Fluidic device for particle analysis and related methods

Abstract

A fluidic device for measuring at least one of a mass density and a weight of a particle is described. The fluidic device includes a sedimentation chamber fluidically connected to an inlet channel configured to be submerged in a liquid. The fluidic device also includes a pumping system connected to the sedimentation chamber. The pumping system is adapted to control a flow of the liquid in the sedimentation chamber. A processor of the fluidic device is configured to obtain particle data related to the particle in at least one region of the sedimentation chamber; and calculate at least one of a mass density and a weight of the particle based on the received data.

Description

[0001] The invention described herein discloses an apparatus and related methods for measuring the mass density, weight, size, and shape of microparticles (such as microspheres, cells, spheres, and organoids). In particular, the architecture of the apparatus is suitable for measuring microparticles with sizes between one micrometer and five thousand micrometers.

[0002] The collection of detailed information on these parameters can have significant implications for industrial and / or research applications in fields such as pharmacology, cell biology, agronomy, and food and environment. This growing demand can be attributed to the widespread use of single cells and cell clusters in a wide range of biomedical applications.

[0003] In particular, cells often exhibit more significant variations in mass density than in size, and obtaining reliable data on these values ​​remains a challenge. Therefore, determining these values ​​could provide an effective analytical tool for monitoring cellular responses to external stimuli such as drugs and environmental changes. Furthermore, obtaining contemporaneous information on particle volume and mass density allows for correlation with biological activity and yields important information, including the organization and viability of cell populations within cell clusters. Background Technology

[0004] With the advent of nanoelectromechanical systems (NEMS), and especially with the development of nanomechanical resonators, methods have been implemented for determining the mass and density of cells, particles, or molecules.

[0005] These systems resonate at specific frequencies that depend on their mass, structure, and rigidity. The sample mass is measured by the change in the oscillation frequency of the resonator as it interacts with it. Initial limitations to the biological applications of this technique arose from the need to operate under vacuum conditions. Suspended microchannel resonators (SMRs) have overcome this application barrier, allowing for the measurement of the density of single cells in solution and thus obtaining statistics on the density distribution.

[0006] However, when using fluid channels with dimensions in the micrometer range, the aforementioned system allows for the analysis of particles smaller than tens of micrometers. This poses a significant technical limitation for applications or research concerning cell clusters (such as spheroids and organoids) that can reach diameters on the millimeter scale.

[0007] Known methods use sedimentation rates to calculate the density of phytoplankton and polystyrene spheres. However, this technique does not allow for measurements to reach the average size of spheres and organoids. More importantly, data is collected in a single step, and repeated measurements are not possible to obtain more accurate output.

[0008] Other techniques employ optically induced electrokinetics systems (OEK) to enhance microparticles previously introduced into microfluidic channels.

[0009] Such a solution is expensive and not suitable for measuring particles larger than 20 micrometers. Summary of the Invention

[0010] A first aspect of the invention describes a fluid apparatus for measuring at least one of the mass density and weight of particles. The fluid apparatus includes a settling chamber fluidly connected to an inlet channel configured to be immersed in a liquid. The fluid apparatus also includes a pumping system connected to the settling chamber. The pumping system is adapted to control the liquid flow in the settling chamber. A processor of the fluid apparatus is configured to acquire particle data related to particles in at least one region of the settling chamber; and to calculate at least one of the particle mass density and weight based on the received data.

[0011] The liquid can be a culture medium or solution, such as a salt solution, suitable for storing and maintaining one or more microparticles or aggregates of microparticles to be introduced into the precipitation chamber.

[0012] Because of the inlet channel directly connected to the precipitation chamber, individual mass density and weight measurements can be performed on a group of particles. This configuration allows for sequential and automated execution of such measurements. The measurements, combined with particle data such as particle size and shape, are important for studies such as cell dynamics or the development of biological models of human organs.

[0013] In one embodiment of the first aspect, the fluid device further includes at least one detection device configured to acquire particle data and provide the particle data to a processor.

[0014] In another embodiment, the fluid device is configured to control the pumping system based on at least a portion of the received particle data.

[0015] In another embodiment, the particle data includes at least one of particle velocity, shape, position, and size.

[0016] In another embodiment, the fluid device further includes a temperature control device configured such that the processor provides a temperature measurement of the liquid in the settling chamber. The processor is configured to calculate at least one of particle mass density and weight based on the temperature measurement.

