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

Abstract

A system (50) for separating particles in a fluid has a first (52) and a second chamber (54) connected via a connector (56) with two ends, the first end (58) connecting to the first chamber (52) and the second end (60) connecting to the second chamber (54). The first chamber is higher than the second chamber and is connected pneumatically to an ambient atmosphere (66). The second chamber (54) is connected pneumatically or fluidically only to the first chamber via the connector only. A first method of operating the system includes adding a liquid to the first chamber to trap air in the connector and the second chamber; centrifuging the system at a first relative centrifugal force (RCF1) such that no part of the liquid enters the second chamber, and some air remains trapped in the connector; centrifuging the system at a second relative centrifugal force (RCF2) higher than RCF1, so at least a part of the liquid is transferred from the first chamber to the second chamber through the connector; and centrifuging the system at a relative centrifugal force (RCF3) lower than RCF2 so at least a part of the liquid is transferred from the second chamber to the first chamber through the connector. A second method of operating the system includes first adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; then, centrifuging the system at a first relative centrifugal force (RCF1) so part of the air remains trapped in the connector and no particles are transported to the second chamber; then, centrifuging the system at a second relative centrifugal force (RCF2) higher than RCF1, so part of the liquid is transferred from the first chamber to the second chamber through the connector; and finally, centrifuging the system at a relative centrifugal force (RCF3) lower than RCF2 so part of the liquid is transferred from the second chamber to the first chamber through the connector.

Description

[0001] CENTRIFUGE-BASED SEDIMENTATION DEVICES FOR SEPARATING BACTERIA AND PARTICLES FROM FLUIDS AND METHODS OF USE

[0002] THEREOF

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to centrifuge-based sedimentation devices and systems, specifically sedimentation containers, for separating bacteria from fluids. Also, related methods of using these centrifuge-based sedimentation containers are disclosed.

[0005] BACKGROUND

[0006] 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 billion However, the current clinical diagnosis of BSI relies on a positive blood culture, which is a slow process that can take several days6, generally 2-3 days42. As the mortality rate of untreated patients increases by 8% per hour7‘39, clinicians often resort to broadspectrum antibiotics. Unfortunately, in approximately 30% of cases, this approach is incorrect and can lead to more fatalities8’40. Furthermore, the use of broad-spectrum antibiotics promotes the growth of antimicrobial resistance9, 41.

[0007] Rapid pathogen identification and antimicrobial susceptibility testing (AST) are preferred methods for diagnosing sepsis 10, 11. Various rapid phenotypic and genotypic methods have been developed for this purpose 12—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

[0008] 17 18 19-21 22 23 24 differentiation ’ , inertial and elastoinertial microfluidics, acoustophoresis ’ , surface acoustic waves (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).

[0009] Size-based filtration is a simple, inexpensive, and highly selective method with successful applications in separating WBCs28or tumor cells ’ from blood and in blood-serum separation . WBCs have typical size 10-20 pm, RBCs 6.2-8.2 pm, platelets 2.0-4.0 pm, and bacteria around 1.5 pm.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 throughput.17

[0010] A need exists for a separation device capable of separating low-abundant bacteria from whole blood at relevant throughput in a single step.

[0011] 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.

[0012] As previously stated, a further problem lies in the length of time needed for identifying pathogens and performing antimicrobial susceptibility testing (AST). This lengthy process is due to the reliance on multiple culture steps for identification and AST.

[0013] In clinical settings, blood samples from patients first undergo a blood culture to increase bacterial count, which takes approximately 4-72 hours to yield a positive result43'45. This step is critical as only 25-38% of blood cultures test positive even in septic patients46'48. Once a culture is confirmed positive, it is essential to identify the microorganism and perform AST to prescribe effective antibiotics. Current bacterial identification methods include genotypic techniques (e.g., polymerase chain reaction), phenotypic methods (e.g., subcultures), and mass spectrometry like matrix-assisted laser desorption ionization time of-flight mass spectrometry (MALDI-TOF), which is one of the fastest and most commonly used methods in well-equipped clinics14. However, identifying bacteria using MALDI-TOF requires pure bacterial colonies, necessitating an additional subculture step that takes at least 6-12 hours, delaying appropriate antibiotic treatment by another day49. For AST, traditional methods involve growing bacteria in broth or on agar plates with antibiotics, a time-consuming process that can take from 18-24 hours due to reliance on macroscopic bacterial growth identification50. Alternatively, microfluidic-based technologies can perform AST within hours by trapping and imaging single bacterial cells, but they mostly require a pure bacterial culture15, 16. Thus, sample preparation remains a bottleneck for these technologies to function directly from blood or positive blood cultures. A robust sample preparation method or device could significantly accelerate the diagnostic process by eliminating the need for subculture for MALDI-TOF and enabling microfluidic-based technologies to be used directly with blood or blood cultures.

[0014] Numerous methods and devices have been developed for sample preparation from blood or blood cultures, tailored to different downstream processing methods14. Common techniques for isolating and concentrating bacterial cells include sedimentation velocity-based differentiation17'18’51filtration52, magnetic bead separation53, acoustophoretic54, inertial19and elastoinertial microfluidics22, surface acoustic waves (SAW)55and dielectrophoresis26. However, except for sedimentation and filtration, these methods often suffer from low throughput or too complex for clinical application. While centrifugation and filtration methods offer high throughput and readily used in clinic for sample preparation for MALDI- TOF or AST56'57, they require multiple steps to prepare samples for downstream processing, making them labor-intensive and susceptible to contamination.

[0015] Thus, a need exists for a high-throughput device for sample preparation that is non- selective, fully automated, and easy to integrate into existing diagnostic pipelines such as MALDI-TOF, single-cell detection, and subculturing.

[0016] SUMMARY OF THE INVENTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In a further embodiment, the sedimentation container may further comprise at least one of a grid, a particle guide, and a siphon.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Desirably, the fluid in the sedimentation container may comprise one of a blood, urine, saliva or fecal sample.

[0028] Preferably, the filter in the sedimentation container may be adapted to separate blood cells from bacteria.

[0029] 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.

[0030] 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.

[0031] In one embodiment the step of removing the larger particles may comprise 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Desirably, in the method according to the second aspect, the liquid sample may be a blood, urine, saliva or fecal sample.

[0037] 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.

[0038] In one embodiment, the centrifugation tube and container device may be centrifuged at approximately 4500g.

[0039] In one embodiment, the centrifugation tube and container device may be centrifuged for 1 or more minutes.

[0040] 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.

[0041] 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.

[0042] Additionally, a system (50) for separating particles 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.

[0043] In a fourth aspect, a system (50) for separating particles in a fluid is provided. The system comprises a first (52) and a second chamber (54) connected via a connector (56). The connector has two ends wherein the first end (58) of the connector connects to the first chamber (52) and the second end (60) of the connector connects to the second chamber (54). The first chamber is positioned higher than the second chamber with respect to a sedimentation direction (62) of the system, and, additionally the first chamber (52) is connected pneumatically to an ambient atmosphere (66). The second chamber (54) is connected only to the first chamber via the connector; the second chamber is connected pneumatically or fluidically to the first chamber.

[0044] In a fifth aspect, a method of operating the system is provided. The method comprises first adding a liquid to the first chamber to trap air in the connector and in the second chamber; then, centrifuging the system at a first relative centrifugal force (RCF1) such that no part of the liquid enters the second chamber, and some air remains trapped in the connector; then, centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that at least a part of the liquid is transferred from the first chamber to the second chamber through the connector; and lastly, centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that at least a part of the liquid is transferred from the second chamber to the first chamber through the connector.

[0045] In a sixth aspect a second method of operating the system is provided. The second method comprises first adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; then, centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; then, centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; and finally, centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that part of the liquid is transferred from the second chamber to the first chamber through the connector.

[0046] In a seventh aspect, a method of boiling through centrifugation in the system is provided. The method of boiling through centrifugation comprises first connecting a first chamber and a second chamber with a connector, the connector extending upwards from the point of connection to the first chamber; then adding a liquid to the first chamber, thereby trapping air within the connector and the second chamber; centrifuging the assembly such that a portion of the liquid moves from the first chamber to the second chamber through the connector, wherein the height of the liquid column in the connector extends above the liquid level in the first chamber such that the resulting negative hydrostatic pressure in the connector results in outgassing or boiling of the liquid in the connector.

[0047] 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.

[0048] 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.

[0049] As used herein, the term “sedimentation direction” is to be understood to refer the direction in which particles particles sediment due to artificial gravity, typically created by rotating devices like centrifuges or lab-on-disk systems using centrifugal force.

[0050] The terms “sedimentation device” and “sedimentation container” are used herein interchangeably.

[0051] As used herein, the term “artificial gravity” refers to a gravitational force that may be created using centrifugal force of a rotating body.

[0052] 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.

[0053] 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.

[0054] 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 pm” is to be understood as 95 - 105 pm.

[0055] Unless otherwise specified, the term “fluid” includes both bodily fluids and non-medical fluids.

[0056] As used herein, the term “biological components” is to be understood to refer to a sample or a diluted sample of one of a blood, stool, saliva, mucus, urine, and other biological fluids that may contain bacteria.

[0057] As used herein, the term “cushion liquid” is to be understood to refer to a liquid used for cushioning or preventing bacterial or particle loss due to excessive centrifugation.

[0058] As used herein, the term “final aliquot” is to be understood to refer to the remaining sample of bacteria or particles extracted from the bottom of the second chamber, at the end of the centrifugation steps.

[0059] Typically, the subject is a human or animal subject.

[0060] 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 for elements 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.

[0061] BRIEF DESCRIPTION OF THE FIGURES

[0062] 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 a with respect to the sedimentation direction.

[0063] Figure IB, shows a sedimentation container comprising a centrifugation container.

[0064] 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.

[0065] Figure ID shows an embodiment of the sedimentation container wherein the sedimentation container further comprises a particle guide for guiding fluid arranged in the sedimentation container.

[0066] Figure IE shows the grid located in the region of the particle guide above the filter.

[0067] Figure IF 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 may be 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).

[0068] Figure 2 shows an example of an outer part of a fluid guide structure, with dimensions in mm.

[0069] Figure 3 shows examples of inner parts of a fluid guide structure, with dimensions in mm.

[0070] 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 sedimentation device. Figure 4D shows the sedimentation device after its usage.

[0071] 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 Figure 5C, after centrifuging, the fluid guide containing the blood cells is lifted away.

[0072] 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 / l-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)

[0073] Figures 7A-7B: Fig. 4 RBC rejection and relative separation efficiency for filters with 2.0 and 3.0 pm pore size, (a) RBC rejection for filters with pore size 2.0 or 3.0 pm 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 pm. Results are for 0.4 mL of whole blood spiked with 104 CFU / mL of K. pneumoniae. Error bars are 68% CI.

[0074] Figures 8A-8D show relative bacteria recovery (CFU in filtrate / CFU in filtrate and retentate) for varying design and operational parameters. Figure 8 A shows fraction of Lymphoprep in Lymphoprep-broth mixtures, Figure 8B shows filter angle a, 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 pm 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.

[0075] 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.

[0076] Figures 10A-10B show photographs of the sedimentation container after one (Figure 10A) and two minutes (Figure 10B) of centrifugation.

[0077] 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 11 A) and with (Figure 1 IB) 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 pm pore size filter, and centrifuging for 30 min at 4500g.

[0078] 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 pm and 0.4 mL, Figure 12B - 2.0 pm and 1.0 mL, Figure 12C - 3.0 pm and 0.4 mL, and Figure 12D - 3.0 pm and 1.0 mL

[0079] 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.

