Sedimentation devices for separating bacteria from fluids and methods of use thereof

Abstract

A sedimentation container in a sedimentation environment with a sedimentation direction, with an inclined filter configured to separate particles of different types in a fluid is disclosed. The sedimentation container may include a grid, a particle guide, and / or a siphon. The sedimentation container may be a centrifuge container. The fluid may be blood, urine, saliva or a fecal sample. A method of using the sedimentation container includes loading a liquid sample having a lower density than a liquid medium onto the top of the liquid medium; centrifuging the sedimentation container, during which the larger particles sediment at a higher velocity than the smaller particles; and removing the larger particles from the sedimentation container, leaving the smaller particles in the remaining liquid in the filtrate collection region. A method of using a sedimentation container, having a fluid guide with an inclined filter and a siphon arranged downstream of a sedimentation field, includes adding a first high density liquid medium inside the siphon, trapping air at a closed end of the siphon; inserting the sedimentation device into a centrifuging tube prefilled with a second liquid medium; adding a liquid sample comprising different types of particles to the top of the fluid guide; centrifuging the liquid sample to move the liquid sample toward the bottom of the sedimentation container, wherein a fraction of the smaller particles passes through the filter, and a rest of the liquid sample moves to the bottom of the siphon and compresses the air trapped in the closed end of the siphon; stopping the centrifuging to allow resuspension of the sediment in the siphon; and repeating the centrifuging process more than once.

