transport.

[0468] [2771 The device 100 integrates immunomagnetic depletion of RBCs with dual membrane sizeexclusion filtration to efficiently remove cellular components from large volumes of whole blood without clogging, hemolysis, or significant dilution. Because RBCs are the most abundant cellular component in blood (comprising 40-45% of blood volume with ~4-6 million RBCs per microliter of blood), it is hypothesized that initial depletion of a significant proportion of RBCs prior to filtration would significantly decrease membrane clogging and increase the blood volume capacity of the system compared to filtration alone.

[0469]

[0278] TEST RESULTS - II

[0470]

[0279] Device Fabrication

[0471]

[0280] The magnet holder, disposable sample chambers, and chamber lids were designed using AutoCAD Fusion360 and 3D printed in VeroWhite resin using a Stratasys Objet 30 3D printer. The prints were cleaned in an alkaline chemical bath for 5 hours after 3D printing to dissolve support material, rinsed with water, and allowed to dry at 30°C for at least two hours. Once dried and removed of all support material, 7 / 16” diameter x 1 / 2” thick N52 neodymium disc magnets (D78-N52, K&J Magnetics) were inserted into the 3D-printed magnet holder, held in place using light-cure adhesive (Loctite 3525, Henkel Adhesives), and cured with 3000 flashes in a UV curing unit (Otoflash G171).

[0472]

[0281] Inlet and outlet pieces of the filter cap were also designed using AutoCAD Fusion360 and 3D printed in VeroWhite resin. The inlet piece was designed with a rectangular seal to connect the filter cap to the chamber and seal it to prevent leakage. Both inlet and outlet pieces included a ringed spoke design printed on the surface in contact with the membranes to help distribute liquid evenly across the membranes. Square-profile 11mm ID O-rings (117 INI 02, McMaster-Carr) were inserted into the inlet and outlet pieces. 2.7 pm and 0.7 pm pore size glass fiber membranes (APFD02500 and APFF04700, Millipore) were cut to a diameter of 14.2875 mm (9 / 16-inch) using a leather hole puncher. The membranes were stacked such that the 2.7 pm pore size membrane was on the bottom, facing the inlet piece, and the 0.7 pm pore size membrane was on top, facing the outlet piece. The pieces were then assembled with the edges sealed using light-cure adhesive and cured with 3000 flashes in a UV curing unit.

[0473] 44

[0474] 4909-5947-0701V.14818-7263-4587V.2

[0475] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0476]

[0282] Steel tube fittings were connected to the inlet and outlet pieces to reach the liquid inside the chamber and interface with the pump tubing. 23G steel tubing (5560K651, McMaster-Carr) was cut to 2.25 mm and inserted into the inlet port such that the steel tubing extended 2 mm from the edge of the filter cap. Pre-cut 23G stainless steel tubing (LVF-KFI-13, Darwin Microfluidics) was inserted into the outlet port. Both fittings were sealed in place using a small drop of light-cure adhesive and cured with 3000 flashes in a UV curing unit.

[0477]

[0283] Filter caps were connected to a miniature peristaltic pump (CPP-1 1000-ZM, Jobst Technologies) using 1 / 16” outer diameter PTFE tubing (BL-PTFE-1605-20, Darwin Microfluidics). Tubing was inserted into the steel tube fittings on the outlet port and at the inlet of the peristaltic micropump.

[0478]

[0284] Finite-element modeling of the device-generated magnetic fields was performed as follows:

[0479]

[0285] Finite Element Modeling

[0480]

[0286] Finite element modeling of the magnetic field generated by device prototypes was performed using COMSOL Multiphysics (Magnetic Fields, No Currents module). Simulations used N52-grade sintered neodymium disc magnets as the magnetic material, matching the dimensions, arrangement, and boundary conditions of each experimental configuration. A fine mesh was applied to capture local variations in the magnetic field around the magnets. Magnetic flux density and field lines were visualized to assess field strength and spatial distribution. Simulated field profiles were qualitatively compared to experimental observations of red blood cell (RBC) separation behavior in response to the magnetic field to evaluate alignment between modeled and observed magnetic effects.

[0481]

[0287] Collection and Storage of Human Whole Blood

[0482]

[0288] Healthy control human whole blood was purchased from HumanCells Biosciences; all specimens were de-identified and tested negative for HIV1 / 2, HBV, HCV, and syphilis prior to receipt. Whole blood was stored at 4°C and handled using BSL-2 facilities and protocols. Blood samples were used within 48 hours of receipt.