[0080] Figures 14A - 14B show a comparison of methods according to the present disclosure and other methods for bacterial isolation from blood. The graphs compare the limit of detection, separation efficiency (ratio of bacteria recovery over RBC recovery in 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 le4 separation efficiency.

[0081] Figures 15A-15E show different embodiments of the fluid guide where the filter is arranged at different angles a with regard to a plane perpendicular to the sedimentation direction. The different angles a 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).

[0082] 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.

[0083] 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 the blood cell and the bacteria pellet in the fluid guide and the centrifugation is repeated.

[0084] 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 at 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.

[0085] Figures 19A-20C show flowchart illustrations of the methods of using a sedimentation container.

[0086] Figure 21 shows a system (50) for separating particles in a fluid which contains a first chamber (52) and a second chamber (54) connected via a connector (56).

[0087] Fig. 22A shows an embodiment of the system (50) where the first chamber (52) contains a grid structure (68).

[0088] Fig. 22B shows an embodiment of the system (50) where the second chamber contains a grid structure (68’).

[0089] Fig. 22C shows an embodiment of the system (50) where the first chamber (52) contains a grid structure (68), where the grid structure comprises walls (70) that are substantially parallel with the sedimentation direction (62)

[0090] Fig. 22D shows an embodiment of the system (50) where the second chamber contains a grid structure (68’) and the grid structure comprises walls (70) that are substantially parallel with the sedimentation direction (62) Fig. 23 A shows an embodiment of the system (50) where the connector (56) is straight and parallel to the sedimentation direction. (Figure 1)

[0091] Fig. 23B shows an embodiment of the system (50) where the connector (56) is bent.

[0092] Fig. 24 shows an embodiment of the system (50) having a cross-section of a lumen (72) of the connector, an inner volume (74) of the connector and an inner volume (76) of the second chamber are configured for a pressure (P Pa), such that liquid entering the connector (56) from the first chamber (52) with the pressure (P Pa) compresses the air in the connector (56) and the second chamber to a pressure being the same pressure (P Pa) such that the liquid enters the lumen (72) but not the second chamber (54).

[0093] Fig. 25 shows an embodiment of the system (50) having a filter (78) for removing impurities from the moving liquid during a transfer.

[0094] Fig. 26A, 26B, 26C show embodiments of the system (50) where at least one of the first chamber (52), the second chamber (54), and the connector (56) comprises at least one compartment or particle collection chamber (80) separated by a neck-like structure (82) from the rest of the respective first chamber (52), second chamber (54), and / or connector (56).

[0095] Fig. 27 shows an embodiment of the system (50) where the at least one compartment (80) comprises a particle collection chamber (82).

[0096] Figs. 28A-28C show various configurations of the closure mechanism. Fig. 28 shows in (A) and (B) different configurations of a system (50) where a closure mechanism (84) is affixed to the second chamber (54), wherein the closure mechanism is configured to open to allow fluidic or pneumatic access to the second chamber, and the closure mechanism comprises an elastic or rubber plug (86). Fig. 28C shows a plugged hole to extract the liquid via that plug.

[0097] Fig. 29 shows an embodiment of the system (50) where the connector (56) connecting to the first chamber (52) is configured to always have a non-zero volume of liquid (88) remaining in the first chamber (52) below the connector connection point of the first chamber (58) when centrifugally transporting liquid from the first chamber (52) into the connector (54). Fig. 30 shows an embodiment of the system (50) where the connector connection point of the second chamber (60) is configured such that anon-zero volume of liquid (88) always remains in the second chamber (54) below the connector connection point of the second chamber (60) when centrifugally transporting liquid from the second chamber (54) back to the first chamber (52).

[0098] Fig. 31 A shows an embodiment of the system (50) where the liquid remaining in the first chamber (52) or the second chamber (54) is distributed in one or more isolated liquid traps (92) at the bottom of the respective chambers (52, 54).

[0099] Fig. 3 IB shows an embodiment of the system (50) where the isolated liquid traps (92) trap air in one or more pocket(s) (94) when their respective chamber (52, 54) is filled with liquid.

[0100] Fig. 31C shows an embodiment of the system (50) where the grid structure (68, 68’) comprises walls (70) that are substantially parallel with the sedimentation direction (64) for ensuring that at least a section of the chamber is compartmentalized in defined volumes (96) when particles settle in the isolated liquid traps (92) (e.g., bacteria).

[0101] Figs. 32 and 33A-33E show a cross-sectional schematic of the system and its operation. Fig. 32 shows a design of the system with different design features highlighted. Fig. 33A shows a cross- sectional schematic of the system with different design features highlighted, and the bottom chamber is pre-filled with a cushion / lysing liquid. Fig. 33B shows sample loading: the top chamber is first filled with density media, and the spiked blood culture placed on top of it, followed by the addition of broth media. Fig. 33C shows a soft spin: the system is centrifuged at 100g to settle the blood cells in the collection pocket while preventing the transport of liquid to the bottom chamber. Fig. 33D shows a hard spin: the system is centrifuged at high g (200g) to move the supernatant to the bottom chamber and sediment the blood cells and bacteria at the bottom. Fig. 33E shows the end state: the centrifuge is stopped to transfer the liquid back from the bottom to the top chamber, leaving a small volume of liquid in the bottom chamber.

[0102] Figs. 34A-C show the different functionalities of the system. Fig. 34A is a schematic of the bacterial isolation for subculture. Fig. 34 B is a schematic of sample preparation for MALDI- TOF. Fig. 34 C is a schematic of sample preparation for microfluidics detection. Figs. 35A-35D show the design and working of the device with integrated filter.

[0103] Figs. 36A-36D show the working of the device with density gradient-based separation.

[0104] Figs. 37A-37D show the design and working of the device for performing antimicrobial susceptibility testing (AST).

[0105] Figs. 38A-38D show the design and working of the device for bacterial identification.

[0106] Figs 39A-39C show centrifugal isolation concatenated with sub-culture. Fig. 39A shows centrifugal process flow for sub-culture from blood culture. Fig. 39B shows the % of bacterial cells refers to the percentage of bacteria recovered in the final liquid relative to the initial number of bacteria in the blood culture. Fig. 39C shows the % rejection of cells refers to the percentage of blood cells removed compared to the total number of blood cells in the sample.

[0107] Figs. 40A-40B shows the centrifugal isolation concatenated with MALDI-TOF. Fig. 40A shows the centrifugal process flow for sample preparation for MALDI-TOF. Fig. 40B shows the MALDI-TOF scores for three bacterial species. Scores between 2 and 3 indicate high-confidence identification; scores between 1.7 and 1.99 indicate low-confidence identification; and scores below 1.7 represent failure to identify any species.

[0108] Figs. 41A-41B shows the centrifugal isolation concatenated with microtrap-based detection. Fig. 41 A shows the centrifugal process flow for sample preparation for micro-trap-based detection. The bottom chamber of the device was prefilled with lysing solution. After centrifugation, the final liquid was sampled out from the device and loaded onto a microfluidic chip. Fig. 41B shows an image of the micro-traps with GFP-labelled E. coli trapped in the micro-traps with three different concentrations of spiked bacteria: 5*106, 5*105, and 5*104CFU / ml.

[0109] Figs 42A -42B show the setup for filming inside a centrifuge tube: Fig. 42A shows a schematics of design of the holder used for filming the device inside the centrifuge, which includes compartments for the camera, the device, and the light source. Fig. 42B shows a photograph of the holder with the camera, the device, and the light source in place. Fig. 43 shows images of liquid movement within the device during centrifugation. The figure includes snapshots of the device inside the centrifuge at 100g (soft spin), 2500g (hard spin), and 1g (centrifuge stopped), illustrating liquid transfer dynamics between the top and bottom chambers.

[0110] Figs. 44A and 44B show the device design for quantifying the volume of liquid transferred to the bottom chamber. Schematic of the device with micro-traps in the bottom chamber. During hard spin, the liquid gets transferred to the bottom chamber and fills the micro-traps. When centrifugation is stopped, the liquid returns to the top chamber, leaving liquid in the traps.

[0111] Figs. 45A-45B show the assembly of the 3D printed device. Fig. 45A is an image showing the three separate 3D-printed components of the device, which are assembled to form the final structure. Fig. 45B shows a CAD model illustrating the complete assembly of the device.

[0112] Fig. 46 shows an image of the device after centrifugal operation, highlighting the sedimentation of blood cells and the final liquid distribution.

[0113] Fig. 47 is a schematic showing the device design for vacuum generation.

[0114] DETAILED DESCRIPTION

[0115] Improved devices and systems for separating different types of particles from a fluid and methods of use thereof will now be described.

[0116] In a first set of 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 a (9) with respect to the sedimentation direction (7). The sedimentation container has atop end (5’) and a bottom end (5”) in the direction of the sedimentation direction (7).

[0117] The sedimentation container (1) is configured to separate particles of different types in a fluid.

[0118] The separation of particles of different types may be based on the difference in cell size and / or terminal velocity between blood cells and bacteria 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.

[0119] 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.

[0120] As shown in Fig. IB, 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.

[0121] 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.

[0122] Fig. ID 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. IE, 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.

[0123] As seen in Fig. IF, 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.

[0124] 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.

[0125] In practice, the sedimentation container is used as follows:

[0126] 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.

[0127] 2. Loading the blood sample onto the top of the medium.

[0128] 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 sina tangential 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 • cosa, after which they sediment into the bacteria collection region.

[0129] 4. Lifting the fluid guide containing the blood cells away from the device, leaving the bacteria in the centrifuge tube.

[0130] Example 1 : Filter-in-Centrifuge Separation of Low-Concentration Bacteria from Blood

[0131] A filter-based centrifugal device for efficiently separating low-abundant bacteria from whole blood at relevant throughput in a single step. Results

[0132] 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 a 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.

[0133] Operating the device proceeded in four steps.

[0134] 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 device was centrifuged for a short duration to remove any air bubbles that may be trapped.

[0135] 2. The blood sample was loaded onto the top of the medium.

[0136] 3. The device was centrifuged, during which the larger blood cells sediment at ~30* higher velocity than the bacteria (according to Stoke’s law!7). 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 sina 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 • cosa, after which they sedimented into the bacteria collection region.

[0137] 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.

[0138] Optimization of Key Parameters

[0139] 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:

[0140] 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 dilferent 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 the filter 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) slowed sedimentation and reduced the artificial gravity (Fg) that drives the bacteria through the filter pores, which also resulted in lower bacteria recovery. Overall, the findings 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.

[0141] Filter inclination angle:

[0142] Filters at 63 °angle resulted in the highest average bacterial recovery, this angle was chosen for the optimal design (Figure 15c). However, the influence of filter inclination angle on bacteria recovery for the range 27° < a < 72° was not significant. The total filter length, along which particles move, scaled with 1 / cosa, whereas the force driving particles through the pores scaled with cosa. It was hypothesized that these two effects canceled each other. The device and method were not limited in terms of the angle of the filter, as long as 0°< a < 90°.

[0143] Filter pore size:

[0144] The fraction of bacteria and RBCs in the filtrate both increased with the filter 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 was 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 pm pores gave the best results. The pore sizes, however, could be of any size between 1 and 5 pm. Furthermore, the pore density in the filter 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 the filter easier than white or red blood cells.

[0145] Centrifugation speed and time:

[0146] To ensure the effective performance of the device, it was important that the bacteria had enough time to sediment to and through the filter. 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.