[0017] Temperature measurement allows for accurate measurements, even when the apparatus is not in a controlled temperature environment. In fact, the movement of particles in the sedimentation chamber also depends on the viscosity of the liquid in which they move, which in turn depends on the properties of the specific liquid used and its temperature.

[0018] In another embodiment, the temperature control device is further configured to adjust the temperature of the liquid in the settling chamber based on at least one of information provided by the processor and a predetermined temperature value.

[0019] Temperature control also allows for the addition of ideal maintenance conditions for particle holding to measurement accuracy. For example, if the particles are made of biological material, the ideal condition is to maintain them at 37 degrees Celsius, and in some applications, the temperature may need to be varied to observe specific biological phenomena.

[0020] In another embodiment, the fluid device further includes a movable support adapted to accommodate at least a portion of at least one detection device. The processor is also configured to guide the movable support based on at least a portion of the received particle data.

[0021] Because of the movable support, the particles can be followed during their movement, thus achieving higher accuracy in the measurement of mass density and weight, and allowing measurements to be performed on particles moving at high speeds in particularly long precipitation chambers. Furthermore, three-dimensional information about the particles can be obtained, for example, by observing the sample from different angles or at different focal planes. The obtained data on the three-dimensional shape of the object allows for better reconstruction of the viscosity coefficient, and therefore allows for better resolution of mass and weight density measurements.

[0022] In another embodiment, the sedimentation chamber further includes a flow channel that is fluidly connected to the inlet channel and has an internal cross-section that narrows in a portion of the flow channel.

[0023] Specifically, the sedimentation chamber includes a flow channel with at least two sections, wherein the internal cross-section of the first section is larger than that of the second section.

[0024] The second part is positioned such that the particles in the settling chamber are adapted to move from the second section to the first section in the absence of flow in the liquid in which the particles are immersed.

[0025] The variation in the cross-section of the sedimentation chamber allows for the formation of converging streamlines. During the flow generation step within the sedimentation chamber, particles follow these streamlines and thus move towards a more central region of the chamber. This phenomenon avoids measurement artifacts dependent on the interaction between the particles and the side surfaces of the sedimentation chamber, resulting in more accurate measurements. It also ensures particle centering, even for long measurement times, thus enabling the retention of samples within the sedimentation chamber for biological experiments. Furthermore, centering is useful for sample extraction, as it reduces the likelihood of sample loss in the fluid system, for example, by adhering to the channel surface.

[0026] In another embodiment, the sedimentation chamber includes flow channels connected in parallel to each other and connected to an inlet channel. Each flow channel is also connected to a flow regulator. In this embodiment, the processor is configured to receive input data related to particles in the inlet channel and to control the flow regulator based on the input data.

[0027] The presence of several settling chambers allows particles to be directed into the appropriate settling chamber based on their size. Therefore, measurements of particles with significantly different sizes (e.g., from one micrometer to 5 millimeters) can be performed in a single instrument. Alternatively, the presence of multiple settling chambers allows for greater flow rates when measuring particle swarms.

[0028] In another embodiment, the fluid device further includes a recirculation channel connected in parallel with the settling chamber. The recirculation channel includes a recirculation device, and the processor is configured to control the recirculation device to recirculate the liquid in the settling chamber.

[0029] Because of the recirculation system, the presence of more than one particle in the fluid system can be avoided, thus enabling repeated, non-destructive analysis of individual particles and their recovery. Furthermore, less analytical liquid is used for measurements that extend over time.

[0030] In another embodiment, the fluid device further includes a secondary channel fluidly connected to the settling chamber. The processor is configured to selectively control the flow in the secondary channel to introduce liquid into and / or drain liquid from the settling chamber.

[0031] The secondary channel can be connected to the sedimentation chamber directly, either through the inlet channel or through the secondary fluid loop.

[0032] The presence of at least one secondary channel allows for the introduction of new liquid into the precipitation chamber during measurement. This enables, for example, the execution of biological experimental protocols. Furthermore, it allows for the introduction of new liquids suitable for precise measurements of specific particles. For example, by selecting a liquid from a set of measurement liquids with a mass density closest to that of the particles, measurement precision and accuracy can be improved. Additionally, the same secondary channel can be used to remove samples and classify them into specific containers. For example, through specialized fluid derivatization, different particles can be collected in different containers, even based on the results of the performed measurement.

[0033] A second aspect of the invention describes a method for measuring at least one of the mass density and weight of particles. According to the method, particles to be analyzed are introduced into a settling chamber of a fluid apparatus through an inlet channel immersed in a liquid. Particle data associated with particles in at least one region of the settling chamber are obtained. The particles move in the liquid stationary within the settling chamber. At least one of the particle mass density and weight is calculated based on the received data.