[0483]

[0289] Automated Device Operation and Workflow

[0484]

[0290] For plasma separation using the device, up to 5 mL of whole blood was added to a sample chamber containing RBC depletion magnetic particles (EasySep RBC Depletion Reagent, Stem

[0485] 45

[0486] 4909-5947-0701V.14818-7263-4587V.2

[0487] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0488] Cell Technologies) at a concentration of 100 pL / mL of blood (i.e., 500 pL reagent for 5 mb of blood). The fdter cap was inserted to seal the sample chamber, and the sealed chamber was inverted to mix for 5-10 seconds, then placed onto the magnetic holder. The pump tubing was connected to the filter cap, and after 5 minutes, the RBC-depleted supernatant was drawn through the filter cap using the peristaltic pump with an applied voltage of 1.8 V (calibrated to an approximate flow rate of 557 pL / minute, expelling cell-free plasma into a clean 5 mL centrifuge tube.

[0489]

[0291] Improvement of Immunomagnetic Separation Workflow

[0490]

[0292] To evaluate the effect of magnetic bead volume, it was varied from 50 to 150 pL / mL of blood sample, with each sample mixed for 10 seconds. After 5 minutes of magnetic separation, the RBC-depleted supernatant was carefully extracted using a micropipette, ensuring the pelleted RBCs remained undisturbed. The results are shown in FIG. 25.

[0491]

[0293] To optimize mixing time, the bead volume was fixed at 100 pL / mL, and samples were mixed for 5, 10, or 30 seconds. Following a 5-minute magnetic separation, the supernatant was again collected and measured using a micropipette. The results are shown in FIG. 26.

[0492]

[0294] Finally, to determine the optimal magnetic separation time, both the bead volume (100 pL / mL) and mixing time (10 seconds) were held constant. Magnetic separation durations ranging from 3 to 7 minutes were tested, after which the RBC-depleted supernatant was extracted and measured. The results are shown in FIG. 27.

[0493]

[0295] Improvement of Immunomagnetic Separation Combined with Filtration

[0494]

[0296] The effect of the flow rate of the supernatant through the filter was evaluated by adjusting the voltage applied to the peristaltic pump, ranging from 1.2 V to 3.0 V. This resulted in flow rates between 335 and 960 pL / min. Flow rates were calibrated by measuring the volume of PBS that passed through the filter over 30 seconds at various pump voltages, then converting these values to pL / min. Filter diameter was fixed at 9 / 16 inches. Magnetic bead volume was again fixed at 100 pL / mL blood, and samples were mixed for 30 seconds. Following a 6-minute magnetic separation, the RBC-depleted supernatant was removed from the chamber through the filter cap at each flow rate. Recovered plasma volume was measured using a micropipette.

[0495]

[0297] To determine the optimal filter diameter, filter caps of various sizes from 12.7 mm to 25.4 mm were manufactured. Magnetic bead volume was again fixed at 100 pL / mL blood, and samples were mixed for 30 seconds. Following a 6-minute magnetic separation, RBC-depleted supernatant

[0496] 46

[0497] 4909-5947-0701V.14818-7263-4587V.2

[0498] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 was removed from the chamber through the filter cap at a flow rate of 557 pL / min. Recovered plasma volume was measured using a micropipette. The degree of hemolysis for each size was determined by measuring the absorbance of each sample separated using the device at 414 nm, which is a known absorbance peak of hemoglobin using a NanoDrop One spectrophotometer (ThermoFisher Scientific). The results are shown in FIGS. 29 and 30.

[0499]

[0298] Evaluation of Cell Separation Efficiency

[0500]

[0299] Gold-standard controls were centrifuged at 1,500 x g for 10 minutes, and the plasma supernatant was removed carefully using a pipette into a clean 1.5 mL tube. 10 pL of each device and centrifugation control sample were then diluted 1 : 1 with Trypan Blue (final volume 20 pL) and added to an Invitrogen Countess cell counting slide (Cl 0228, ThermoFisher Scientific). Input whole blood comparison samples were diluted 1000-fold prior to the addition of Trypan Blue. Cell counts were obtained using an automated cell counter (Invitrogen Countess 3, ThermoFisher Scientific). The results are shown in FIGS. 31 and 32.

[0501]

[0300] Measurement of Hemolysis

[0502]

[0301] The degree of hemolysis was determined by measuring the absorbance of each sample separated using the device and gold-standard centrifugation at 414 nm, which is a known absorbance peak of hemoglobin using a NanoDrop One spectrophotometer (ThermoFisher Scientific). 2 pL of each sample was measured using the spectrophotometer, using PBS as a blank. Gold-standard centrifugation and fully lysed whole blood samples were used as controls. The obtained absorbance values were baseline-corrected to achieve only positive values. The results are shown in FIG. 33.