[0147] Bacteria recovery and blood cell rejection:

[0148] 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 in filtrate / 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 the filtrate for all tests (n=3 for each bacterial species). RBC rejection for filters with 2.0 and 3.0 pm pore size was studied and it was found that 2.0 pm pores provided the highest RBC rejection and relative separation efficiency (relative bacteria recovery / [l -RBC rejection]). WBC and platelet rejection for a filter with 3.0 pm pore size were also studied (Figure 13).

[0149] Comparison with state-of-the-art:

[0150] 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 clinical application 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.

[0151] Role of grid in bacterial recovery:

[0152] The upper section of the fluid guide was designed for terminal velocity-based particle separation. However, the Coriolis force caused a flow 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 horizontal flow components, strongly diminishing the effect of the Coriolis vortex. By blocking the horizontal flow 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.

[0153] Design of the grid:

[0154] 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.

[0155] Coriolis mixing:

[0156] 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 horizontal flow 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.

[0157] Inclined filter:

[0158] Filter inclination was critical to the separation of bacteria. Because of sufficient filter angle the blood cells did not sit on the filter and rolled over and enabled centrifugation and filtration based separation. Inventors have developed the device with different filter angles 27 °, 48 °, 630and 72°. (Figures 15A-15D). However, other angles of filters were also possible. The length of the filter was directly proportional to the angle of inclination whereas the force that drove the particle through the filter pore was inversely proportional to the inclination angle.

[0159] Length of device:

[0160] The length of the device above the filter was critical in the separation of bacteria as the centrifugation-based separation was happening in the region above filter because different sedimentation velocity of bacteria and RBC. If the above filter was not long enough then bacteria and blood cell arrived simultaneously on the filter and interacted with each other on the filter, which could decrease the bacterial recovery. The device and method were not limited in terms of the device length.

[0161] Centrifugal separation + Filtration and Up-concentration:

[0162] 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 the fluid 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.

[0163] Oscillating device for increasing bacterial recovery

[0164] 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 it can’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.

[0165] Example 2 - Bacterial Separation from Blood Through Automated Repeated Filtration in a Centrifuge

[0166] 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 pm, 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.

[0167] 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 ImL 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).

[0168] 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.

[0169] 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 18 A).

[0170] 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).

[0171] Bacterial recovery

[0172] The 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 the initial 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.

[0173] 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.

[0174] Characterization of re-suspension

[0175] The operation of the pump was characterized by loading the device with blood without bacterial cells, as depicted in Figure 18A.

[0176] 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.

[0177] 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.

[0178] Blood cells rejection

[0179] 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.

[0180] Methods

[0181] 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 (IP A) 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 pm was affixed using epoxy glue (Loctite, USA). Subsequently, the separation device with the filter was inserted into a 50 mL centrifuge tube.

[0182] Medium preparation

[0183] 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, US A) were used as density media.

[0184] Spiked blood preparation

[0185] 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 pL of it was mixed with 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).

[0186] Post-centrifugation analysis

[0187] 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.

[0188] Bacteria counting

[0189] 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.

[0190] Blood cells counting

[0191] 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 equation \

[0192] , Cell count in filtrate rejection rate [%] = 100- 100 x - — - — — — - —

[0193] Cell count of the blood added

[0194] (1)

[0195] In Examples 1 and 2, this study developed an automated device for repeated blood filtration to isolate bacterial cells. Utilizing only a centrifuge, this hands-off method efficiently performs repeated filtration. The resuspension of sediment is passively achieved by trapping air in the device’s micro-pump, activated through the starting and stopping of the centrifuge. This repeated filtration system resuspends over 90% of the red blood cell (RBC) sediment and increases bacterial recovery in the filtrate from 4% to 24% over 1 to 10 cycles. The filtrate contained minimal blood cells, with approximately 99.7% of RBCs being removed during filtration. This simple yet robust device offered significant potential for sample preparation in diagnostic and microbiological studies. Additionally, its reliance solely on a centrifuge enhances its applicability in low-resource settings.

[0196] Second Set of Embodiments:

[0197] In a second set of embodiments, a system (50) for separating particles in a fluid contains a first (52) and a second chamber (54) connected via a connector (56). As shown in Fig. 21, the connector has two ends, wherein the first / top end (58) of the connector connects to the first chamber (52) and the second / bottom end (60) of the connector connects to the second chamber (54). The first chamber is positioned higher than the second chamber with respect to a sedimentation direction (62) of the system so that when the system is placed in a centrifugal field, fluid in the first chamber can be transferred to the second chamber in a controlled fashion by centrifugal force. Additionally, the first chamber (52) is connected pneumatically to an ambient atmosphere (66), while the second chamber (54) is connected only to the first chamber via the connector; the second chamber is connected pneumatically or fluidically to the first chamber.

[0198] A grid (68) may be located in the first chamber (Fig. 22A). Alternatively, the grid (68’) may be in the second chamber (Fig. 22B) or in both the first and second chambers (Figs. 22C, 22D). The grid (68, 68’) may comprise walls (70, 70’) that are either substantially parallel with, or at an angle with respect to the sedimentation direction (62), for preventing mixing of liquid during centrifugal acceleration or deceleration.

[0199] The connector (56) may comprise a lumen (72) or a siphon, shown in Fig. 24; the connector’s shape may be straight and parallel to the sedimentation direction, as shown in Fig. 23 A, or bent, as shown in Fig. 23B.

[0200] Fig. 24 shows the system where a cross-section of the lumen (72) of the connector, an inner volume (74) of the connector and an inner volume (76) of the second chamber (54) are configured for a pressure (P Pa) within the first chamber (52), such that liquid entering the connector (56) from the first chamber (52) with a critical pressure value P (Pa) compresses the air in the connector (56) and the second chamber (54) to a pressure that is equal to the pressure P (Pa) such that the liquid can enter the lumen (72) but not the second chamber (54).

[0201] Fig. 25 shows the system having a filter (78) removing impurities from the moving liquid during a transfer; the filter may be placed between the first chamber (52) and the second chamber (54). The first chamber (52) may have a volume capacity between 1-100 mL, preferably 10-25 mL.

[0202] As shown in Figs. 26A-26C, in the system at least one of the first chamber (52), the second chamber (54), and the connector (56) comprises at least one compartment (80) that is separated by a neck-like structure (82) from the rest of the respective first chamber (52), second chamber (54), and / or connector (56). In Fig. 26A the compartment (80) and neck-like structure (82) are part of the first chamber; in Fig. 26B, they are part of the first chamber (52) and the connector (56); and in Fig. 26C, they are part of the first chamber (52), the connector (56) and the second chamber (54). As shown in Fig. 27, the at least one compartment (80) may be a particle collection chamber (83).

[0203] The second chamber may have a closure mechanism (84) that is affixed to the second chamber (54) and configured to open to allow fluidic or pneumatic access to the second chamber. Figs. 28A-28C show various configurations of the closure mechanism. The closure mechanism (84) may be an elastic or rubber plug (86). As shown in Fig. 28C, one purpose of the closure mechanism is to be able to extract liquid or particles from the second chamber after centrifuging. Since the second chamber is only connected to the first chamber, it is typically hard to reach; therefore, a plugged hole is added in it to extract the liquid via that plug.

[0204] Fig. 29 shows that the connector (56) connecting to the first chamber (52) is configured so that, during use, the first chamber (52) always has anon-zero volume of liquid (88) remaining below the first / top end (58) of the connector when centrifugally transporting liquid from the first chamber (52) into the connector (56).

[0205] In the system, a part of the liquid may move from the first chamber (52) to the second chamber (54) by increasing the centrifuging at a relative centrifugal force (RCF2). The liquid may transfer back from the second chamber (54) to the first chamber (52) by decreasing the centrifugation relative centrifugal force (RCF3) or stopping the centrifuging. Also, the liquid can be moved back and forth repeatedly by changing the relative centrifugal force.

[0206] Fig. 30 shows that the bottom end (60) of the connector to the second chamber (54) is configured such that a non-zero volume of liquid remains in the second chamber (54) below the bottom end (60) of connector in the direction of sedimentation (62) when centrifugally transporting liquid from the second chamber (54) back to the first chamber (52). The bottom end (60) of the connector to the second chamber (54) is configured such that a non-zero volume of liquid (88) always remains in the second chamber (54) below the bottom end of the connector connection (90), and the volume of the liquid can be metered.

[0207] In the system the second chamber (54) may contain at least one of a liquid and a solid. The liquid may comprise at least one of a lysing solution, a viability indicator, a cushion liquid, a density separation liquid, a colorimetric agent, and a protein extractor liquid. The solid may comprise at least one of an agar, a biochemical analyte, and a chromogenic agent.

[0208] In the system shown in Fig. 31 A, the first chamber (52) or the second chamber (54) may contain one or more liquid traps (92) at the bottom of the respective chamber (52, 54) for collection of liquid or particles in the chamber (52, 54), wherein the liquid traps (92) are isolated from other liquid in their respective chamber with an air pocket (94). As shown in Fig. 3 IB, the isolated liquid traps (92) may contain one or more pockets for trapping air (94), when their respective chamber (52, 54) is filled with liquid.

[0209] The liquid or particles in the liquid traps are isolated from their respective chambers with an air pocket after centrifugation. During centrifugation, air needs to be trapped and compressed somewhere inside the isolated liquid trap such that during deceleration, this air expands and partly flows out of the isolated trap, thereby remaining at the entrance of the trap such that the liquid remaining in the trap is isolated from the liquid above by an air pocket. That creates a small pocket of isolated liquid in each of the traps.

[0210] Fig. 31C shows the grid structures (68, 68’) placed above the liquid traps (29), such that each isolated trap receives all particles sedimenting from a specific grid region with a well-defined volume. The grid structures have walls (70) that are substantially parallel with the sedimentation direction (62) to ensure that at least a section of the chamber is compartmentalized in pre-defined volumes (96) when particles settle in the isolated liquid traps (92). In some embodiments, some compartments may differ in volume, typically with a ratio 2An(1, 2, 4, 8, etc) or 10An(1, 10, 100). In that manner, certain liquid traps will have 10, 100, etc more particles than others, which can be used for automated quantitative colorimetric testing on the different resulting bacterial concentrations in the different traps.

[0211] The system may be configured to isolate blood cells. Additionally, the system may be configured to isolate pathogenic organisms such as bacteria or fungi.

[0212] A method of operating the system described above comprises the steps of: a) adding a liquid to the first chamber to trap air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) such that no part of the liquid enters the second chamber, and some air remains trapped in the connector; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that at least a part of the liquid is transferred from the first chamber to the second chamber through the connector; and d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that at least a part of the liquid is transferred from the second chamber to the first chamber through the connector.

[0213] At the end of step d), one of three scenarios may take place. In most cases, a first part of the liquid remains in the first chamber and a second part of the liquid remains in the second chamber. Under ideal circumstances, a first part of the liquid remains in the first chamber and no part of the liquid remains in the second chamber. Under hard centrifuging conditions, no part of the liquid remains in the first chamber and a part of the liquid remains in the second chamber; some liquid may also remain in the connector.

[0214] As shown in Figs. 33A-33D, in a second method of operating the system, the steps comprise: a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; and d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that part of the liquid is transferred from the second chamber to the first chamber through the connector.

[0215] At the end of step d), one of three scenarios may take place. In most cases, a first part of the particles remains in the first chamber and a second part of the particles remain in the second chamber. Under ideal circumstances, a first part of the particles remains in the first chamber and no part of the particles remain in the second chamber. Under hard centrifuging conditions, no part of the particles remain in the first chamber and a part of the particles remain in the second chamber; some particles may also remain in the connector.