Description

SEDIMENTATION DEVICES FOR SEPARATING BACTERIA FROM FLUIDS AND METHODS OF USE THEREOF FIELD OF THE INVENTION The present invention relates to sedimentation devices, specifically sedimentation containers, for separating bacteria from fluids. Also, related methods of using these sedimentation containers are disclosed. BACKGROUND Severe bloodstream infections represent a significant global healthcare challenge, resulting in high mortality rates and medical expenses. Sepsis alone is responsible for over 11 million deaths annually worldwide, accounting for one in every five deaths, with an estimated cost to the US economy of approximately $20 billion1–5. However, the current clinical diagnosis of BSI relies on a positive blood culture, which is a slow process that can take several days6. As the mortality rate of untreated patients increases by 8% per hour7, clinicians often resort to broad- spectrum antibiotics. Unfortunately, in approximately 30% of cases, this approach is incorrect and can lead to more fatalities8. Furthermore, the use of broad-spectrum antibiotics promotes the growth of antimicrobial resistance9. Rapid pathogen identification and antimicrobial susceptibility testing (AST) are preferred methods for diagnosing sepsis10,11. Various rapid phenotypic and genotypic methods have been developed for this purpose12–16. However, the bottleneck in sepsis diagnosis lies in sample preparation, particularly in separating bacteria from whole blood, given that the concentration of bacteria is as low as 1-10 CFU / mL, whereas blood cells have concentrations in the range of 4-6 billion / mL for red blood cells (RBCs) and 5-10 million / mL for white blood cells (WBCs). Various methods have been investigated for bacterial separation, including sedimentation velocity-based differentiation17,18, inertial19–21and elastoinertial22microfluidics, acoustophoresis23,24, surface acousticwaves (SAW)25, dielectrophoresis (DEP)26, and magnetic beads based separation27. However, these methods suffer from low throughput (DEP, SAW), low separation efficiency (density-based separation, inertial microfluidics), or require high bacterial concentration for efficient separation (inertial and elastoinertial microfluidics, DEP, SAW, acoustophoresis) or are selective to specific bacterial strains (magnetic beads) (Figure 5). Size-based filtration is a simple, inexpensive, and highly selective method with successful applications in separating WBCs28or tumor cells29,30from blood and in blood-serum separation31. WBCs have typical size 10–20 µm, RBCs 6.2–8.2 µm, platelets 2.0-4.0 µm, and bacteria around 1.5 µm.17Nonetheless, filter caking can impede filtration, particularly during dead-end filtration, where abundant RBCs accumulate on the filter surface to form a clogging layer that blocks bacteria and limits volumetric throughput17A need exists for a separation device capable of separating low-abundant bacteria from whole blood at relevant throughput in a single step. Since more than half of the bacteria settle with the blood cell sediments, a further need exists to separate the bacteria trapped in these blood cell sediments. SUMMARY OF THE INVENTION It is an object of the present disclosure to provide improved devices for separating particles of different types in a fluid, and particularly for separating bacteria from blood, urine, saliva, and / or fecal samples. To better address these one or more concerns, a sedimentation container for separating particles of different types in a fluid, and methods of use having the features defined in the independent claims are provided. Preferable embodiments are defined in the dependent claims.These, and other objects which are evident to the skilled person from the present disclosure, are met by the different aspects and embodiments of the invention as claimed in the appended claims and as generally disclosed herein. Hence, according to a first aspect, a sedimentation container in a sedimentation environment with a sedimentation direction is provided. The sedimentation container comprises a filter having an inclination angle to the sedimentation direction, wherein the filter is configured to separate particles of different types in a fluid. In one embodiment, the inclined filter may be positioned at an angle between substantially 27 and 72 degrees with regard to a plane perpendicular to the sedimentation direction. In a further embodiment, the sedimentation container may further comprise at least one of a grid, a particle guide, and a siphon. In one embodiment, in a sedimentation field and the grid may be placed higher compared to the filter, and wherein the grid may comprise walls that are substantially parallel with the sedimentation direction. The sedimentation container according to the first aspect may comprise at least a filtrate collection region for collecting filtrate in a lower region of the sedimentation container and a separate retentate collection region for collecting retentate particles. Preferably the sedimentation environment of the sedimentation container may be configured by creating artificial gravity to separate the different particles in a fluid based on their type. Desirably, the sedimentation container may comprise a centrifuge container or a centrifuge tube.In one embodiment, the sedimentation container may have a centrifugal acceleration and the centrifugal acceleration may be configured to increase and decrease at least once during centrifugation. In another embodiment, the sedimentation container may have an angular velocity and the angular velocity may be configured to increase and decrease at least once during centrifugation. In a further embodiment of the sedimentation container, an open end of the siphon may be placed in fluidic connection with the retentate collector and the siphon may be configured to fill up with liquid during centrifugal acceleration, thus compressing the air, and decompress during centrifugal deceleration to resuspend retentate particles back to the container. Desirably, the fluid in the sedimentation container may comprise one of a blood, urine, saliva or fecal sample. Preferably, the filter in the sedimentation container may be adapted to separate blood cells from bacteria. Desirably, the sedimentation container may be configured to isolate and up- concentrate bacteria from body fluid, the body fluid comprising at least one of blood, urine, and a fecal sample, and swab in one centrifugation step. In a second aspect , a method of using the sedimentation container according to the first aspect and its embodiments is disclosed. The method comprises loading a liquid sample having a lower density than a liquid medium onto the top of the liquid medium; centrifuging the sedimentation container, during which the larger particles sediment at a higher velocity than the smaller particles; and removing the larger particles from the sedimentation container, leaving the smaller particles in the remaining liquid in the filtrate collection region. In one embodiment the step of removing the larger particles may comprise lifting the retentate collection region comprising the larger particles away fromthe sedimentation container, leaving the smaller particles in the remaining liquid in the centrifuge container or tube. Desirably, the method may include filling the sedimentation container with a liquid medium to reach the top of the grid, before loading the liquid sample. Desirably, the method may include centrifuging the sedimentation container to remove any air bubbles that may be trapped in the liquid medium, before loading the liquid sample. Preferably, the step of centrifuging the sedimentation container may comprise centrifuging the sedimentation container at a first speed during which a first relative fraction of the larger particles reach the filter earlier than a first relative fraction of the smaller particles; and centrifuging the sedimentation container at a second speed, during which a second relative fraction of the larger particles move over the surface of the filter without crossing the filter, which second relative fraction of the larger particles is larger than the relative fraction of smaller particles moving across the filter without crossing the filter; Desirably, in the method according to the second aspect, the liquid medium may be configured to have a density above that of the liquid sample for promoting efficient layered loading of the liquid sample on top of the liquid medium and sedimentation of the particles of the liquid sample through the filter pores. Desirably, in the method according to the second aspect , the liquid sample may be a blood, urine, saliva or fecal sample. In one embodiment, the liquid sample may be blood and the liquid medium may comprise a mixture