[0503]

[0302] Evaluation of Plasma Separation Across a Range of Hematocrit Levels

[0504]

[0303] To evaluate the device at a range of blood volumes and various hematocrit levels, blood samples were prepared at artificial blood hematocrit levels (40%, 45%, 50% and 55%) by centrifuging whole blood at 1,500 x g for 10 minutes, removing the separated plasma to calculate the leftover volume of packed blood cells (while hematocrit refers to red blood cells percentage only, this is a close estimate, as the packed blood cells are >99% RBC). Plasma was then added back at appropriate volumes to generate the correct total% RBC in each sample. The results are shown in FIGS. 34-36.

[0505]

[0304] Live Subject Statement

[0506] 47

[0507] 4909-5947-0701V.14818-7263-4587V.2

[0508] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0509]

[0305] All blood samples used were purchased as de-identified specimens from HumanCells Biosciences. All human blood samples were collected by HumanCells Biosciences from fully consented, IRB-approved donors who have tested negative for HIV1 / 2, HBV, HCV, and syphilis, under IRB #2023-0213 approved by Pearl IRB, in adherence with Good Clinical Practices outlined by the U.S. Food and Drug Administration.

[0510]

[0306] It has been observed that, when attempting to process 5 mL of whole blood using the filter alone, there was minimal plasma yield (mean plasma volume = 45 pL) due to rapid membrane clogging, halting the flow of blood through the filter almost instantly (FIG. 41). However, when RBCs were initially immunomagnetically depleted and then passed through the dual-membrane filter, plasma yield increased significantly (1.96 mL, p<0.0001).

[0511]

[0307] RBC separation efficiency of immunomagnetic depletion was assessed to determine whether this step alone was sufficient to remove RBCs from the sample. It was observed that immunomagnetic depletion of RBCs alone enabled -1000-fold depletion of blood cells; however, this still left, on average, 4878 cells / pL in the recovered plasma sample (FIG. 41). Filtration following depletion further reduced the number of blood cells to a mean concentration of 56 cells / pL. As a result, the integration of both immunodepletion and filtration is important for efficient, high-quality separation of plasma from large volumes of whole blood in this system.

[0512]

[0308] To enable the device for diagnostic applications requiring large plasma volumes with high sensitivity, key workflow and design parameters are needed to maximize plasma yield. Immunomagnetic RBC depletion efficiency is strongly influenced by the geometry of the sample chamber and the configuration of the magnetic separator. Effective magnetic separation requires a magnetic field that is both sufficiently strong and spatially optimized to rapidly isolate RBCs from the entire chamber while maintaining a distinct plasma zone for extraction that is largely free from RBC-bound magnetic beads.

[0513]

[0309] To achieve this, three chamber and magnetic separator prototypes were tested to optimize both the speed and spatial resolution of magnetic separation. Finite element modeling (COMSOL) was used to simulate the applied magnetic field on the chamber for each prototype, followed by validation through visual observation of RBC separation. Across all designs, observed RBC migration correlated well with regions of high magnetic field strength, while RBC-depleted plasma accumulated in areas of low field strength.

[0514] 48

[0515] 4909-5947-0701V.14818-7263-4587V.2

[0516] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0517]

[0310] It was observed that separating RBCs using Prototype #1 resulted in multiple small plasma accumulation zones due to uneven magnetic field distribution, which complicated automation, as the plasma must be drawn from a single fixed point. The vertical orientation also resulted in slower RBC separation from the upper chamber regions. Prototype #2, with a horizontal configuration, improved separation kinetics but offered only a limited plasma accumulation area near the chamber front. Prototype #3, with a nearly square orientation, demonstrated the most effective separation performance, enabling both rapid RBC clearance and a clearly defined plasma collection zone. Quantitative analysis of RBC-depleted supernatant recovery after 5 minutes of magnetic separation confirmed the superior performance of Prototype #3, which yielded 1.990 mL of supernatant, compared to 1.696 mL and 1.348 mL for Prototypes #1 and #2, respectively. Prototype #3 also demonstrated the most efficient RBC separation, with 3,233 remaining cells in the supernatant, compared to 53,417 and 17,624 cells for Prototypes #1 and #2, respectively. See Fig. 43 for prototype designs.