[0216] In the second method of operating the system, the liquid-containing particles may contain at least two types of particles, type 1 particles and type 2 particles, that differ in their sedimentation behavior. After step d), the ratio of number of type 1 particles over type 2 particles in the first chamber is different from the ratio of number of type 1 particles over number of type 2 particles in the second chamber. This is an example of enrichment.

[0217] In any of the previous methods, air in the connector can be retained after stopping the centrifugation such that liquids in the first chamber and liquids in the second chamber are not in contact with each other.

[0218] The air retained in the connector after stopping the centrifugation can be transported to the connector from the second chamber during the deceleration of the system.

[0219] Alternatively, the second method of operating the system may comprise the steps of: a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; d) comprises centrifuging the system such that the particles sediment in the second chamber; and e) centrifuging the system such that the liquid is transferred from the second chamber to the first chamber through the connector such that some volume of liquid and / or particles remains in the second chamber.

[0220] In this method, the particles with different sizes or density may be separated through their differential sedimentation during centrifugation. Furthermore, the liquid may contain particles that comprise at least one of bacteria, fungi, tissue, cells, vesicles, and biological components. In some embodiments the liquid may be blood and the particles that sediment in the first chamber are blood cells. In some embodiments, the liquid may be blood and the particles may include bacteria, wherein the bacteria may sediment in the second chamber after transferring the liquid from the first chamber to the second chamber.

[0221] In yet another embodiment, the volume of liquid remaining in the second chamber may be metered by changing the position of the bottom end of the connector (60) after transfer and adjusted to increase or optimize a concentration of smaller particles such as bacteria.

[0222] In another embodiment, the second method of operating the system comprises the steps of: a. first pre-filling the second chamber with a liquid or solid and then adding a liquid that contains particles in the first chamber, thereby trapping air in the connector and in the second chamber; b. centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c. centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; d. centrifuging the system such that said particles sediment in the second chamber in the prefilled liquid or solid; and e. centrifuging the system such that said liquid is transferred from the second chamber to the first chamber through the connector in a manner that leaves some volume of liquid in the second chamber.

[0223] The liquid in the second chamber may comprise at least one analyte for lysing, a viability indicator, a cushion liquid, a density separation liquid, a protein extractor liquid, and an agar.

[0224] Additionally, this method may also comprise the steps of: f. centrifuging the system such that the liquid is transferred from the first chamber to the second chamber through the connector; and g. repeatedly decelerating and accelerating the centrifuge to repeatedly transport at least part of the liquid between the first and second chamber for mixing part of the liquid. The second method of operating the system when used for improved separation of particles by increasing the sedimentation length of particles, comprises the steps of: a) providing a layer of liquid having a higher density than the liquid containing particles in the first chamber, such that the liquid of higher density is positioned below the liquid containing particles, for trapping air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; d) centrifuging the system such that the particles sediment in the second chamber; and e) centrifuging the system such that the liquid is transferred from the second chamber to the first chamber through the connector such that some volume of liquid and / or particles remains in the second chamber.

[0225] In some embodiments, a lysing solution may be provided in the second chamber, such that blood cells undergo lysis when delivered into the second chamber. The lysing solution may be provided as a mixture with a density gradient medium, so that during centrifugation, low density components do not get displaced upwards when the liquid is transferred from the second chamber to the first chamber, thereby allowing efficient pelleting and lysis of blood cells.

[0226] As shown in Fig. 34C, the method of operating the system for preparing a blood sample for microfluidic analysis in the system, comprises the steps of: before step a), optionally, second chamber is filled with a lysing solution to allow direct sedimentation of both bacteria and blood cell to perform selective cell lysis; step a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; step b) centrifuging the system the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and air remain trapped in the connector; step c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria transferred from the first chamber to the second chamber through the connector before step d) centrifuging to sediment the bacteria and remaining blood cell in the second chamber, where the second chamber can be pre-filled with a lysing solution to facilitate direct sedimentation of both bacteria and blood cell and also the lysis of blood cells; step d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that the supernatant liquid is transferred from the second chamber to the first chamber through the connector in a manner that leaves some volume of liquid in the second chamber; and step e) transferring the remaining volume comprising bacteria directly to a microfluidic device for bacterial detection.

[0227] As shown in Fig. 34B, the method of operating the system for preparing a blood sample for MALDI-TOF analysis in the system, comprises the steps of: a. adding blood or a blood culture containing bacteria to the first chamber, thereby trapping an air pocket within the connector and the second chamber b. centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and trap the air pocket in the connector; c. centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria transferred from the first chamber to the second chamber through the connector; d. centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) to sediment the bacteria and the remaining blood cells in the second chamber, where the second chamber is one of an empty or contains an analyte, wherein the analyte is a protein extraction or cell lysis reagent where the cells can sediment; e. optionally varying the spin relative centrifugal force (RCF) of the device to mix a supernatant with the liquid in the second chamber and allowing liquid to move between the first chamber and the second chamber as spin relative centrifugal force (RCF) changes; f. placing a final aliquot on a MALDI plate for bacterial identification; and g. analyzing an aliquot of the sample from the second chamber by mass spectrometry. As shown in Fig. 33A-33D, the method of operating the system for preparing a blood sample for a generic downstream usage in the system comprises the steps of: before step a), optionally, the second chamber is filled with a lysing solution to allow direct sedimentation of both bacteria and blood cell to perform selective cell lysis; step a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; step b) centrifuging the system the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and air remain trapped in the connector step c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria transferred from the first chamber to the second chamber through the connector before step d) centrifuging to sediment the bacteria and remaining blood cell in the second chamber, where the second chamber can be pre-filled with a lysing solution to facilitate direct sedimentation of both bacteria and blood cells for lysis of blood cells; step d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that the supernatant liquid is transferred from the second chamber to the first chamber through the connector in a manner that leaves some volume of liquid in the second chamber, wherein the second chamber contains a density gradient media (mixture of density gradient medium and sodium chloride) to facilitate the density-based separation, wherein white blood cells (wbcs) and platelets concentrate in an upper layer and bacteria and red blood cells (rbcs) concentrate in a lower layer; and step e) transferring the remaining volume comprising bacteria directly to a device for detection or identification; step f) sampling the bottom lower layer having high concentration of the bacteria.

[0228] A method of operating the system for performing antimicrobial susceptibility testing in the system comprises the steps of: step a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; step b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and air remains trapped in the connector step c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria is transferred from the first chamber to the second chamber through the connector; step d) centrifuging the bacteria and remaining bloods cells at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2), to sediment in isolated liquid traps in the second chamber, the liquid traps containing antibiotics and a viability indicator; and step e) centrifuging the system to transfer the supernatant liquid from the second chamber to the first chamber resulting in isolation of the traps in the second chamber by decompressing the trapped air within the isolated liquid traps; and step f) analyzing bacterial growth within the isolated traps by monitoring color change, fluorescence, or optical density to assess bacterial viability in the presence and absence of antibiotics.

[0229] A method of operating the system for bacterial identification, comprises the steps of: step a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; step b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and air remain trapped in the connector step c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria transferred from the first chamber to the second chamber through the connector; step d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) to sediment the bacteria into isolated liquid traps containing chromogenic agar in the second chamber; step e) centrifuging the system to transfer the supernatant liquid from the second chamber to the first chamber and isolate the traps in the second chamber through the decompression of trapped air within the isolated liquid traps; and step f) incubating the bacteria on the chromogenic agar and analyzing color changes to identify the bacterial species.

[0230] A method of operating the system for isolating and culturing bacteria from blood culture using the system, comprises the steps of: step a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; step b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber and air remain trapped in the connector step c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria transferred from the first chamber to the second chamber through the connector; step d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that the supernatant liquid is transferred from the second chamber to the first chamber through the connector in a manner that leaves some volume of liquid in the second chamber having concentrated bacteria; and step e) transferring the remaining volume comprising bacteria directly to a microfluidic device for bacterial detection; and step 1) sampling an aliquot from the second chamber, diluting the aliquot in growth media and then culturing it.

[0231] A method of operating the system for filtering a biological sample in the system, wherein the connecting connector includes a filter comprises the steps of: a) adding a biological sample containing bacteria to the first chamber, thereby trapping an air pocket within the connector and the second chamber. b) centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector to sediment larger particles, for example, blood cells or big particles, within the first chamber, such that no particles are transported to the second chamber. c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant liquid containing bacteria is transferred from the first chamber to the second chamber through the connector and therefore passes through the filter to filter out the bigger particles; d) centrifuging the system centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) to sediment the bacteria in the second chamber; and e) centrifuging the system such that supernatant liquid containing bacteria is transferred from the second chamber to the first chamber through the connector, which comprises a filter to filter out larger particles in a manner that leaves some volume of liquid having concentrated bacteria in the second chamber.

[0232] In this method of filtering a biological sample, the biological sample may be blood, urine, stool, or mucus. The pore size of the filter may range from 0.2 to 5 um.

[0233] A method of operating the system for isolating lymphocytes or platelets from a blood sample in the system comprises the steps of: a) adding blood or blood mixture sample to the first chamber or over a layer of density media, such as fi coll or lymphoprep or another medium of lesser density, in the first chamber, thereby trapping an air pocket within the connector and the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment red blood cells in the first chamber while retaining lymphocytes and platelets in the supernatant; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), to sediment red blood cells in the first chamber while retaining lymphocytes or platelets in the supernatant; d) centrifuging the system at a relative centrifugal force (RCF3) which is lower than the second relative centrifugal force (RCF2) to sediment the lymphocytes or platelets in the second chamber; and e) centrifuging the system to transfer the supernatant liquid from the second chamber to the first chamber through the connector to leave some volume of liquid having concentrated lymphocytes or platelets in the second chamber. A method of operating the system for filtering for isolating extracellular vesicles (EVs) from a blood sample in the system, wherein the connecting connector includes a filter, comprises the steps of: a) adding blood or a blood mixture directly to the first chamber or over a layer of density media in the first chamber to trap an air pocket within the connector and the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber, such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), liquid wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that the supernatant contains smaller particles, the smaller particles comprising EVs, and the filter filters out the larger particles; d) centrifuging the system to sediment the EVs in the second chamber in the liquid or the solid; and, e) centrifuging the system such that the liquid is transferred from the second chamber to the first chamber through the connector such that some volume of liquid having concentrated EVs remains in the second chamber.

[0234] A method of operating the system for testing blood plasma in the system, comprises the steps of: a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) to sediment most of the blood cells in the first chamber, leaving plasma in the supernatant such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the relative connector; and move it into the isolated liquid traps containing analytes d) centrifuging the system at a centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) to transfer the extra liquid from the second chamber to the first chamber through the connector and to isolate the traps in the second chamber by decompressing the trapped air within the isolated liquid traps; and e) analyzing color changes in the liquid traps to test for various biochemical components in the plasma.

[0235] A method of boiling through centrifugation in the system, comprises the steps of: a) connecting a first chamber and a second chamber with a connector, the connector extending upwards from the point of connection to the first chamber; b) adding a liquid to the first chamber, thereby trapping air within the connector and the second chamber; c) centrifuging the assembly such that a portion of the liquid moves from the first chamber to the second chamber through the connector, wherein the height of the liquid column in the connector extends above the liquid level in the first chamber such that the resulting negative hydrostatic pressure in the connector results in outgassing or boiling of the liquid in the connector.