of substantially 75% Lymphoprep and substantially 25% broth. In one embodiment, the centrifugation tube and container device may be centrifuged at approximately 4500g. In one embodiment, the centrifugation tube and container device may be centrifuged for 1 or more minutes.In a third aspect, a method of using a sedimentation container, the sedimentation container comprising a fluid guide having an inclined filter configured to separate different particles in a fluid and a siphon arranged downstream of a sedimentation field of the sedimentation container is disclosed. The method comprises adding a first high density liquid medium inside the siphon for avoiding sedimentation of retentate particles at the bottom of the fluid guide, trapping air at a closed end of the siphon; inserting the sedimentation device into a centrifuging tube prefilled with a second liquid medium; adding a liquid sample comprising different types of particles to the top of the fluid guide; centrifuging the liquid sample to move the particles toward the bottom of the sedimentation container, wherein a fraction of the smaller particles passes through the filter, and a rest of the particles moves to the bottom of the siphon and compresses the air trapped in the closed end of the siphon ; stopping the centrifuging to allow resuspension of the sediment in the siphon; and repeating the centrifuging process more than once. In one embodiment, the siphon may be placed at a lower end of the inclined filter. In one embodiment, the centrifuging process is repeated between 1 to 10 times. As used herein, the term the "sedimentation environment" refers to a setup where particles sediment due to artificial gravity, typically created by rotating devices like centrifuges or lab-on-disk systems using centrifugal force. As used herein, the term “sedimentation field” is to be understood to refer to the direction of the force acting on particles that causes them to settle, typically in systems using centrifugal or gravitational forces. The terms “sedimentation device” and “sedimentation container” are used herein interchangeably.As used herein, the term “artificial gravity” may be created using centrifugal force of a rotating body. As used herein, the term “centrifugal acceleration” is to be understood to refer as the rate at which the spin speed of a centrifuge increases. As used herein, the term “siphon” is to be understood to refer to a passive pump that moves a fluid back and forth during centrifugal acceleration and deceleration. As used in this description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “about”, as used herein, indicates the value of a given quantity can include quantities ranging within 10% of the stated value, or optionally within 5% of the value, or in some embodiments within 1% of the value. As used herein, the terms “approximately” and “substantially”” are to be interpreted as ± 5% in relation to a numeric value. Consequently, the term “approximately 100 µm” is to be understood as 95 - 105 µm. Unless otherwise specified, the term “fluid” includes both bodily fluids and non-medical fluids. Typically, the subject is a human or animal subject. In particular, it is noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” also include plural referents unless the context clearly dictates otherwise. While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular embodiment to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims. BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows a sedimentation container placed in a sedimentation environment where sedimentation takes place in a direction, the sedimentation direction. The filter forms an inclination angle α with respect to the sedimentation direction. Figure 1B, shows the sedimentation container comprising a centrifugation container. Figure 1C shows an embodiment of the sedimentation container wherein the sedimentation container further comprises a grid placed in a higher in the sedimentation container than the filter. Figure 1D shows an embodiment of the sedimentation container wherein the sedimentation container further comprises a particle guide for guiding fluid arranged in the sedimentation container. Figure 1E shows the grid located in the region of the particle guide above the filter. Figure 1F shows the sedimentation container comprising a retentate collection region for collecting the filter retentate particles, and a filtrate collection region for collecting the filtrate, for example the bacteria, in the lower / bottom region of the centrifuge tube. The sedimentation container maybe a centrifuge container. An air vent may also be provided in the particle guide to mitigate air trapping during liquid priming. Figure 1G shows the sedimentation container comprising fluid guide (27) comprising a siphon (23) arranged in the sedimentation container with an open end (25) in fluidic connection with the retentate collector (17). The siphon may comprise air (or another compressible fluid). Figure 2 shows an example of an outer part of the fluid guide structure, with dimensions in mm. Figure 3:shows examples of inner parts of the fluid guide structure., with dimensions in mm. Figures 4A-4E illustrate the fabrication and assembly of the device. Schematics and photographs of the fluid guide and its assembly. Figure 4A shows a CAD image of the fluid guide main part with a section view. Figure 4B shows the integration of the filter into the fluid guide by sliding the fluid guide sheath over the filter and fluid guide main part. Figure 4C illustrates the mounting of the fluid guide into a centrifuge tube. Figure 4D shows the complete sedimentaton device. Figure 4D shows the sedimentation device after its usage. Figures 5A-5C show cross-sectional schematics of the device and its operation. In Figure 5A the 3D-printed fluid guide comprises a filter and is inserted in a centrifuge tube. The fluid guide is subsequently filled with Lymphoprep-broth medium and blood infected with bacteria. In Figure 5B, during centrifuging, blood cells sediment at a higher velocity than bacteria (top inset). Blood cells cannot pass the filter (low left inset) and sediment into the blood cell collection pocket. Bacteria sediment through the filter pores (bottom right inset) into the bottom region of the centrifuge tube. In Fiugure 5C, after centrifuging, the fluid guide containing the blood cells is lifted away.Figures 6A-6C present data relating to the sedimentation container’s performance. Figure 6A shows the total bacteria recovery (CFU in filtrate / CFU in blood), Figure 6B shows RBC rejection (RBCs in retentate / RBCs in filtrate and retentate), and Figure 6C shows the total separation efficiency (total bacteria recovery / 1-RBC rejection) when processing 1 mL whole blood spiked with E. coli, K.pneumoniae, and S. aureus at 100 and 1000 CFU / mL. Error bars are sd (a, b) or 68% CI (c) Figures 7A-7B: Fig.4 RBC rejection and relative separation efficiency for filters with 2.0 and 3.0 μm pore size. (a) RBC rejection for filters with pore size 2.0 or 3.0 μm and 0.4 and 1 mL blood sample volume. Error bars are sd. p- value (tails 2, type 2) indicates significance: ns is not significant, * is p<0.05, ** is p<0.01. (b) Relative separation efficiency (ratio of relative bacteria recovery over RBC recovery in the filtrate, where relative bacteria refers to the ratio of bacteria in the filtrate over bacteria in the filtrate and retentate combined) of filters with pore size 2.0 and 3.0 μm. Results are for 0.4 mL of whole blood spiked with 104 CFU / mL of K. pneumoniae. Error bars are 68% CI. Figures 8A-8D show relative bacteria recovery (CFU in filtrate / CFU in filtrate and retentate) for varying design and operational parameters. Figure 8A shows fraction of Lymphoprep in Lymphoprep-broth mixtures, Figure 8B shows filter angle α, Figure 8C shows filter pore size, and Figure 8D shows centrifugation acceleration and duration. Results are for 104 CFU / mL of K. pneumoniae spiked in 0.4 mL of whole blood. Unless indicated otherwise, we used 75% Lymphoprep in a Lymphoprep-broth mixture, 63° filter angle, 60 min centrifuging at 4500g, and filters with 3.0 μm pore size. Error bars are sd. p-value (tails 2, type 2) indicates significance: ns is not significant, ps is p<0.1, * is p<0.05, ** is p<0.01.Figures 9A-9D are photographs of whole blood loaded on media with different densities: Figure A - 100 % broth, Figure B - 50% broth + 50% Lymphoprep, Figure C - 25% broth + 75% Lymphoprep, and Figure D - 100% Lymphoprep. Figures 10A-10B show photographs of the sedimentation container after one (Figure A) and two minutes (Figure 10B) centrifugation. Figures 11A-11C show the effect of grid structures on Coriolis mixing during centrifugal acceleration. Schematic (left) and