[0518]

[0311] To further improve immunomagnetic RBC depletion, the volume of anti-RBC magnetic beads added per milliliter of whole blood was optimizes. A range of bead concentrations from 50 to 150 pL / mL were tested and the corresponding RBC-depleted supernatant recovery was tested. Supernatant yield increased with bead volume; however, the improvement plateaued beyond 100 pL / mL, with no statistically significant difference between 100 and 150 pL / mL (p = 0.3625).

[0519]

[0312] The impact of mixing duration on plasma yield was also assessed. Mixing whole blood with anti-RBC magnetic beads is the only manual step in the device workflow and thus a potential source of user-to-user variability. To evaluate the robustness of this step, RBC-depleted supernatant yield after mixing for 5, 10, or 30 seconds was compared. No statistically significant differences were observed among the groups (p = 0.4935; Fig. 2C), indicating that plasma recovery is insensitive to moderate variations in mixing time.

[0520]

[0313] The magnetic separation step is a major rate-limiting component of the workflow. To reduce total protocol time while maintaining high plasma yield, separation durations ranging from 3 to 6 minutes were evaluated. Plasma yield increased with longer separation times, though differences beyond 3 minutes were not statistically significant. Based on the observed trend and yield plateau beyond 6 minutes, 6 minutes may be selected as the optimal magnetic separation time.

[0521] 49

[0522] 4909-5947-0701V.14818-7263-4587V.2

[0523] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0524]

[0314] With the immunomagnetic RBC depletion step established, integration with the dualmembrane filtration process is needed. Specifically, the pump flow rate may be tuned to maximize plasma yield while minimizing total protocol time. Since flow rate is directly proportional to pump speed (RPM), which in turn is regulated by the applied voltage, plasma yield across a range of voltages corresponding to flow rates of approximately 335, 557, 758, and 960 iiL / min were evaluated, following 5 minutes of magnetic separation.

[0525]

[0315] A significant negative linear correlation was observed between flow rate and plasma yield (R2= 0.71, p = 0.0084), indicating that higher flow rates reduced plasma recovery. However, oneway ANOVA revealed no statistically significant differences in plasma yield among individual flow rate groups (p = 0.1338). Based on these results, a flow rate of 557 pL / min (corresponding to 1.8 V applied voltage) may be selected as the optimal condition, balancing plasma recovery with the goal of completing separation from 5 mL of blood in under 10 minutes (the typical duration of centrifugation).

[0526]

[0316] To assess whether membrane size affects plasma yield via changes in dead volume, the impact of reducing filter membrane diameter was investigated. Although smaller diameters reduce membrane surface area — potentially increasing the risk of red blood cell (RBC) clogging — it was hypothesized that prior immunomagnetic depletion of RBCs would mitigate this risk, allowing for reduced membrane size without increased clogging or hemolysis. A range of filter diameters were tested, from 25.4 mm down to 12.7 mm, and both plasma volume recovery and hemolysis were evaluated, by measuring absorbance at 414 nm, the characteristic peak for free heme released from lysed blood cells.

[0527]

[0317] Contrary to the hypothesis, no statistically significant differences in recovered plasma volume between filters of different diameters (one-way ANOVA, p = 0.778; Fig. 2F) were observed. Similarly, hemolysis did not differ significantly across diameter groups (one-way ANOVA, p = 0.2294; Fig. 2G). However, the 25.4 mm filters exhibited slightly greater variability in both plasma yield and hemolysis, which may be due to a higher incidence of trapped air bubbles in the larger membrane area, contributing to inconsistent performance. Based on these findings, a 14.3 mm membrane diameter may be selected for further studies, balancing performance consistency and minimizing the likelihood of air bubble formation.

[0528] 50

[0529] 4909-5947-0701V.14818-7263-4587V.2

[0530] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0531]

[0318] After optimizing the device design and workflow parameters, plasma yield from the device was compared to that obtained using gold-standard centrifugation. From an initial blood volume of 5 mL, the device recovered an average of 2.12 mL of plasma, compared to 2.64 mL from centrifugation (p < 0.0001), corresponding to an average recovery efficiency of 80.3%. To assess plasma quality, the residual cellular content of plasma obtained by the device was compared to that obtain by centrifugation. The device achieved high blood cell removal efficiency (>99.999%), yielding an average residual cell count of 19.3 cells / pL, with no significant difference from centrifuged plasma (19.0 cells / pL, p > 0.999), from an initial whole blood concentration of 4.26 x 106cells / pL. Hemolysis was also measured by measuring absorbance at 414 nm. Plasma separated by the device exhibited minimal hemolysis (2.24 a.u.), with no significant difference compared to centrifugation (2.11 a.u., p = 0.2321) (Fig. 3c). Together, these results demonstrate that the device produces high-quality plasma comparable to centrifugation, with acceptable yield and minimal cellular contamination or hemolysis.