[0236] Example 3 - Rapid Isolation and Concentration of Bacteria from Blood Samples

[0237] System design

[0238] A centrifuge-based device was developed for the automated sample preparation of whole blood or blood culture samples of septic patients for (1) bacterial subculturing, (2) MALDI-TOF-based bacteria identification, or (3) microtrap-based bacteria detection. The system has the form factor of a standard 50 mL centrifuge tube and comprises a top and a bottom chamber connected by a siphon. The top chamber contains a grid, a cup-like structure, and two blood cell collection pockets. The bottom chamber features a rubber stop in its base. Bacterial separation and concentration is achieved by combining velocity -based sedimentation, automated transport of supernatant and subsequent sedimentation.

[0239] System Preparation

[0240] The systems were 3D printed. The bottom / second chamber was pre-filled with 0.4 mL of either a lysing solution or a cushion liquid, depending on the intended downstream application. The top / first chamber was pre-filled with 5 mL of a density medium, which is a mixture of Lymphoprep and blood culture medium (BCM) in ratio 3:5. Air trapped in the cup and bottom / second chamber prevented the density medium from flowing down. 7.5 ml of blood culture sample, a mixture of 3 mL spiked blood and 4.5 mL BCM, was layered on top of the density medium. 5 mL broth medium was layered on top of the sample.

[0241] Centrifuge Operation

[0242] Centrifugation proceeds by a first soft spin step followed by a second hard spin step. During the soft spin at 100 g for 12 min, the fluid moves into the cup, compressing the trapped air to a level that allows filling the cup but not the bottom chamber. Blood cells exhibit terminal velocities 20-30 times higher than bacterial cells17, resulting in the blood cells sedimenting into the blood collection pockets while the bacteria remain in the supernatant.

[0243] During the hard spin, the device is first accelerated to 200g for 1 min and then 2500g for 28 min. Increasing the spin speed above the critical centrifugal acceleration, g*, initiates fluid transfer to the bottom chamber. For a fluid density p, g* can be designed to be in the range 100g < g* < 200g by choosing an appropriate cup volume, Vc, bottom chamber volume, Vb, and hydrostatic height, h, (Figure 32), such that p h ■ g*= (Vb + Vc) / Vb (1) where p is the fluid density.

[0244] Governing equation for fluid

[0245] The system automates differential centrifugation by utilizing air compression as a valve and a siphon to control fluid flow based on centrifugal spin speed.

[0246] Preventing siphon activation in soft-spin: The top chamber consists of a cup-like structure filled with air, with a volume denoted as Vc (Fig. 32). The remaining volume in the top chamber is Fr, and the bottom chamber has a volume VB.

[0247] During centrifugation at 100g, after the liquid samples were fully loaded, the hydrostatic pressure head increased by 100-fold given by equation 2, driving the liquid towards the bottom chamber. This movement compressed the air within the cup-like structure. The resulting air compression generated a back pressure given by equation (3). The system geometry was designed such that the hydrostatic pressure would not exceed the pressure generated by air compression in the cup given by equation (4).

[0248] P =p g h (2)

[0249] \P = Palm Fr F« (3) pgh < P^Vc / PB (4)

[0250] Liquid transfer in hard spin: Once most of the blood cells had sedimented, the system was subjected to a higher spin speed. At this stage, the hydrostatic pressure overcame the counteracting pressure from air compression, thereby activating the siphon and transferring the supernatant from the top chamber to the bottom chamber. The equilibrium position of the liquid during high-speed centrifugation was governed by equation (5), and the system’s geometry ensured that most of the liquid was transferred to the bottom / second chamber, except for the residual fluid in the cup and the blood cell collection chambers, as depicted in Fig. 33C. pgh ■ 2500g > atmf / Vb+Vc -Vf(5)

[0251] The centrifugation at 200g gently transfers part of the supernatant to the bottom chamber without stirring the blood cell sediment in the collection pockets. Increasing the centrifugation to 2500g ensures the transfer of most of the supernatant and facilitates the faster sedimentation of bacterial cells at the bottom. During the process, the broth medium fills the cup, ensuring that all bacteria-containing liquid is transferred to the bottom chamber. When the centrifuge stops, air decompression in the bottom chamber drives all the liquid above the siphon tube back to the top chamber, leaving the concentrated bacteria in the bottom chamber, separated from the cup by air in the siphon. The device was then vortexed to resuspend the bacteria in the bottom chamber, after which the liquid was sampled from the bottom chamber using a syringe that pierced through the rubber stopper at the bottom

[0252] Liquid transfer during operation

[0253] The liquid transfer to the bottom chamber was visualized with a camera placed inside the centrifuge tube during operation, see Figs 42A-B, and 43. Specifically, a wireless camera was integrated into the centrifuge setup to monitor liquid movement under varying centrifugation profiles, such as soft and hard spin conditions. A custom-designed centrifuge holder was 3D-printed to accommodate the camera (Global Tsolar Lights Electrical, China), a light source (Ledlenser, Germany), and the fluidic system. The design of the holder, shown in Figure 42, features dedicated compartments for securely housing these components during operation. During centrifugation, the wireless camera transmits real-time images of the process to an external computer. Figure 42A shows the setup for filming inside a centrifuge tube, a CAD design of the holder used for filming the system inside the centrifuge, which includes compartments for the camera, the system, and the light source. Fig. 42B is a photograph of the holder with the camera, the system, and the light source in place.

[0254] Liquid motion in the system

[0255] The system was filled with blood culture and centrifuged at 100g (soft spin). It was observed that the liquid level moved up inside the cup-like structure, but the liquid did not transfer to the bottom / second chamber. However, during hard spin centrifugation at 2500g, the liquid was transferred to the bottom / second chamber, leaving only the residual volume in the cup-like structure. When the centrifuge was stopped, the liquid returned to the top / first chamber, leaving only a small volume of liquid in the bottom / second chamber. Fig. 43 shows images of liquid movement within the system during centrifugation. The figure includes snapshots of the system inside the centrifuge at 100g (soft spin), 2500g (hard spin), and 1g (centrifuge stopped), illustrating liquid transfer dynamics between the top and bottom chambers.

[0256] Vacuum creation in the centrifugal system

[0257] The system shown in Fig. 47 consists of two chambers: a cup-like structure and a tube-like connector structure connecting the chambers. During hard spin (centrifugation at 2500g), the liquid is transferred to the bottom chamber via the siphon (a combination of the cup and tube). Throughout the transfer process, the liquid surface in the top chamber remains at atmospheric pressure. When the liquid level in the top chamber drops below the top point of the cup, the pressure inside the cup is described by the equation 6.

[0258] P = atm pgh (6)

[0259] Fig. 47 shows a system design for vacuum generation. When the pressure inside the cup, as described by the equation, reaches zero, the liquid begins to boil. This boiling prevents further transfer of the liquid, resulting in the state shown in the Fig. 47.

[0260] Calculation of Volume transferred to the bottom / second chamber

[0261] To quantify the liquid transfer, grooves were introduced in the outer wall of the bottom chamber of one test system; the purpose of the grooves was to trap a small volume of sample when submerged. This allowed observing the maximum liquid height in the chamber post-operation.

[0262] To evaluate the volume of liquid transferred from the top to the bottom chamber during a hard spin, small liquid traps were designed in the bottom chamber. Fig. 44A shows the system design for quantifying the volume of liquid transferred to the bottom chamber, specifically a schematic of the system with micro-traps in the bottom chamber. Fig. 44B shows that, during a hard-spin, the liquid gets transferred to the bottom chamber and fills the micro-traps. When centrifugation is stopped, the liquid returns to the top chamber, leaving liquid in the traps.

[0263] The volume beneath these traps was determined manually by incrementally adding liquid to the chamber, as highlighted in the Fig. 44A-44B. Starting with 17.5 mL of liquid (mixed with ink) in the top chamber, the system was centrifuged at 2500g. Following centrifugation, liquid was observed in the first, second, and third traps from the bottom, but not in the fourth trap. This indicates that the volume of liquid transferred to the bottom chamber is between 8.6 mL and 10 mL.

[0264] Concatenating bacterial isolation with sub-culturing

[0265] Bacterial subculturing following isolation is a key step for obtaining pure colonies, enabling accurate identification and AST. To evaluate the potential of our new isolation approach, we combined the novel centrifugal isolation method with subsequent subculturing (Figure 39A- C). The bottom chamber was prefilled with 0.4 mL cushion fluid. Centrifuging resulted in approximately 0.7 mL of up-concentrated bacteria in the bottom chamber. The entire device was vortexed to resuspend the bacteria in the bottom chamber. The sample was extracted with a syringe and plated on agar plates. The recovery rates, i.e., the number of colonies on the subculture plates relative to the number of colonies in the spiked blood sample, were 33 ± 3% (n=3), 30 ± 2% (n=3), and 48 ± 13% (n=3) for concentrations of 500, 100, and 10 CFU / mL of E. coli, respectively. The recovery rates for K. pneumoniae and E. faecalis were 31 ± 2% (n=3) and 34 ± 4% (n=3) respectively. The difference in recovery is significant only between E. coli and K. pneumoniae; other differences are not significant. Furthermore, the rejection percentages of blood cells are 99.97 ± 0.00% (n=4) for red blood cells, 97 ± 1% (n=4) for white blood cells, and 94 ± 4% (n=4) for platelets (Figure 39A-C). Concatenating bacterial isolation with MALDI-TOF-based identification

[0266] The potential of the new isolation approach for MALDI-TOF sample preparation (Figure 40 A-B) was evaluated. The bottom chamber was prefilled with 0.4 mL of cushion fluid. A volume of 3 mL of blood was mixed with 4.5 mL of overnight bacterial culture, with each bacterial species (E. coli, K. pneumoniae, and P. piersonii) tested separately. The resulting solution for each test had a bacterial concentration of approximately 5 * 108CFU / mL. The 0.7 mL sample retrieved after centrifugation was mixed with 0.5 mL of deionized (DI) water and incubated at 37°C for 10 min. The mixture was centrifuged at 4000g for 5 min, and the resulting pellet was resuspended in 150 pL of solution. A 1 pL aliquot was transferred onto a MALDI target plate and air-dried at room temperature followed by the application of the matrix solution and subsequently analyzed using the MALDI-TOF MS system after the matrix solution air-dried. The identification score, or log score, was calculated by the MALDI-TOF MS machine based on the match between the sample’s spectrum and reference spectra (Figure 40 B). The E. coli and K. pneumoniae were identified with high confidence, and the P. piersonii with low confidence (n = 3).