side-view photographs of density medium in a centrifuge tube after loading of blood (middle) and 3 s after the onset of centrifugation (right) for structures without (Figure 11A) and with (Figure 11B) a grid. Figure 11C shows the relative bacteria recovery from the separation device with and without the grid structure. Results are for 104 CFU / mL of K. pneumoniae spiked in 0.4 mL of whole blood, using 75% Lymphoprep + 25% broth medium, 63 °filter angle, 3.0 μm pore size filter, and centrifuging for 30 min at 4500g. Figures 12A-12D show light absorbance by Hb for different mixtures of retentate or filtrate sample and DIW. Measurements 1, 2, and 3 refer to three different experiments performed with the blood of the same donor. Concentration means the volume of sample / total volum6e. The filter pore size and blood volume were, respectively, Figure 12A - 2.0 μm and 0.4 mL, Figure 12B - 2.0 μm and 1.0 mL, Figure 12C - 3.0 μm and 0.4 mL, and Figure 12D - 3.0 μm and 1.0 mL Figure 13: WBC and platelet rejection for a filter with 3.0 m pore size and 1 mL blood sample volume. Error bars are sd. Figures 14A - 14B show a comparison of the inventors’ work with other methods for bacterial isolation from blood. The graphs compare the limit of detection, separation efficiency (ratio of bacteria recovery over RBC recoveryin the filtrate) in Figure 14A, and in Figure 14B, throughput. Methods reporting 100% RBC rejection (i.e., infinite separation efficiency) are plotted at the arbitrary value of 1e4 separation efficiency. Figures 15A-15E show different embodiments of the fluid guide where the filter is arranged at different angles α with regard to a plane perpendicular to the sedimentation direction. The different angles α are shown as follows: Figure 15A - 27°, Figure 15B - 48°, Figure 15C - 63°, Figure 15D - 72°. Figure 15E shows the introductory part of the grid structure (unit: mm). Figures 16A-16D show cross-sectional schematics of the device involving centrifugation based separation and filtration and up-concentration of bacterial cell. In Figure 16A, the 3D-printed fluid guide contains a filter and is inserted in a centrifuge tube. The fluid guide is subsequently filled with Lymphoprep-broth medium and blood infected with bacteria. In Figure 16B, during centrifuging, blood cells sediment at a higher velocity than bacteria (top inset). Blood cells cannot pass the filter (low left inset) and sediment into the blood cell collection pocket. Bacteria sediment through the filter pores (bottom right inset) into the bottom region of the centrifuge tube. In Figure 16C, after centrifuging, the fluid guide containing the blood cells is lifted away. In Figure 16D, the supernatant liquid is removed to recover the concentrated bacterial liquid at the bottom. Figures 17A-17D show cross-section views of the oscillating device for higher bacterial recovery. Figure 17A shows the fluid guide, comprising an integrated filter and an air pocket, kept in a centrifuge tube . The device is filled with a mixture of blood spiked with bacteria and broth. In Figure 17B, after starting the centrifugation, the air in the air pocket gets compressed and particles start settling. In Figure 17C, the blood cells stay inside the fluid guide whereas bacteria pass the filter and settle in the centrifuge tube. In Figure 17D, when the centrifugation is stopped the compressed air expands and resuspends theblood cell and the bacteria pellet in the fluid guide and the centrifugation is repeated. Figures 18A-18C are cross-sectional views showing how the bacterial separation from blood through repeated filtration works. Figure 18A shows the fluid guide having an integrated filter and a siphon is kept in a centrifuge tube. The device is filled with a mixture of blood spiked with bacteria and broth. In Figure 18B, after starting the centrifugation, the air in the closed end of the siphon gets compressed and particles start settling. The blood cells stay inside the fluid guide whereas bacteria pass the filter and settle in the centrifuge tube. In Figure 18C, when the centrifugation is stopped, the compressed air expands and resuspends the blood cell and the bacteria pellet in the fluid guide and the centrifugation is repeated. Figures 19A-20C show flowchart illustrations of the methods of using the sedimentation container according to the second and third aspects of the invention. DETAILED DESCRIPTION Improved devices for separating different types of particles from a fluid and methods of use thereof will now be described. In embodiments, a sedimentation container includes a sedimentation container (1) comprising an inclined filter (3). As shown in Fig.1A the sedimentation container (1) is placed in a sedimentation environment where sedimentation takes place in a direction, the sedimentation direction (7). The filter (3) forms an inclination angle α (9) with respect to the sedimentation direction (7). The sedimentation container has a top end (5’) and a bottom end (5’’) in the direction of the sedimentation direction (7). The sedimentation container (1) is configured to separate particles of different types in a fluid. The separation of particles of different types may be based on the difference in cell size and / or terminal velocity between blood cells andbacteria that utilizes centrifuge-driven cross-filter particle transport. Other forms of separating particles of different types based on mechanical, chemical or electrical interactions in a fluid are also contemplated. Having the filter arranged at an inclination angle (9) enables efficient separation of different particles in a fluid, particularly separation of low- abundant bacteria from whole blood at relevant throughput in a single step. As shown in Fig.1B, shows the sedimentation container (1) comprises a centrifugation container (11). The centrifugation container may be of a volume known and used by persons of skill in the art. As a non-limiting example the centrifugation tube may be a 50 mL centrifuge tube. Fig.1C shows an embodiment of the sedimentation container (1) wherein the sedimentation container further comprises a grid (13) placed in a higher in the sedimentation container than the filter (13). By higher it may be meant closer to the top end (5’) of the sedimentation container. The grid (13) comprises walls that are substantially parallel to the sedimentation direction. The grid may have vertical walls placed in a square mesh to prevent liquid convection induced by the Coriolis effect during centrifuge acceleration. Various vertical wall thicknesses and square mesh pitches may be possible, in accordance with the types of particles to be separated from a given liquid. In one embodiment used to separate bacteria from blood components, a grid of vertical walls of thickness 0.4 mm are placed in a square mesh with a pitch of 1.6 mm. Fig.1D shows an embodiment of the sedimentation container (1) wherein the sedimentation container further comprises a particle guide (15) for guiding fluid arranged in the sedimentation container. The inclined filter (3) may be attached to the particle guide (15). As shown in Fig.1E, in some embodiments, the grid (13) may be located in the region of the particle guide above the filter. The particle guide (15) may take one of various shapes to optimize the separation of different particles in a fluid.As seen in Fig.1F , the sedimentation container (1) may also comprise a retentate collection region (17) for collecting the filter retentate particles, and a filtrate collection region (19) for collecting the filtrate, for example the bacteria, in the lower / bottom region of the centrifuge tube. The sedimentation container may be a centrifuge container. An air vent (21) may also be provided in the particle guide (15) to mitigate air trapping during liquid priming. As seen in Figure 1G, the sedimentation container may comprise a fluid guide (27) comprising a siphon (23) arranged in the sedimentation container with an open end (25) in fluidic connection with the retentate collector (17). The siphon may comprise air (or another compressible fluid) and the open end 25) of the siphon is configured to fill up with liquid during centrifugational acceleration, thus compressing the air. During centrifugal deceleration the air is decompressed to resuspend fluid back to the container. The fluid for separation in the sedimentation container may comprise one of a blood, urine, saliva or fecal sample. The fluid may also comprise a non- physiologic fluid sample or a fluid not for medical use. In some embodiments of the sedimentation container (1), the filter is adapted to separate blood cells from bacteria. In practice, the sedimentation container is used as follows: 1. (Optionally) Filling the sedimentation container with a liquid medium, (for example, a density medium comprising a mixture of 75% Lymphoprep and 25% broth) until it reaches a top of the grid and centrifuging the device for a short duration to remove any air bubbles that may be trapped. 