[0532]

[0319] To evaluate the performance of the device under physiologically relevant conditions, the device was validated across a range of hematocrit (HCT) levels. Hematocrit, the volume fraction of RBCs in whole blood, varies widely between individuals and can significantly impact the degree of RBC clogging in microfluidic and filtration-based systems. Hematocrit typically ranges from 35-45% in healthy females and 39-50% in healthy males and can be further altered by factors such as hydration status, anemia, polycythemia, or underlying disease. Plasma yield and hemolysis for blood samples were thus evaluated with hematocrit levels ranging from 40-55%, which are at and beyond the normal physiological range.

[0533]

[0320] As expected, a significant negative linear correlation was observed between hematocrit and plasma yield from the device (R2= 0.91, p < 0.0001), consistent with the fact that higher hematocrit levels reduce the plasma fraction in whole blood. Plasma recovery efficiency did not differ significantly among the 40%, 45%, and 50% hematocrit groups (p = 0.8052, 0.9587, and 0.9755, respectively). However, the 55% hematocrit group showed a significant decrease in recovery efficiency compared to the 40%, 45%, and 50% groups (p = 0.0086, 0.0287, and 0.0165, respectively). Importantly, there were no significant differences in hemolysis across hematocrit levels (one-way ANOVA, p = 0.5007).

[0534] 51

[0535] 4909-5947-0701V.14818-7263-4587V.2

[0536] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0537]

[0321] These results indicate that the device performs consistently across a physiologically relevant range of hematocrit values (40-50%), supporting its reliability for use in diverse patient populations. Although recovery efficiency is slightly reduced at very high hematocrit (55%), the quality of the plasma remains uncompromised, suggesting the device is still suitable for use in patients with elevated hematocrit, including those with polycythemia, dehydration, or other atypical conditions, when yield requirements are not stringent.

[0538]

[0322] To validate the quality of plasma produced by the separation device, the recovery of representative plasma components — proteins, lipids, and metabolites — in device-separated plasma was compared to plasma obtained via gold-standard centrifugation. Accurate recovery of these components is essential to ensure that the immunomagnetic depletion and filtration system does not selectively retain or adsorb biomolecules during separation.

[0539]

[0323] As an initial assessment, total protein concentration was measured as a broad indicator of plasma protein retention. The mean total protein concentration in the device-separated plasma was 3.20 g / dL, which was not significantly different from that of centrifuge-separated plasma (3.30 g / dL, p = 0.5337)(FIG. 37).

[0540]

[0324] Metabolite preservation was evaluated by measuring glucose concentration. Glucose was selected as a representative metabolite due to its high clinical relevance and its routine use as a biomarker in plasma-based diagnostics. Accurate glucose measurement is critical for diagnosing and managing diabetes, as well as for identifying acute metabolic disturbances such as hypoglycemia or hyperglycemia. No significant difference in glucose levels were observed between centrifuged plasma (mean concentration = 0.99 mg / dL) and device- separated plasma (1.31 mg / dL, p = 0.4546) (Fig. 38).

[0541]

[0325] Lipid preservation was assessed by measuring total cholesterol concentration, a standard clinical biomarker used to evaluate cardiovascular risk. This measurement requires preservation of both free and lipoprotein-bound cholesterol. Again, no significant difference was observed between centrifuged plasma (mean concentration = 163.3 mg / dL) and device-separated plasma (152.7 mg / dL, p = 0.5725) (Fig. 39).

[0542]

[0326] Collectively, these findings indicate that the PlasmaLIFT system preserves key plasma biomarkers without significant loss, producing plasma that is comparable in analyte composition to that obtained via gold-standard centrifugation.

[0543] 52

[0544] 4909-5947-0701V.14818-7263-4587V.2

[0545] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427

[0546]

[0327] The compatibility of the device with downstream applications involving cell-free nucleic acid detection, such as liquid biopsy, was assessed. Experiments were conducted to measure recovery of both endogenous and spiked nucleic acids in the separated plasma. Specifically, total cell-free nucleic acid yield as an indicator of analyte preservation was evaluated, along with recovery of spiked synthetic nucleic acids and microRNAs (miRNAs) to quantitatively assess retention and loss during processing.

[0547]

[0328] No significant difference was observed in endogenous DNA recovery between centrifuged plasma (mean concentration = 17.3 ng / mL) and device-separated plasma (16.4 ng / mL, p = 0.7371) (Fig. 40).The device 100 is a novel large-volume plasma separation device that leverages immunomagnetic RBC depletion followed by dual-membrane filtration to isolate plasma from whole blood without centrifugation. This technology is the only centrifuge-free plasma separation method reported thus far that is capable of processing blood volumes up to 5 mb, substantially higher than existing POC devices, which are currently limited to processing volumes under 1.8 mL.