[0267] Concatenating bacterial isolation with microtrap-based bacteria detection Microtrap-based detection is a fast and effective method for identifying bacteria directly from blood, but it requires efficient removal of blood cells and bacterial concentration51. To evaluate the potential of our new isolation approach for microtrap-based isolation, we prefilled the bottom chamber with 0.4 mL of lysing solution dissolved in Percoll at a 5:3 volumetric ratio. Blood spiked with three different concentrations (5x l06, 5xl05and 5x l04CFU / ml) of Green Fluorescent Protein (GFP)-labelled E. coli were used as blood samples. Sample liquid transferred to the bottom chamber remained on top of the denser lysing solution. The centrifugation process caused the bacterial cells and any remaining blood cells, which are denser than the lysing solution, to settle, leading to the selective lysis of the blood cells. It is noteworthy that Percoll, a density gradient medium, forms a density gradient during centrifugation, while the lysing solution remains uniformly distributed due to proper mixing at the outset. The 0.7 mL sample retrieved after centrifugation was resuspended, incubated for 10 min and loaded into a previously developed microtrap platform15’51. For all bacterial concentrations, the E. coli cells were successfully trapped (Fig. 41B). The trapped bacteria were detected microscopically for all experiments. Methods

[0268] System fabrication

[0269] The system was designed using SolidWorks CAD software and fabricated in three distinct parts: top, bottom, and cap, as detailed in Fig. 45. Fabrication was performed using a Form 3+ 3D printer (Formlabs, USA) with Clear V4 resin. Post-fabrication, the components were cleaned using a Form Wash (Formlabs, USA) with isopropanol (IP A) to remove residual resin from the surfaces. The internal structures of the system were manually cleaned using a needle and IPA to ensure thorough removal of excess resin. Subsequently, the parts were cured in a Form Cure (Formlabs, USA) using ultraviolet (UV) light for 30 minutes at 60 °C. To address challenges during 3D printing, a hole was incorporated into the blood cell collection pocket II of the top part to prevent cupping. This hole was sealed post-fabrication using Clear V4 resin to prevent leakage during operation.

[0270] System assembly

[0271] The top and bottom 3D-printed components were adhered using Clear V4 resin, which was solidified through curing with UV light in the Form Cure for 10 minutes. A polyisoprene- rubber plug was inserted into the cap and glued using ClearSeal Glass Clear adhesive (Casco, Switzerland), then allowed to dry overnight. Subsequently, the cap was attached to the bottom component using Clear V4 resin. Prior to this step, the bottom chamber was prefilled with a cushion or lysing liquid according to the use case.

[0272] The system consists of three distinct 3D-printed components that are assembled together. The two larger parts namely Top and Bottom part, as shown in Fig. 45 A, were bonded using clear v4 resin and subsequently cured under ultraviolet light in a Form Cure system (Formlabs, USA) for 10 minutes. The bottom cap incorporates a polyisoprene-rubber plug sourced from 3 mL Megro™ SOFT-JECT™ disposable syringes (Henke-Sass, Wolf GmbH). This plug was attached to the 3D-printed cap using ClearSeal Glass Clear adhesive (Casco, Switzerland) and allowed to dry overnight. The cap was then affixed to the system using clear v4 resin and cured under ultraviolet light for an additional 10 minutes. Fig. 45B illustrates the CAD model of the assembly of the system.

[0273] Fig. 45A shows an assembly of the 3D printed system, where the three separate 3D-printed components of the system are assembled to form the final structure. Fig. 45B is CAD model illustrating the complete assembly of the system.

[0274] Fig. 46 shows an image of the system after centrifugal operation, highlighting the sedimentation of blood cells and the final liquid distribution.

[0275] Medium preparation

[0276] The density medium used in this study was a mixture of Lymphoprep (STEMCELL Technologies, Canada), a medium with a density of 1.077 g / mL, and blood culture medium (BCM) (BD BACTEC Plus Aerobic medium, BD, USA) in a 3:5 ratio.

[0277] The broth was prepared by dissolving Luria low salt powder (L3397, Sigma-Aldrich, USA) in deionized water (DIW) at a concentration of 25 g / L, followed by autoclaving to ensure sterility. The cushion liquid employed was a combination of Percoll (Sigma- Aldrich, USA), a density medium with a density range of 1.125-1.135 g / mL, and BCM mixed in a 3:5 ratio. The lysing solution used in the study was prepared by mixing Percoll with a lysing liquid in a 3:5 ratio. The lysing liquid itself consisted of 2% (w / v) sodium cholate hydrate (Sigma- Aldrich, USA) and 1% (w / v) saponin (Sigma-Aldrich, USA), both dissolved in BCM.

[0278] Bacterial strains

[0279] Four different bacterial strains were used in this study. The E. coli strain carried a plasmid expressing mVenusNB fluorescence proteins. The other bacterial strains, E. faecaUis. and Pantoea piersonii. were clinical strains randomly collected from a clinical microbiology laboratory in Sweden. For long-term storage, the bacteria were maintained at -80 °C in standard glycerol solution. Prior to use, the strains were incubated overnight at 37 °C in BD BACTEC Plus Aerobic medium (BD, USA), referred to as Blood Culture Medium (BCM). After incubation, the bacterial cultures were diluted in BCM to approximately 104, 103, and 102CFU / mL, which were subsequently used to spike blood samples at varying concentrations for the experiments. Further, the concentration of spiking solution was determined by plate counting of bacterial solution of 103CFU / ml.

[0280] Spiked blood preparation

[0281] In the experiments, healthy donor blood was obtained from the blood bank (Blodcentralen, Stockholm, Sweden). The blood samples were used within two days of collection and stored at 4 °C prior to use. The blood was diluted with BCM in a 2:3 ratio and spiked with bacteria. During spiking, the volume of the liquid added was always less than 4% of the total volume of the blood culture.

[0282] Bacteria counting

[0283] Bacterial counting was conducted by plating the bacterial solution on agar plates, followed by overnight incubation at 37 °C. The agar plates were prepared by dissolving LB broth with agar (Miller) (Sigma-Aldrich, USA) in deionized water at a concentration of 40 g / L. The mixture was autoclaved, poured into Petri dishes, and allowed to cool. For all subculture experiments, 100 pL of the final aliquot was plated in triplicates. However, for the 10 CFU / mL experiments, the entire final aliquot was plated to ensure accurate quantification.

[0284] Blood cell quantifiation

[0285] Blood cell counts in whole blood and final aliquots were measured using a hematology analyzer (Swelab Alfa Plus, Boule Diagnostics, Sweden).

[0286] Process for MALDI-TOF

[0287] A I pL aliquot of the final sample was applied to the MALDI-TOF plate and air-dried at room temperature. Subsequently, the matrix solution was applied, and the sample was analyzed using the MALDI-TOF MS system after the matrix solution had air-dried. Microfluidic platform fabrication

[0288] The design and the fabrication of the microfluidic chip were reported by15. The silicon mold used for the chip was manufactured by Conscience AB, Sweden, and was silanized for 30 minutes prior to replication with poly dimethylsiloxane (PDMS) (Sylgard 184, DOW, USA). A mixture of PDMS and curing agent in a 10: 1 w / w ratio was poured onto the mold and cured by incubation at 80 “Covemight. Openings for the PDMS ports (2.0, 2.1, 2.2, 5.1, and 5.2; see chip design in15) were created using a 0.5 mm puncher. The fabricated PDMS stamps were cleaned with isopropanol (IP A) before being bonded to a glass coverslip (No. 1.5, Menzel-Glaser, Germany) using plasma treatment. The bonded assembly was subsequently heat-cured for 1 hour at 80 °C.

[0289] Flow control

[0290] The microfluidic system was set up on a microscope and connected to reservoirs using tubing (TYGON, Saint-Gobain, North America). The flow controller used for pressurizing the reservoirs was the FlowEZ (Fluigent, France). For priming, the reservoirs connected to ports 2.1, 2.2, 5.1, and 5.2 were initially pressurized at 500 mbar with water containing 0.085 g / L of Pluronic F108 (Sigma- Aldrich, USA), after which the pressure was reduced to 0 mbar. Subsequently, the reservoir connected to inlet port 2.0 was pressurized at 500 mbar for priming and then increased to 1000 mbar for sample loading.

[0291] Optical setup

[0292] Images of the microtraps were captured at lOOx magnification using a Nikon Eclipse Ti-U inverted microscope. Fluorescence images were also taken at lOOx magnification using a CFI Plan Fluor DLL lOOx (1.30 NA, oil) objective. The fluorescence images were acquired with a filter cube comprising a FF497-Di01 dichroic mirror (Semrock, USA), a FF01- 469 / 35 excitation filter (Semrock, USA), and a FF01-525 / 39 emission filter (Semrock, USA).

[0293] Discussion

[0294] System Design

[0295] A centrifuge-based system for high-throughput sample preparation, enabling the isolation and concentration of bacteria from whole blood in a single automated step using only a standard centrifuge. The system uses compressed air as a valve and siphon to control liquid flow. Previously, air-compression-based valves have been used in lab-on-disk systems58; however, these systems were limited by low throughput, incompatibility with standard laboratory centrifuges, and the need for active valves. Our system addresses these limitations by handling 7.5 mL of blood culture, ensuring compatibility with standard centrifuges, and operating without active valves. However, increasing the volume or size of the device while using passive valves presents challenges such as vacuum formation (Figure 47), blood cell resuspension at higher spin speeds, and large dead volumes, as discussed below. These challenges were addressed through the careful selection of design parameters for the cup, siphon, blood cell collection pockets, and grid, as detailed below.

[0296] Position of the Cup and Siphon: The siphon is connected to the top of the cup, utilizing the entire air volume in the cup (K) for compression as described in equation 1. This prevents the transfer of liquid to the bottom chamber during soft spin. The siphon top is positioned below the liquid surface during high-speed spinning, avoiding liquid boiling.

[0297] Blood Cell Collection Pocket Opening: The connections to the blood collection pockets are narrowed to minimize the swirling of blood cells during centrifugal acceleration or deceleration, hence avoiding unwanted resuspension and blood cell transfer to the bottom chamber.

[0298] Cup Volume: Increasing the cup volume allows increasing the RCF during the soft spin without transferring liquid to the bottom chamber as described in equation 1. A large cup volume, however, forms a dead volume for the liquid transferred from the top chamber to the bottom chamber, risking incomplete bacteria transfer to the bottom chamber. The broth layer added above the spiked blood mixture mitigates such incomplete bacterial transfer. During hard spin, the broth fills the cup volume, thereby pushing the bacteria-rich supernatant into the bottom chamber.

[0299] Grid Design: To prevent the broth from mixing with the supernatant during acceleration from low-speed to high-speed centrifugation, a rectangular grid-like structure was introduced at the top of the upper chamber52. Additionally, the cross-sectional area of the lower part of the top chamber was reduced. These modifications minimize the effects of Coriolis and Euler forces, which can otherwise mix the sample and reduce the efficiency of velocity-based sedimentation and mix the broth which further decreases the recovery of bacteria cells.

[0300] Liquid transfer analysis

[0301] The top chamber was filled with 17.5 mL of liquid. During the hard spin, only 8.6 to 10 mL of liquid was transferred to the bottom chamber, while 8-9 mL remained in the top chamber (Fig. 44). Of the liquid remaining in the top chamber, around 4 mL was retained in the blood cell collection pockets, approximately 4 mL in the cup-like structure, and the rest constituted the dead volume above the blood cell collection pockets. The transfer of only about half of the liquid to the bottom chamber does not significantly affect bacterial recovery. This is because the remaining liquid in the top chamber contains fewer bacteria, as the blood collection pockets primarily collect blood cells, while the cup is filled with broth media.

[0302] Isolation of bacteria for sub-culture

[0303] To isolate bacteria for subculture, the spiked blood mixture was layered over density media, facilitating separation based on velocity differentiation. The falling distance through the density media enhances the separation of bacteria from blood cells. Further, a cushion liquid in the bottom chamber minimizes bacterial loss due to excessive centrifugation. The primary objective of bacterial isolation for subculture is to directly obtain solid bacterial colonies, bypassing the traditional blood culture process. This is particularly advantageous for detection methods such as MALDI-TOF and genotypic techniques, which require solid bacterial cultures or pure colonies for accurate identification. The method described here streamlines the process by enabling blood processing directly in the device, followed by plating to yield solid colonies, effectively eliminating the need for the blood culture step. Notably, this approach remains effective even at very low bacterial concentrations, down to 10 CFU / mL, underscoring its potential for clinical applicability.