2. Loading the blood sample onto the top of the medium. 3. Centrifuging the sedimentation container, during which the larger blood cells sediment at ∼30× higher velocity than the bacteria (according to Stoke’s law). Blood cells move along the filter surface before the bacteria and sediment in the blood cell collector at the bottom edge of the filter. Bacteria that land on the solid fraction of the filter experience a force of Fg sinαtangential to the filter surface, where Fg is the artificial gravity acting on the bacteria. Bacteria that reach the filter pores are dragged into the pore and across the filter with force Fg · cosα, after which they sediment into the bacteria collection region. 4. Lifting the fluid guide containing the blood cells away from the device, leaving the bacteria in the centrifuge tube. Example 1: Filter-in-Centrifuge Separation of Low-Concentration Bacteria from Blood A filter-based centrifugal device for efficiently separating low-abundant bacteria from whole blood at relevant throughput in a single step. Results A separation container was developed, based on the difference in cell size and terminal velocity between blood cells and bacteria 17, that utilized centrifuge-driven cross-filter particle transport (Figures 2 and 3). The separation container comprised a 3D-printed fluid guide inserted into a 50 mL centrifuge tube. The fluid guide incorporated a filter, inclined at angle α with respect to the centrifugal acceleration, and had pores smaller than blood cells but larger than bacteria. The region of the fluid guide above the filter featured a grid of vertical walls of thickness 0.4 mm placed in a square mesh with a pitch of 1.6 mm to prevent liquid convection induced by the Coriolis effect during centrifuge acceleration (Figures 11A-11C)34. At the lower side of the filter, the fluid guide included a blood cell collection pocket for the filter retentate, while the filtrate was collected in the lower region of the centrifuge tube. An air vent in the fluid guide mitigated air trapping during liquid priming. Operating the device proceeded in four steps. 1. The fluid guide was filled with density medium (a mixture of 75% Lymphoprep and 25% broth) until it reaches the top of the grid. The devicewas centrifuged for a short duration to remove any air bubbles that may be trapped. 2. The blood sample was loaded onto the top of the medium. 3. The device was centrifuged, during which the larger blood cells sediment at ∼30× higher velocity than the bacteria (according to Stoke’s law17). Blood cells moved along the filter surface before the bacteria and sedimented in the blood cell collector at the bottom edge of the filter. Bacteria that landed on the solid fraction of the filter experienced a force of Fg sinα tangential to the filter surface, where Fg was the artificial gravity acting on the bacteria. Bacteria that reached the filter pores were dragged into the pore and across the filter with force Fg · cosα, after which they sedimented into the bacteria collection region. 4. The fluid guide containing the blood cells was lifted away from the device, leaving the bacteria in 18 mL of Lymphoprep-broth medium in the centrifuge tube. The sample was then ready for downstream processing. Optimization of Key Parameters The key parameters associated with the working of the device were investigated and optimized for two variables, bacteria recovery and blood cell rejection. Density of media: The density media was made by mixing lymphoprep (1.077 g / cc) and LB broth media (1.000 g / cc). Mixing lymphoprep with broth created different densities of medium. The optimal medium mixture was found to be 75% lymphoprep in broth, which has a density slightly above that of blood , thus promoting both efficient layered loading (Figure 8c) and sedimentation of particles through thefilter pores (Figure 8a). In contrast, using a lower-density mixture (0 or 50% lymphoprep) prevented layered loading and resulted in the uncontrollable sinking of blood (Figures 8a and Figures 8b), causing cells to be transported downward by convection rather than sedimentation. This led to lower bacteria recovery and compromised the efficiency of the device. On the other hand, using a higher density mixture (100% lymphoprep) slowedsedimentation and reduced the artificial gravity (Fg) that drives the bacteria through thefilter pores, which also resulted in lower bacteria recovery. Overall, thefindings highlight the importance of choosing the right density for the medium to optimize performance and improve efficiency. The device and method are not limited in terms of the media density. Filter inclination angle: Filters at 63 °angle resulted in the highest average bacterial recovery, this angle was chosen for the optimal design (Figure 15c). However, the influence offilter inclination angle on bacteria recovery for the range 27◦ < α < 72◦ was not significant. The totalfilter length, along which particles move, scaled with 1 / cosα, whereas the force driving particles through the pores scaled with cosα. It was hypothesized that these two effects canceled each other. The device and method were not limited in terms of the angle of thefilter, as long as 0◦< α < 90◦. Filter pore size: The fraction of bacteria and RBCs in thefiltrate both increased with thefilter pore size (Figure 8C and Figure 7A). The optimal pore size depended on what parameter one wanted to optimize. Small pores were beneficial if maximal blood cell rejection wa targeted. When maximal bacteria recovery was targeted, large pores provided a better result. Among the pore sizes tested for maximal separation efficiency, it was observed that 2 μm pores gave the best results. The pore sizes, however, could be of any size between 1 and 5 μm. Furthermore, the pore density in thefilter could vary. The cross- sectional shape of the pores could be different, including circular pores, elliptical pores, pores in the shape of a slit, square pores, or other pores. The pores could be regular in shape, or could be random, as in a porous material. The device and method were not limited in terms of pore size, as long as bacteria could move through thefilter easier than white or red blood cells. Centrifugation speed and time:To ensure the effective performance of the device, it was important that the bacteria had enough time to sediment to and through thefilter. The sedimentation length was directly proportional to the product of the artificial gravity and centrifugation time. Observations revealed that most RBCs sedimented to the collection pocket within 1-2 minutes at 4500g (Figures 10A- 10B). Given that RBCs have an estimated sedimentation velocity 30 times larger than that of bacteria, it was expected for the bacteria to typically sediment in 30-60 min. Consistent with this expectation, it was found that extending the centrifugation time beyond 60 min did not significantly increase bacteria recovery (Figure 8D). The optimal conditions for centrifugation were therefore 60 min at 4500g, which provided a relative bacteria recovery of 65%. The device and method were not limited in terms of centrifugation force or centrifugal acceleration or deceleration. Bacteria recovery and blood cell rejection: Using these optimal design parameters, these bacteria were separated from 1 mL of spiked blood in 1 h with 32 ± 4% (sd, n=18) total bacteria recovery (CFU infiltrate / CFU in blood) and 99.4 ± 0.1% (sd, n=3) RBC rejection(Figure 5), 98.4 ± 1.4% WBC removal, and 90.0 ± 2.6% platelet removal. No significant difference was found in bacteria recovery between 100 and 1000 CFU / mL concentration of spiked blood, nor between different bacterial species. Blood spiked with 10 CFU / mL of the three bacteria species resulted in positive overnight culture of thefiltrate for all tests (n=3 for each bacterial species). RBC rejection forfilters with 2.0 and 3.0 μm pore size was studied and it was found that 2.0 μm pores provided the highest RBC rejection and relative separation efficiency (relative bacteria recovery / [1-RBC rejection]). WBC and platelet rejection for afilter with 3.0 μm pore size were also studied (Figure 13). Comparison with state-of-the-art: The inventors’ method uniquely provided the combined separation of bacteria at low concentration, high separation efficiency and relevant throughput, which were the three most important performance parameters from a clinicalapplication perspective (Figure 13). Other sedimentation-based methods have enabled a low bacterial concentration detection but feature limited RBC[17, 18] or WBC