[0548]

[0329] In exemplary embodiments, the system yields about 2 mL of plasma from 5 mL of whole blood, which meets or exceeds the input volume requirements for several molecular diagnostic assays, including the Cepheid GeneXpert Viral Load tests for HIV, HBV, and HCV, and the cobas® EGFR Mutation Test v2, thereby potentially enabling usage with several clinically validated platforms.

[0549]

[0330] Designed as a modular and automated system, the device 100 can operate as a standalone tool for existing diagnostic assays or be integrated with in-development microfluidic sample preparation platforms, such as those for on-chip nucleic acid extraction. Its simple workflow and electronics are easily adaptable to be battery-powered and operable via a basic on / off switch, enabling use in decentralized or mobile diagnostic settings. Compared to conventional centrifugation and existing POC plasma separation tools, the device’s automated operation improves biosafety and reduces variability from manual handling.

[0550]

[0331] The immunomagnetic depletion and filtration concept is inherently modular, scalable, and adaptable to a broad range of clinical needs. Both the magnetic separator and sample chamber can be reconfigured to support varying input volumes, making the system feasible for low-volume capillary collections as well as higher-volume venous draws required for more sensitive diagnostic

[0551] 53

[0552] 4909-5947-0701V.14818-7263-4587V.2

[0553] 4909-5947-070 lv.1 Atty. Dkt. No. 93597-7427 applications. While the design can be optimized for venous blood processing of ~5 mb, the underlying technology may be scaled down to handle smaller or sub-milliliter volumes, enabling use in settings without access to trained phlebotomists. This opens the door for integration with emerging minimally invasive, user-friendly blood self-collection devices such as OnFlow and MaxFlow (Loop Medical), TAPMicro (YourBio), RedDrop One (RedDropDx), and Tasso (Tasso, Inc.), which can collect 500 pL to 1.5 mb of capillary whole blood. Such tools further extend the potential reach of the PlasmaLIFT platform to decentralized, at-home, and point-of-care diagnostics.

[0554]

[0332] While particular embodiments of the present disclosure have been shown and described in detail, it would be obvious to those skilled in the art that various modifications and improvements thereon may be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover all such modifications and improvements that are within the scope of this disclosure.

[0555] 54

[0556] 4909-5947-0701V.14818-7263-4587V.2

[0557] 4909-5947-070 lv.1

Claims

Atty. Dkt. No. 93597-7427What is claimed is:

1. A biological sample processing device, comprising: a first axis having disposed in order there along: a first sample-receiving chamber; one or more intermediate second chambers; a third processed-sample chamber; the first, second, and third chambers being sequentially in fluid communication with adjacent chamber(s), the chambers having a top surface and a bottom surface, wherein all chambers are configured to contain a fluid so as to form a continuous fluid pathway from the first chamber through the intermediate second chambers to the third chamber; a first magnet; a second magnet; the first magnet and the first, second and third chambers being configured for movement of the first magnet relative to the chambers, the second magnet and the first, second and third chamber being configured for movement of the second magnet relative to the chambers, independent of the movement of the first magnet, wherein relative movement of either the first and / or second magnet along the first axis from the first chamber to the third chamber gives rise to transfer of magnetic material from the first chamber, when contained therein, through the second chambers and into the third chamber.

2. The sample processing device of claim 1, further comprising a blood filtration component (a) in fluid connection with said first chamber, or (b) not in fluid connection with said first chamber, said blood filtration component configured to separate plasma from a blood sample moved therethrough.554909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-74273. The sample processing device of claim 2, wherein the blood fdtration component comprises a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough.

4. The sample processing device of claim 3, wherein the blood filtration component comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

5. The sample processing device of claim 1, wherein the first magnet is present in or on a first track or guide, via which track or guide the first magnet is slidable across the first, second and third chambers, and wherein the first track or guide is adjacent to a top surface of the first, second and third chambers.

6. The sample processing device of claim 1, wherein the second magnet is present in or on a second track or guide, via which track or guide the second magnet it is slidable across the first, second and third chambers, and wherein the second track or guide is adjacent to a bottom surface of the first, second and third chambers.

7. The sample processing device of claim 2, wherein the blood filtration component is adapted for blood sample volumes of 100 uL or less.