[0304] MALDI-TOF-based identification

[0305] Another powerful and clinically relevant application of the device is its use in sample preparation for MALDI-TOF analysis. In most clinical settings, the standard workflow requires subculturing positive blood cultures, a process that takes approximately 6-12 hours and can delay the initiation of appropriate antibiotic therapy by up to a day. The purified bacterial aliquot obtained using our device was well-suited for MALDI-TOF-based identification, accurately determining the causative bacterial species with high score values in under an hour (Table 1). This method involves fewer centrifugation steps and requires less hands-on time compared to other kits, such as the Sepsityper60’61and Accelerate Arc System61’62thereby offering a more efficient and streamlined alternative.

[0306] Microtrap based detection

[0307] We have also demonstrated the downstream application of our system on a microfluidic platform for bacterial detection. Microfluidics is a powerful technology with the potential to revolutionize diagnostics, particularly in its ability to perform rapid phenotypic testing by analyzing the growth of single bacterial cells, unlike traditional clinical practices that rely on macroscopic growth. Additionally, bacteria can be identified using genotypic methods such as FISH59.

[0308] However, microfluidic systems typically require pure bacterial cultures, as any interfering cells can clog the micro-traps — especially those with dimensions of just a few microns — thereby compromising the assay. While there are existing methods and systems for sample preparation from whole blood for microfluidic-based detection, they generally involve multiple steps such as isolation, concentration, and lysing, as demonstrated in our previous work51and other studies18. These methods are labour-intensive and require trained personnel. In contrast, the current system simplifies this process by performing multiple centrifugation steps and even mixing (for lysing blood cells) passively, using only a centrifuge. This system automates these tasks and can be fine-tuned for similar processing applications. Bacterial detection was successfully demonstrated using a microfluidic system starting from a concentration of 5 x 104CFU / ml, which is significantly lower than the bacterial concentration typically found in positive blood cultures. Comparison with the state-of-the-art methods

[0309] In this study, a centrifuge-based system for high-throughput sample preparation was developed, enabling the isolation and concentration of bacteria from whole blood in a single step using only a standard centrifuge. The clinical applicability of the system was demonstrated through various applications, including sample preparation for subculturing, MALDI-TOF-based bacterial identification, and microfluidic-based detection. These applications were successfully performed with both high bacterial concentrations from positive blood cultures and clinically relevant lower bacterial concentrations.

[0310] The key parameters for sample preparation methods from blood or blood culture for downstream detection include up concentration factor, minimum concentration of bacteria, blood processing capability, throughput, and cell removal efficiency. These parameters are critical for the detection of bacterial presence, identification, and AST in the clinical management of sepsis and bloodstream infections (BSIs).

[0311] Up-Concentration Factor: The up-concentration factor is defined as the ratio of the bacterial concentration in the final aliquot to the initial concentration in the blood or blood culture. A higher up-concentration factor is advantageous for downstream detection, as the primary challenge in diagnosing sepsis or BSIs lies in the extremely low bacterial concentrations typically present in clinical samples. The developed system exhibits a significantly high up- concentration factor, making it well-suited for these applications.

[0312] Minimum Concentration of Bacteria: Given the low bacterial concentration in blood or blood culture during sepsis or BSI, it is crucial for any method or system to perform efficiently at such low levels. The developed system has been demonstrated to function effectively at bacterial concentrations as low as 10 CFU / mL, which is clinically relevant. This highlights its potential utility in addressing one of the most challenging aspects of sepsis diagnosis. Throughput and Blood Volume Processing: Due to the low bacterial concentrations in clinical scenarios, processing large volumes of blood with high throughput is essential. The developed system demonstrates a superior throughput compared to many existing methods, as illustrated in the accompanying comparative data table. This capability enhances its applicability for routine clinical use.

[0313] Cell Removal Efficiency: In addition to bacterial concentration, the number of residual blood cells in the final aliquot significantly impacts the accuracy of bacterial detection. Residual blood cells can interfere with downstream detection processes, making blood cell removal efficiency a critical parameter. The developed system achieves a red blood cell (RBC) removal efficiency of 99.99%, which is exceptionally high and comparable to, if not better than, other existing methods. In summary, the system demonstrates excellent performance across all critical parameters for sample preparation from blood or blood culture, underscoring its potential for enhancing the detection of sepsis and bloodstream infections in clinical settings.

[0314] Table 1. Key performance parameters of smart centrifugation and other separation methods.

[0315] Most existing methods have a low up-concentration factor17’51, 52 52 12 2255— defined as the ratio of bacterial concentration in the final aliquot to the initial concentration in the blood or blood culture — as well as low throughput52'54, 19, 22,55, 26, making them less scalable. A higher up-concentration factor and throughput are critical for downstream detection, given that the primary challenge in diagnosing sepsis or bloodstream infections (BSIs) lies in the extremely low bacterial concentrations typically found in clinical samples. Although high- throughput centrifugation methods exist, they are often labor intensive and require multiple centrifugation and lysing steps51, 18. In contrast, the developed system retains the advantages of centrifugation-based methods while simplifying the process by automating all liquid handling, mixing, and lysing steps through adjustments in the centrifuge’s spin speed. Moreover, many existing methods are effective only at high bacterial concentrations54, 19, 55, 26, whereas the current system demonstrates robust performance even at low bacterial concentrations, such as 10 CFU / mL.

[0316] The system is user-friendly and highly adaptable for clinical implementation, as it operates with a standard swinging bucket centrifuge. Additionally, it supports parallelization, with up to 16 systems processed simultaneously in a centrifuge equipped with 16 holders, enabling efficient high-throughput processing. This combination of efficiency, scalability, and simplicity positions the system as a practical and innovative solution for bacterial isolation and concentration in clinical settings.

[0317] Conclusion

[0318] In conclusion, the system functions as a versatile centrifuge tube capable of performing multiple applications in a single centrifugal step. This innovation not only reduces the labour intensity of sample preparation but also facilitates the integration of advanced diagnostic technologies into clinical settings, particularly those requiring complex or lengthy sample preparation processes. The system can be further enhanced to include additional functionalities such as filtration or density -based separation by incorporating a membrane filter or adding density gradient media in the bottom chamber. Additionally, the system can be easily adapted for sample preparation from various body fluids or solids, such as stool, urine, mucus, or other biological samples, further broadening its clinical utility.

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Claims

CLAIMS1. A system (50) for separating particles in a fluid, the system comprising:- a first (52) and a second chamber (54) connected via a connector (56), the connector having two ends wherein the first end (58) of the connector connects to the first chamber (52) and the second end (60) of the connector connects to the second chamber (54), wherein- the first chamber is positioned higher than the second chamber with respect to a sedimentation direction (62) of the system,- the first chamber (52) is additionally connected pneumatically to an ambient atmosphere(66)- the second chamber (54) is connected only to the first chamber via the connector, wherein the second chamber is connected pneumatically or fluidically to the first chamber.

2. The system according to claim 1, wherein the first chamber (52) contains a grid structure (68).

3. The system according to any of the above claims wherein the second chamber contains a grid structure (68’).

4. The system according to claim 2 or 3, wherein the grid structure comprises walls (70) that are substantially parallel with the sedimentation direction (62) for preventing mixing of liquid during centrifugal acceleration or deceleration.

5. The system according to any of the above claims, wherein the connector (56) is straight and parallel to the sedimentation direction.

6. The system according to any of the above claims, wherein the connector (56) is bent.

7. The system according to any of the above claims, wherein a cross-section of a lumen (72) of the connector, an inner volume (74) of the connector and an inner volume (76) of the second chamber (54) are configured for a critical pressure value P (Pa) such thatliquid that enters the connector (56) from the first chamber (52) without entering the second chamber (54) compresses the air in the connector (56) and the second chamber (54) to a pressure that is below the critical pressure value P (Pa).

8. The system according to any of the above claims, wherein the system comprises a filter (78).

9. The system according to any of the above claims, wherein the first chamber, has a volume capacity between 1-100 mL.

10. The system according to any of the above claims, wherein at least one of the first chamber (52), the second chamber (54), and the connector (56) comprises at least one compartment or particle collection chamber (80) that is separated by a neck-like structure (82) from the rest of the respective first chamber (52), second chamber (54), and / or connector (56).

11. The system according to any of the above claims, wherein a closure mechanism (84) is affixed to the second chamber (54), wherein the closure mechanism is configured to open to allow fluidic or pneumatic access to the second chamber.

12. The system according to any of the above claims, the system comprising a closure mechanism (84), wherein the closure mechanism comprises an elastic or rubber plug (86).

13. The system according to any of the above claims wherein the connector (56) connecting to the first chamber (52) is configured so that, during use, the first chamber (52) always has anon-zero volume of liquid (88) remaining below the first end (58) of the connector when centrifugally transporting liquid from the first chamber (52) into the connector (56).

14. The system according to any of the above claims, wherein the bottom end (60) of the connector to the second chamber (54) is configured such that a non-zero volume of liquid remains in the second chamber (54) below the bottom end of connector (60) in thedirection of sedimentation (62) when centrifugally transporting liquid from the second chamber (54) back to the first chamber (52).

15. The system according to any of the above claims, wherein the bottom end of the connector (60) to the second chamber (54) is configured such that a non-zero volume of liquid (88) always remains in the second chamber (54) below the bottom end of connector (60), and the volume of the liquid can be metered.

16. The system according to any of the above claims, wherein the second chamber (54) contains at least one of a liquid and a solid.

17. The system according to claim 16, wherein the liquid comprises at least one of a lysing solution, a viability indicator, a cushion liquid, a density separation liquid, a colorimetric agent, and a protein extractor liquid.

18. The system according to claim 16 or 17, wherein the solid comprises at least one of an agar, a biochemical analyte, and a chromogenic agent.

19. The system according to claim 13 or 15, wherein the first chamber (52) or the second chamber (54) contain one or more liquid traps (92) at the bottom of the respective chamber (52, 54) for collection of liquid or particles in the chamber (52, 54) after centrifugation, wherein the liquid traps (92) are isolated from other liquid in their respective chamber with an air pocket (94).

20. The system according to claim 19, wherein the isolated liquid traps (92) contain one or more pockets for trapping air (94), when their respective chamber (52, 54) is filled with liquid.

21. The system according to claim 19, wherein liquid or particles in the liquid traps are isolated from their respective chambers with an air pocket after centrifugation.

22. The system according to any of the above claims, wherein the grid structure (68, 68’) comprises walls (70) that are substantially parallel with the sedimentation direction (62)for ensuring that at least a section of the chamber is compartmentalized in pre-defined volumes (96) when particles settle in the isolated liquid traps (92), wherein the predefined volumes can comprise different ratios.

23. The system according to any of the above claims configured to isolate blood cells.

24. The system according to any of the above claims configured to isolate pathogenic organisms such as bacteria or fungi.

25. A method of operating the system according to any of the previous claims, comprising: a) adding a liquid to the first chamber to trap air in the connector and in the second chamber; b) centrifuging the system at a first relative centrifugal force (RCF1) such that no part of the liquid enters the second chamber, and some air remains trapped in the connector; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that at least a part of the liquid is transferred from the first chamber to the second chamber through the connector; and d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that at least a part of the liquid is transferred from the second chamber to the first chamber through the connector.