[0018] removal, affecting potential downstream sample processing. A separation efficiency of 100 or above is typically desirable to avoid interference of blood cells during microfluidic handling (clogging) or optical readout. Several methods reported high separation efficiency [22, 24- 26] or high throughput [22, 24, 19, 37], but these methods have been shown only for high bacteria concentration, 1000 CFU / mL or above, which would necessitate 'me-consuming bacterial culture of the clinical sample prior to processing with these methods. To allow rapid sepsis diagnostics necessitated a sample processing throughput of at least 10 mL blood (considering the low bacterial count) in one hour (considering the need for rapid results). Inventors’ approach allowed reaching this requirement through parallelization, e.g., by dividing a 10 mL blood sample into 10 aliquots in different devices during the same centrifugation. The downstream processing required blood cell removal to avoid interference with the blood cells in the identification of pathogens and AST. Achieving high RBC, WBC, and platelet removal would be significant for implementing easy downstream processing

[0037] . Inventors’ approach achieved 99.4 ± 0.1% RBC removal, 98.4 ± 1.4% WBC removal, and 90.0 ± 2.6% platelet removal, positively addressing the clinical need. Role of grid in bacterial recovery: The upper section of thefluid guide was designed for terminal velocity-based particle separation. However, the Coriolis force caused aflow vortex during centrifuge acceleration, which can cause the mixing of the separating particles and reduce the bacteria recovery (Figures 11A-11C). The vertical grid walls effectively blocked horizontalflow components, strongly diminishing the effect of the Coriolis vortex. By blocking the horizontalflow component using the vertical wall grid, the vertical sedimentation transport of the bacteria and blood cells was not impeded. The size of the grid used was sufficient to prevent the mixing. The device and method were not limited in terms of the design, size and placement of the grid.Design of the grid: The higher value of pitch of the grid structure like 2, 3, or 4 mm could also prevent the Coriolis mixing at a large extent and also give less surface area to bacteria to stick to the surface. Coriolis mixing: During device development, Coriolis mixing was observed to convectively mix the sample during centrifugal acceleration and deceleration. This phenomenon was studied by integrating a wireless camera (Global Tsolar Lights Electrical, China) and light source (Ledlenser, Germany) next to a centrifuge tube during centrifuge operation (Figures 11A-11C). During centrifuging, the camera sent images of the centrifugation wirelessly from within the centrifuge to a computer outside the centrifuge. Despite the suboptimal image quality in this cramped optical configuration, the obtained results were sufficient for visual inspection of the centrifugation process. Centrifuge tubes with and without vertical grid structures were compared. The centrifuge was accelerated to 10000 g and visualized continuously in real- time. In centrifuge tubes without a grid, the blood was convectively mixed with the density medium within seconds. In contrast, in centrifuge tubes with a grid, most blood cells moved by sedimentation rather than convection. Inventors concluded that the grid efficiently reduced the mixing by blocking horizontalflow components. The inventors made an attempt to recover bacteria from the separation device without the grid structure and received fewer bacteria recovery (around 26%). The device and method were not limited in terms of the amount of Coriolis mixing occurring. Inclinedfilter: Filter inclination was critical to the separation of bacteria. Because of sufficientfilter angle the blood cells did not sit on thefilter and rolled over and enabled centrifugation andfiltration based separation. Inventors have developed the device with differentfilter angles 27 º, 48 º, 63 º and 72º. (Figures 15A-15D). However, other angles offilters were also possible. Thelength of thefilter was directly proportional to the angle of inclination whereas the force that drove the particle through thefilter pore was inversely proportional to the inclination angle. Length of device: The length of the device above thefilter was critical in the separation of bacteria as the centrifugation-based separation was happening in the region abovefilter because different sedimentation velocity of bacteria and RBC. If the abovefilter was not long enough then bacteria and blood cell arrived simultaneously on thefilter and interacted with each other on thefilter, which could decrease the bacterial recovery. The device and method were not limited in terms of the device length. Centrifugal separation + Filtration and Up-concentration: The method of using the device could be modified slightly to do three processes in one device at a time i.e centrifugation-based separation, filtration, and up-concentration of bacteria at the end (Figures 16A-16D). The additional up-concentration step involves removing the supernatant from above the bacteria after removing thefluid guide. Advantageously, this technology concatenates the three methods of sample preparation in one step, which makes it very powerful. The up-concentrated bacterial liquid are usually required for downstream processing like for microfluidic application or genotypic identification technique like PCR. Starting from 1 ml of blood around 35% of the bacteria could be collected in small volume (around 0.5 ml) in a single step removing most of the blood cells. Oscillating device for increasing bacterial recovery The oscillating device for the separation of bacteria from whole blood consists of a fluid guide having an integrated filter and a siphon is kept in a centrifuge tube as shown in Figures 17A-17D. The device is filled with a mixture of blood spiked with bacteria and broth and centrifuged. At the onset of centrifugation air in the air pocket gets compressed and particles start settling. The blood cells along with some bacteria settle in the bottom part of the fluid guide as itcan’t cross the filter whereas some bacteria pass the filter and settle in the centrifuge tube. When the centrifugation is stopped the compressed air expands and resuspends the blood cell and the bacteria pellet in the fluid guide. In this manner, bacteria that were trapped between the blood cells are resuspended again. In a next centrifugation step they can pass through the filter. The acceleration and deceleration of the centrifuge can be repeated several times, each time resuspending the settled particles, and each time increasing the chance for the bacteria to go through the filter rather than getting trapped among the blood cells. After more than one acceleration- deceleration cycles, bacteria can be recovered from below the filter. The device and method are not limited in terms of the siphon / air pocket placement, volume and geometry. Example 2 - Bacterial Separation from Blood Through Automated Repeated Filtration in a Centrifuge A separation device capable of performing repeated filtration by utilizing trapped air acting as a passive pump to re- suspend blood cell sediment (Figure 1G) was developed. The device was fabricated using a 3D printer and inserted into a 50 mL centrifuge tube. The device had an attached membrane filter inclined at 63 degrees. The filter had pores of size 3 μm, smaller than blood cells but larger than bacteria. a siphon or passive pump having trapped air was placed on the lower side of the inclined filter,. To use the separation device, the following four steps were followed: 1. Loading media: Add 0.8 mL of Percoll (high-density medium) were added inside the passive pump to avoid sedimentation of blood cells at the bottom of the passive pump. The separation device was then inserted into a 50 mL centrifuge tube prefilled with 1mL of lymphoprep at the bottom and approximately 30 mL of broth over it. Lastly, 1 mL of infected blood(mixed with 10 mL of broth) was added inside the sample reservoir (Figure 18A). 