8. The sample processing device of claim 2, wherein the blood filtration component comprises a dual membrane filter comprising a first top membrane filter having a first and second face on opposite sides of the first filter, and a second bottom membrane filter having a first and second face on opposite sides of the second filter, the second filter adjacent to the first filter such that a fluid applied to a first face of the first top membrane filter under negative pressure will flow through the first face of the top membrane filter, out of the second face of said top filter, through a first face of the second bottom filter which faces the second face of the first564909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-7427 filter, and through the adjacent second bottom filter membrane and exit through the second face thereof.

9. The sample processing device of claim 8, wherein a pore size of the first filter is larger than the pore size of the second filter.

10. The sample processing device of claim 9, wherein the pore size of the first filter is 2.0 microns to 5.0 microns, optionally about 2.7 microns.

11. The sample processing device of claim 9, wherein the pore size of the second filter is about 0.1 microns to 3.0 microns, optionally about 0.7 microns.

12. The sample processing device of claim 3, wherein the filters of the dual membrane size-exclusion filter comprise glass fiber.

13. The sample processing device of claim 1, comprising channels between adjacent chambers for fluid communication with the adjacent chamber(s), wherein said channels narrow triangularly from one chamber to an adjacent subsequent chamber in sequential order along the first axis of the device from the first sample-receiving chamber to the third processed-sample chamber.

14. The sample processing device of claim 1, wherein the chambers are circular.

15. The sample processing device of claim 1, wherein the first chamber contains a plurality of magnetic particles.

16. The sample processing device of claim 15, wherein the plurality of magnetic particles is functionalized with a capture molecule.574909-5947-0701V.14818-7263-4587V.2 4909-5947-070 lv.1Atty. Dkt. No. 93597-742717. The sample processing device of claim 1, wherein the first chamber contains a liquid immiscible with a liquid in an adjacent intermediate second chamber.

18. The sample processing device of claim 1, wherein the first chamber contains an aqueous solution and the adjacent intermediate second chamber contains an oil.

19. The sample processing device of claim 1, wherein the intermediate second chambers are a plurality of chambers and are an odd number of chambers in total.

20. The sample processing device of claim 1, wherein the intermediate second chambers are five chambers in total.

21. The sample processing device of claim 1, wherein, in sequential order between the first sample-receiving chamber and the third processed-sample chamber, a first intermediate second chamber contains an oil phase, a second intermediate second chamber contains an aqueous phase, a third intermediate second chamber contains an oil phase, a fourth intermediate second chamber contains an aqueous phase, and a fifth intermediate second chamber contains an oil phase.

22. The sample processing device of claim 1, wherein the third chamber contains an aqueous solution.

23. The sample processing device of claim 1, wherein the first magnet is moveable in an automated fashion.

24. The sample processing device of claim 1, wherein the first magnet is moveable in an manual fashion.

25. The sample processing device of claim 1, wherein the second magnet is moveable in an automated fashion.584909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-742726. The sample processing device of claim 1, wherein the second magnet is moveable in an manual fashion.

27. The sample processing device of claim 1, wherein said first and / or second magnet comprises a handle attachment for manually moving the magnet along a track or guide which is parallel to the first axis, and wherein the handle is substantially perpendicular to the first axis of the device.

28. The sample processing device of claim 1, further comprising a supply of nanoparticles disposed in a chamber of the device, the nanoparticles optionally comprising at least one of plasmonic outer shell and a magnetic core.

29. The sample processing device of claim 1, wherein the sample processing device comprises agents in chambers thereof to effect any one or more of lysis, binding of target material to capture beads, washing, elution, and / or nucleic acid amplification.

30. The sample processing device of claim 1, further comprising a source of illumination configured to illuminate the contents of a chamber of the device.

31. The sample processing device of claim 1, further comprising in the first samplereceiving chamber magnetic material in the form of functionalized nanoparticles.

32. The sample processing device of claim 31, wherein the functionalized nanoparticles are functionalized to bind one or more nucleic acids.

33. The sample processing device of claim 32, wherein the functionalized nanoparticles are functionalized to bind a virus, a viral antigen, a bacteria, a bacterial antigen, a protein or a cell.594909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-742734. A method of processing a sample, comprising operating a device according to claim 1, 2 or 27, so as to process a sample liquid supplied thereto.

35. The method of claim 34, comprising at least moving the first and second magnet in an alternate fashion relative to each other and the first chamber in order to effect mixing of the sample in the first chamber by movement of magnetic material therein.

36. The method of claim 34, comprising at least moving the first and second magnet relative to each other and the third chamber in order to effect mixing of the processed sample in the third chamber by movement of magnetic material therein.