26. The method according to claim 25, wherein, at the end of step d), a first part of the liquid remains in the first chamber and a second part of the liquid remains in the second chamber, or a first part of the liquid remains in the first chamber and no part of the liquid remains in the second chamber, or no part of the liquid remains in the first chamber and a part of the liquid remains in the second chamber.

27. A method of operating the system in any one of the claims 1-24, comprising the steps of: a) adding a liquid containing particles to the first chamber to trap air in the connector and in the second chamber;b) centrifuging the system at a first relative centrifugal force (RCF1) such that part of the air remains trapped in the connector and no particles are transported to the second chamber; c) centrifuging the system at a second relative centrifugal force (RCF2), wherein the second relative centrifugal force (RCF2) is higher than the first relative centrifugal force (RCF1), such that part of the liquid is transferred from the first chamber to the second chamber through the connector; and d) centrifuging the system at a relative centrifugal force (RCF3) lower than the second relative centrifugal force (RCF2) such that part of the liquid is transferred from the second chamber to the first chamber through the connector.

28. The method of operating the system according to claim 27, wherein, after step d), a first part of the particles remain in the first chamber and a second part of the particles remain in the second chamber, or a first part of the particles remain in the first chamber and no part of the particles remain in the second chamber, or- no part of the particles remain in the first chamber and a part of the particles remain in the second chamber.

29. The method of operating the system according to claim 27 or 28, wherein, the liquid containing particles contains at least two types of particles, type 1 particles and type 2 particles, that differ in their sedimentation behavior, and wherein after step d), the ratio of number of type 1 particles over type 2 particles in the first chamber is different from the ratio of number of type 1 particles over number of type 2 particles in the second chamber.

30. The method according to any one of claims 25 to 29, wherein air in the connector is retained after stopping the centrifugation such that liquids in the first chamber and liquids in the second chamber are not in contact with each other.

31. The method according to claim 30, wherein the air retained in the connector after stopping the centrifugation is transported to the connector from the second chamber during the deceleration of the system.

32. The method according to claim 27-31, wherein the particles with different size or density are separated through difference in their sedimentation behaviour during centrifugation.

33. The method according to claim 32, wherein the liquid contains particles that comprise at least one of bacteria, fungi, tissue, cells, vesicles, and biological components.

34. The method according to claim 33, wherein the liquid is blood and the particles that sediment in the first chamber are blood cells.

35. The method according to claim 33, wherein the liquid is blood and the particles include bacteria, wherein the bacteria sediment in the second chamber after transferring the liquid from the first chamber to the second chamber.

36. The method according to claim 33, wherein the volume of liquid remaining in the second chamber after transfer adjusted to increase or optimize concentration of particles such as bacteria, present in the second chamber after centrifugation.

37. The method according to any one of claims 27-36, wherein step a) comprises first providing the second chamber with a liquid or solid and then adding a liquid that contains particles in the first chamber, thereby trapping air in the connector and in the second chamber, and following step c), the method comprises step d) centrifuging the system such that the particles sediment in the second chamber in the liquid or the solid; and, step e) centrifuging the system such that the liquid is transferred from the second chamber to the first chamber through the connector such that some volume of liquid remains in the second chamber.

38. The method according to claim 37, wherein the liquid in the second chamber comprises at least one of an analyte for lysing, a viability indicator, a cushion liquid, a density separation liquid, a protein extractor liquid and an agar.

39. The method according to claim 37 or 38, further comprising, following step e):f) centrifuging the system such that the liquid is transferred from the first chamber to the second chamber through the connector; and g) repeatedly decelerating and accelerating the centrifuge to repeatedly transport at least part of the liquid between the first and second chamber for mixing at least part of the liquid.

40. The method according to any one of claims 27-39, wherein step a) further comprises providing to the first chamber a layer of low-density liquid having a lower density than the liquid containing particles such that the liquid of lower density is positioned above the liquid containing particles.

41. The method according to any one of claims 31-40, wherein a lysing solution is provided in the second chamber, such that blood cells undergo lysis when delivered into the second chamber.

42. The method according to claim 41, wherein the lysing solution is provided as a mixture with a density gradient medium, ensuring that it remains denser than the transferred supernatant liquid. This allows it to stay at the bottom of the second chamber, facilitating efficient pelleting and lysis of blood cells.

43. The method of operating the system according to claim 27 for preparing a blood sample for microfluidic analysis in the system, wherein: before step a) optionally, a second chamber is filled with a lysing solution to allow direct sedimentation of both bacteria and blood cell to perform selective cell lysis in step a) the liquid is blood or a blood culture containing bacteria that is added to a first chamber, to trap an air pocket within the connector and a second chamber; step b) comprises centrifuging the system to sediment most of the blood cells in the first chamber while maintaining the air pocket trapped in the connector; step c) comprises centrifuging the system to transfer a supernatant liquid containing bacteria from the first chamber to the second chamber through the connector; following step c) and before step d), centrifuging to sediment the bacteria and remaining blood cells in the lysing solution in the second chamber;step d) comprises centrifuging the system such that the supernatant liquid is transferred from the second chamber to the first chamber through the connector such that some volume of liquid remains in the second chamber; and following step d), the method further comprises: step e) transferring the remaining volume comprising bacteria directly to a microfluidic device for bacterial detection.

44. The method of operating the system according to claim 27 for preparing a blood sample for MALDI-TOF analysis in the system, wherein: in step a) the liquid is blood or a blood culture containing bacteria that is added to a first chamber, to trap an air pocket within the connector and a second chamber; step b) comprises centrifuging the system to sediment most of the blood cells in the first chamber while maintaining the air pocket trapped in the connector; in step c) the transferred liquid is supernatant liquid containing bacteria; step d) comprises centrifuging to sediment the bacteria and the remaining blood cells in the second chamber, where the second chamber is one of an empty chamber or a chamber containing an analyte, wherein the analyte is a protein extraction or cell lysis reagent where the cells can sediment, and wherein following step d), the method additionally comprises the steps of: step e) optionally varying the spin relative centrifugal force (RCF) of the system to mix a supernatant with the liquid in the second chamber and allowing liquid to move between the first chamber and the second chamber as spin relative centrifugal force (RCF) changes; step f) extracting a sample from the second chamber and placing a final aliquot on a MALDI plate for bacterial identification; and step g) analyzing an aliquot of the sample from the second chamber by mass spectrometry.

45. The method of operating the system according to claim 43, wherein the method is for preparing a blood sample for a generic downstream usage in the system, and wherein: in step d) the second chamber contains a density gradient media (mixture of density gradient medium and sodium chloride) to facilitate the density-based separation,wherein white blood cells (wbcs) and platelets concentrate in an upper layer and bacteria and red blood cells (rbcs) concentrate in a lower layer; in step e) transferring the remaining volume comprising bacteria directly to a device for detection or identification; and following step e), the method comprises: step f) sampling the bottom lower layer having high concentration of the bacteria.

46. The method of operating the system according to claim 43, wherein the method is for performing antimicrobial susceptibility testing in the system, and wherein: in step d) the bacteria and remaining blood cells are centrifuged to sediment in isolated liquid traps in the second chamber, wherein the liquid traps contain antibiotics and a viability indicator; following step d), the method comprises the steps of: e) centrifuging the system to transfer the supernatant liquid from the second chamber to the first chamber resulting in isolation of the traps in the second chamber by decompressing the trapped air within the isolated liquid traps; and f) analyzing bacterial growth within the isolated traps by monitoring color change, fluorescence, or optical density to assess bacterial viability in the presence and absence of antibiotics.

47. The method of operating the system according to claim 43, wherein the method is for bacterial identification in the system, and wherein: in step d) the system is centrifuged to sediment the bacteria into isolated liquid traps containing chromogenic agar in the second chamber; in step e) the system is centrifuged to transfer the supernatant liquid from the second chamber to the first chamber and isolate the traps in the second chamber through the decompression of trapped air within the isolated liquid traps; and following step e) In step f) incubating the bacteria on the chromogenic agar and analyzing color changes to identify the bacterial species.

48. The method of operating the system according to claim 43, wherein the method is for isolating and culturing bacteria from blood culture using the system, and wherein: in step e) the second chamber contains concentrated bacteria; and following step e), the method comprises:f) sampling an aliquot from the second chamber, diluting the aliquot in growth media and then culturing it.

49. The method of operating the system according to claim 32 for filtering a biological sample in the system, wherein- the connecting connector includes a filter, and wherein: in step a) the liquid is a biological sample containing bacteria; step b) comprises centrifuging the system to sediment larger particles (blood cells or big particles) within the first chamber; after step d), the method comprises step e) centrifuging the system such that the liquid is transferred from the second chamber through the connector such that some volume of liquid and / or particles remains in the second chamber; and in steps c) and e) the liquid is supernatant liquid containing bacteria which passes through the filter to filter out the bigger particles.

50. The method of filtering a biological sample according to claim 49, wherein the pore size of the filter ranges from 0.2 to 5 urn.

51. The method of operating the system according to claim 32 for isolating lymphocytes or platelets from a blood sample in the system, wherein: in step a) blood or blood mixture sample is added to the first chamber or over a layer of density media); step b) comprises centrifuging the system to sediment red blood cells in the first chamber while retaining most of lymphocytes or platelets in a supernatant liquid; in step c) the supernatant liquid transferred from the first chamber to the second chamber contains lymphocytes or platelets; in step d) the centrifuging sediments the lymphocytes or platelets in the second chamber; and step e) comprises centrifuging the system to transfer the supernatant liquid from the second chamber to the first chamber through the connector to leave some volume of liquid having concentrated lymphocytes or platelets in the second chamber.

52. The method of operating the system according to claim 37 for filtration for isolating extracellular vesicles (EVs) from a blood sample in the system, wherein the connecting connector includes a filter, wherein: step a) comprises adding blood or a blood mixture directly to the first chamber or over a layer of density media in the first chamber to trap an air pocket within the connector and the second chamber; step b) further comprises centrifuging the system to sediment most of the blood cells in the first chamber; in step c) the supernatant liquid transferred from the first chamber to the second chamber contains smaller particles, the smaller particles comprising EVs, and the filter filters out the bigger particles; in step d) centrifuging the system sediments the EVs in the second chamber; and in step e) the volume of liquid left in the second chamber comprises concentrated EVs.

53. The method of operating the system according to claim 27 for testing blood plasma in the system, wherein:- step a) comprises adding blood to the first chamber to trap an air pocket within the connector and the second chamber;- step b) further comprises centrifuging the system to sediment most of the blood cells in the first chamber, leaving plasma in the supernatant;- step c) comprises centrifuging the system to transfer the supernatant plasma from the first chamber to the second chamber through the connector;- step d) centrifuging the system to direct and move the plasma into isolated liquid traps containing an analyte, and following step d), the method further comprises the steps of: e) centrifuging the system to transfer the extra liquid from the second chamber to the first chamber through the connector and to isolate the traps in the second chamber by decompressing the trapped air within the isolated liquid traps; f) analyzing color changes in the liquid traps to test for various biochemical components in the plasma.

54. A method of boiling through centrifugation in the system according to any one of claims 1- 28, comprising:a) connecting a first chamber and a second chamber with a connector, the connector extending upwards from the point of connection to the first chamber; b) adding a liquid to the first chamber, thereby trapping air within the connector and the second chamber; c) centrifuging the assembly such that a portion of the liquid moves from the first chamber to the second chamber through the connector, wherein the height of the liquid column in the connector extends above the liquid level in the first chamber such that the resulting negative hydrostatic pressure in the connector results in outgassing or boiling of the liquid in the connector.

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