2. Centrifugation for filtration: Centrifugal force compressed the trapped air in the passive pump and moved the blood cells and bacteria to the bottom of the tube (Figure 18B). Two different centrifugation speeds were applied to filter bacteria efficiently. First, low-speed centrifugation at 200 g for 2 minutes was used to sediment the blood cells as they moved faster due to their higher sedimentation velocity from the larger size. Next, high-speed centrifugation at 1000 g for 5 minutes was applied to sediment the bacteria. As the bacteria sedimented and moved over the filter, some passed through the filter and were collected in the filtrate at the bottom of the tube, while others settled at the bottom of the sample reservoir along with most of the blood cells. 3. Resuspension process: Stopping centrifugation released compressed air in the passive pump, pushing up the sediment and resulting in its resuspension (Figure 18C). The centrifuge was started and stopped three times at 1000 g to ensure complete resuspension of all sedimented blood cells and bacteria. After resuspension, the device returned to its initial state as it was before centrifugation (Figure 18A). 4. Repeating the centrifugation: The centrifugal cycle was repeated to repeatedly resuspend the sediment and filter the bacterial cells to enrich them in the filtrate (Figures 18B-18C). BacterialThe filtering cycling was repeated 1, 3, 5 and 10 times, and the total and relative recovery of bacteria in the filtrate is shown in Figure 18C. Total recovery was the ratio of the number of bacteria recovered in the filtrate to theinitial number of bacteria, whereas relative recovery is the ratio of bacteria recovered in the filtrate to the total bacteria recovered (sum of bacteria in filtrate, reservoir, and the passive pump). The results indicated that the percentage of bacterial recovery increased with the number of filtration cycles, rising from 4% to 18% until 5 cycles (Figure 2A). However, the total bacterial recovery decreased from 5 cycles to 10 cycles, in contrast to the relative recovery, which continues to increase. We hypothesized that the observed decrease in total bacterial recovery from 5 to 10 cycles is due to bacterial loss from excessive centrifugation. To address this, we increased the volume of Lymphoprep (a high density medium) at the bottom to 20 ml to increase the effective path length. As a result, the total recovery increased from 18% to 24% at 5 to 10 cycles. Characterization of re-suspension The operation of the pump was characterized by loading the device with blood without bacterial cells, as depicted in Figure 18A. The centrifugation cycle was repeated 5 times, and the blood cell count in the sample reservoir was measured using a hematology analyzer. More than 90% of the blood cells were resuspended by the passive pump. The results suggested that re-suspension was not much affected by the number of centrifugation cycles. This was because the trapped air did not escape the passive pump due to the small diameter of the tube connecting the sample reservoir with the passive pump, which prevented bubbling. Additionally, pre-filling the device with Percoll, which had a higher density than blood cells, resulted in the blood cells settling at the top of the Percoll rather than at the bottom of the pump. This facilitates the resuspension of the blood cells.Blood cells rejection The quantity of blood cells in the filtrate was measured after the experiment. Following 5 cycles of filtration, the filtrate contained approximately 0.3% of red blood cells (RBCs), 9% of white blood cells (WBCs), and 30% of platelets. This indicated that 99.7% of RBCs, 91% of WBCs, and 70% of platelets were effectively rejected during filtration. Methods Device fabrication The device was designed using Solidworks 2021 and fabricated with a Form 3+ 3D printer (Formlabs, USA) employing clear V4 resin material. Post- fabrication, it was cleaned in a Form Wash system (Formlabs, USA) using isopropanol (IPA) for 45 minutes, followed by curing in a Form Cure (Formlabs, USA) under ultraviolet (UV) light at 60 ◦C for 45 to 60 minutes. A Nuclepore track-etched polycarbonate membrane filter (Whatman, Cytiva, UK) with a pore size of 3.0 μm was affixed using epoxy glue (Loctite, USA). Subsequently, the separation device with the filter was inserted into a 50 mL centrifuge tube. Medium preparation The broth was prepared by dissolving Luria low salt LB broth powders (L3397, Sigma-Aldrich, USA) in deionized water (DIW) at a concentration of 25 g / L, followed by autoclaving. Lymphoprep (STEMCELL Technologies, Canada) and Percoll (Sigma-Aldrich,USA) were used as density media. Spiked blood preparation Fresh blood from healthy donors was obtained from the blood bank (Blodcentralen, Stockholm, Sweden) and used for experiments within three days of sampling. E. coli ATCC 11775 was obtained from Uppsala University and stored at -80 ◦C in a glycerol solution. The bacteria were cultured overnight in broth media, diluted to 105 CFU / mL, and 700 μL of it was mixedwith 1 mL of healthy blood. This spiked blood (1 mL) was combined with 10 mL of broth in the sample reservoir. The precise bacterial concentrations in the samples were determined by agar plate counting (n=3). Post-centrifugation analysis After centrifugation, the separation device was removed from the centrifuge tube, and liquid from the reservoir and the passive pump was sampled. All three liquid samples were carefully resuspended and inoculated onto agar plates for bacterial quantification. The volumes of the liquid samples were also measured to calculate the total bacterial count. Bacteria counting Agar plates were used for bacterial counting. They were prepared by dissolving LB Broth with agar (Miller) (Sigma-Aldrich, USA) in DIW at a concentration of 40 g / L, then autoclaved and transferred to clean Petri dishes. A small volume of each of the three liquid samples—filtrate, reservoir, and pump—was plated on the agar separately (n=3 for each). The plates were incubated overnight at 37 ◦C, and the colonies were counted to evaluate the bacterial cell concentration. Blood cells counting The blood cell count was determined using a hematology analyzer, Swelab Alfa Plus (Boule Diagnostics, Sweden), which measures RBCs, WBCs, and platelets. Blood cell counts were obtained for both the blood used in the experiment and the filtrate post-experiment to calculate the rejection rate using the following equation1. Conclusions This study developed an automated device for repeated blood filtration to isolate bacterial cells. Utilizing only a centrifuge, this hands-off methodefficiently performs repeated filtration. 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Claims