37. The method of claim 34, comprising at least moving the first and / or second magnet along the track or guide from the first chamber through the intermediate second chambers to the third processed-sample chamber in order to effect the passing of the sample through the contents of the intermediate second chambers.

38. A blood filtration device, the device comprising a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough, the device configured for blood sample volumes of 100 uL or less, and wherein the dual membrane size-exclusion filter comprises two membranes, with a first membrane having a pore size larger than a second membrane.

39. The blood filtration device of claim 38, wherein the pore size of the first membrane is 2.5 to 5 microns, optionally about 2.7 microns.

40. The blood filtration device of claim 38, wherein the pore size of the second membrane is 0.1 to 3.0 microns, optionally about 0.7 microns.

41. The blood filtration device of claim 38, wherein the membranes comprise glass fiber.604909-5947-0701V.14818-7263-4587V.2 4909-5947-070 lv.1Atty. Dkt. No. 93597-742742. The blood filtration device of claim 38, wherein the device comprises (a) a port for applying negative suction pressure, such that a blood sample applied to the blood filtration component is pulled therethrough, and / or (b) an inlet port for applying the blood sample and, optionally, a flush through solution.

43. The blood filtration device of claim 38, wherein the device is configured for blood volumes of 50 uL or less.

44. A biological sample processing device, comprising: a container for placing in a chamber which chamber is magnetic or through which a magnetic force can be applied, the container containing magnetic beads having thereon a molecule which attaches to human red blood cells; a chamber which is magnetic or through which a magnetic force can be applied for receiving the container; a blood filtration component (a) in fluid connection with said container, or (b) not in fluid connection with said container, said blood filtration component configured to separate plasma from a blood sample moved therethrough by suction from a syringe or pump component.

45. The sample processing device of claim 44, wherein the blood filtration component comprises a dual membrane size-exclusion filter which separates plasma from a blood sample moved therethrough.

46. The sample processing device of claim 44, wherein the blood filtration component comprises a outlet port on an external face thereof for applying negative suction pressure and can be attached to the sample container, and on an opposite face thereof an inlet port such that when the blood filtration component is placed on the container, a blood sample in the sample container can be pulled therethrough.

47. The sample processing device of claim 45, wherein the pore size of the first membrane of the dual membrane is 2.5 to 5 microns, optionally about 2.7 microns and / or614909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-7427 wherein the pore size of the second membrane of the dual membrane is 0.1 to 3.0 microns, optionally about 0.7 microns.

48. The sample processing device of claim 44, wherein the magnetic beads comprise attached thereto one or more antibodies which bind red blood cell surface markers.

49. The sample processing device of claim 44, wherein the membranes comprise glass fiber.

50. The sample processing device of claim 44, wherein the chamber which is magnetic or through which a magnetic force can be applied is a chamber comprising permanent magnets.

51. A method of processing a blood sample, comprising: applying the blood sample into the chamber of the sample processing device of any of claims 44- 50 and incubating the sample therein for a time sufficient for red blood cells to attach to the magnetic beads via the molecule which attaches to human red blood cells and sealing the container with a lid having the blood filtration component attached thereto or configured as part of said lid; and placing the container prior to, during, or after incubating, in the chamber which is magnetic or through which a magnetic force can be applied so as to thereby hold the red blood cells bound to the magnetic beads in the chamber and extracting via suction pressure, through the blood filtration component, a red blood cell-depleted supernatant, whereby passage of the supernatant through the blood filtration component and through the outlet removes a majority of any remaining cells and platelets in the red blood cell-depleted supernatant.

52. The method of claim 51, wherein the sample is incubated for a time sufficient for red blood cells bound to a magnetic bead to settle at a bottom of the chamber, optionally for about 3 to about 6 minutes.

53. The method of claim 51, wherein the blood sample is 0.5 to 7.5 ml.624909-5947-0701V.14818-7263-4587V.24909-5947-070 lv.1Atty. Dkt. No. 93597-742754. The method of claim 51, wherein the molecule is an antibody to a red blood cell surface antigen.

55. The method of claim 51, wherein the chamber which is magnetic or through which a magnetic force can be applied surrounds a lower portion of the container when the container is placed therein and whereby red blood cells bound to the magnetic beads are held in the lower portion of the container when the supernatant is removed.

56. The method of claim 51, wherein the method effects a processed blood sample having 102cells / ml or less once processed.

57. The method of claim 51, wherein the method effects a processed blood sample having less than 102cells / ml once processed.634909-5947-0701V. 14818-7263-4587V.24909-5947-070 lv.1

Images