CLAIMS 1. A sedimentation container in a sedimentation environment with a sedimentation direction, comprising a filter having an inclination angle to the sedimentation direction, wherein the filter is configured to separate particles of different types in a fluid.

2. The sedimentation container according to claim 1, wherein the inclined filter is positioned at an angle α between substantially 27 and 72 degrees with regard to a plane perpendicular to the sedimentation direction.

3. The sedimentation container according to claims 1 or 2, further comprising at least one of a grid, a particle guide, and a siphon.

4. The sedimentation container according to claim 3, wherein, in a sedimentation field the grid is placed higher compared to the filter, and wherein the grid comprises walls that are substantially parallel with the sedimentation direction.

5. The sedimentation container according to any of the previous claims, further comprising at least a filtrate collection region for collecting filtrate in a lower region of the sedimentation container and a separate retentate collection region for collecting retentate particles.

6. The sedimentation container according to any of the previous claims, wherein the sedimentation environment is configured by creating artificial gravity to separate the different particles in a fluid based on their type.

7. The sedimentation container according to claim 6, wherein the sedimentation container comprises a centrifuge container or a centrifuge tube.

8. The sedimentation container according to claim 6 or 7, the sedimentation container having a centrifugal acceleration, wherein the centrifugal acceleration is configured to increase and decrease at least once during centrifugation.

9. The sedimentation container according to claim 6 or 7, the sedimentation container having an angular velocity, wherein the angular velocity is configured to increase and decrease at least once during centrifugation. 10.The sedimentation container according to claim 8 or 9, wherein an open end of the siphon is placed in fluidic connection with the retentate collector and the siphon is configured to fill up with liquid during centrifugational acceleration, thus compressing the air, and decompress during centrifugal deceleration to resuspend retentate particles back to the container. 11.The sedimentation container according to any of the previous claims, wherein the fluid comprises one of a blood, urine, saliva or fecal sample. 12.The sedimentation container according to claim 11, wherein the filter is adapted to separate blood cells from bacteria. 13.The sedimentation container according to any one of claims 1-12, wherein the sedimentation container is configured to isolate and up- concentrate bacteria from body fluid, the body fluid comprising at least one of a blood, urine, fecal sample, and swab in one centrifugation step. 14.A method of using the sedimentation container according to any of the previous claims, comprising: - Loading a liquid sample having a lower density than a liquid medium onto the top of the liquid medium; - Centrifuging the sedimentation container, during which the larger particles sediment at a higher velocity than the smaller particles; - Removing the larger particles from the sedimentation container, leaving the smaller particles in the remaining liquid in the filtrate collection region 15.The method according to claim 14, wherein the step of removing the larger particles comprises lifting the retentate collection region comprising the larger particles away from the sedimentation container,leaving the smaller particles in the remaining liquid in the centrifuge container or tube. 16.The method according to claim 14 or 15, comprising filling the sedimentation container with a liquid medium to reach the top of the grid, before loading the liquid sample. 17.The method according to any of claims 14 -16, comprising centrifuging the sedimentation container to remove any air bubbles that may be trapped in the liquid medium, before loading the liquid sample. 18.The method according to any of claims 14-17, wherein the step of centrifuging the sedimentation container comprises - Centrifuging the sedimentation container at a first speed during which a first relative fraction of the larger particles reach the filter earlier than a first relative fraction of the smaller particles; and - Centrifuging the sedimentation container at a second speed, during which a second relative fraction of the larger particles move over the surface of the filter without crossing the filter, which second relative fraction of the larger particles is larger than the relative fraction of smaller particles moving across the filter without crossing the filter; 19.The method according to any one of claims 14-18, wherein the liquid medium is configured to have a density above that of the liquid sample for promoting efficient layered loading of the liquid sample on top of the liquid medium and sedimentation of the particles of the liquid sample through the filter pores. 20.The method according to claim 14-18, wherein the liquid sample is blood, urine, saliva or a fecal sample. 21.The method according to claim 20, wherein the liquid sample is blood and the liquid medium comprises a mixture of substantially 75% Lymphoprep and substantially 25% broth. 22.The method according to claim 21, wherein the centrifugation tube and container device are centrifuged at approximately 4500g. 23.The method according to claim 22, wherein the centrifugation tube and container device are centrifuged for 1 or more minutes.A method of using a sedimentation container, the sedimentation container comprising a fluid guide having an inclined filter configured to separate different particles in a fluid and a siphon arranged downstream of a sedimentation field of the sedimentation container, the method comprising: - Adding a first high density liquid medium inside the siphon, trapping air at a closed end of the siphon; - Inserting the sedimentation device into a centrifuging tube prefilled with a second liquid medium; - Adding a liquid sample comprising different types of particles to the top of the fluid guide; - Centrifuging the liquid sample to move the liquid sample toward the bottom of the sedimentation container, wherein a fraction of the smaller particles passes through the filter, and a rest of the liquid sample moves to the bottom of the siphon and compresses the air trapped in the closed end of the siphon ; - Stopping the centrifuging to allow resuspension of the sediment in the siphon; - Repeating the centrifuging process more than once. The method according to claim 24, wherein the siphon is placed at a lower end of the inclined filter. The method according to claim 24, wherein the centrifuging process is repeated between 1 to 10 times.

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