[0034] Measurements can be taken while the particles are moving in a still liquid. A still liquid is defined as one in which there is no flow in the settling channel. In one embodiment, the particles move under the acceleration of a force field. For example, the settling chamber can be positioned such that the particles move in a still liquid that is accelerated by gravity.

[0035] In one embodiment of the second aspect, obtaining particle data includes acquiring particle data by means of at least one detection device and providing the acquired particle data to a processor.

[0036] In another embodiment, the method further includes controlling the flow in the sedimentation chamber based on at least a portion of the received particle data.

[0037] In another embodiment, the method further includes measuring the temperature of the liquid in the sedimentation chamber; and even calculating at least one of the particle mass density and weight based on the temperature measurement.

[0038] In another embodiment, the method further includes adjusting the temperature of the liquid in the settling chamber based on at least one of information provided by the processor and a predetermined temperature value.

[0039] In another embodiment, the method further includes guiding at least a portion of at least one detection device based on at least a portion of the received particle data.

[0040] In another embodiment, the method further includes recirculating the liquid in which the particles to be analyzed are not immersed in a recirculation channel connected in parallel with the fluid in the sedimentation chamber.

[0041] In another embodiment, the method further includes replacing the liquid in which the particles to be analyzed are not immersed with a second liquid different from the liquid before and / or after measuring at least one of the particle mass density and weight.

[0042] In another embodiment, the method further includes selecting particles based on one of mass density, weight, size, and shape; and collecting the selected particles in a predetermined container based on at least one of mass density, weight, size, and shape.

[0043] The advantages of the fluid device described above also apply to the method described with respect to the second aspect and its embodiments. Attached Figure Description

[0044] Figure 1 : A front view of the sedimentation chamber containing the analytical medium and particles. A depiction of the forces involved and the final velocities.

[0045] Figure 2 : Illustrative depiction of the present invention.

[0046] Figure 3 : Illustrated depiction of an implementation related to a temperature control device.

[0047] Figure 4 Linear regression data obtained from the vertical variation of particle position as a function of time.

[0048] Figure 5 Experimental results for the average density obtained on seven different PS microspheres for each batch (20, 50 and 90 μm) analyzed, and the corresponding standard deviation obtained for each measurement on seven replicates.

[0049] Figure 6: A) Experimental results of the average density obtained on three batches of PS microspheres, shown in comparison with values ​​claimed by the manufacturer and the corresponding standard deviations. B) Experimentally determined final velocity for the three quantities of the tested microspheres, compared with the theoretical velocity.

[0050] Figure 7 : A schematic description of the centering device (A). An enlargement of part 100c (A, B) is provided for a schematic depiction of particle centering via pulsed flow (B) and modulated flow (C).

[0051] Figure 8 : A schematic description of a sedimentation chamber comprising several flow channels with varying amounts, wherein the flow channels are connected to independent flow regulators to meet particle diversity requirements.

[0052] Figure 9A graphic depiction of a technical solution related to the measurement of the movement of large particles, wherein the measurement is performed by identifying the passage of particles from different target locations at adjustable and known distances.

[0053] Figure 10 : A graphic depiction of a dual-camera system used for bilateral morphological particle characterization.

[0054] Figure 11 : An illustrative depiction of an implementation method related to the acquisition of three-dimensional information about particles, wherein three-dimensional information about particles is acquired based on the acquisition of images reflected from multiple angles.

[0055] Figure 12 : An illustrative depiction of an implementation method related to 3D image acquisition, wherein 3D image acquisition is performed based on a detection device that orbits relative to a cylindrical sedimentation chamber.

[0056] Figure 13 : Illustrated description of an implementation including a recirculation channel and an associated secondary pumping system.

[0057] Figure 14 : An illustrative depiction of an implementation that includes a secondary channel for introducing and exchanging additional analytical media.

[0058] Figure 15 A graphical depiction of the diameter and density of spheroids over time, where the monitoring was used to assess their maturity prior to drug treatment.

[0059] Figure 16 : An illustrative depiction of an implementation method specifically for particle classification. Detailed Implementation

[0060] For individual medical or biological laboratories, it is becoming increasingly common to study particles of different properties (such as cells, microspheres, spheroids, organoids, and / or other forms of particle aggregates).

[0061] Given the wide variety of samples and the focus on measuring weight, density, size, and shape, the embodiments and implementations described below present a technical solution that allows for the measurement of such parameters using a single device and the various methods of use associated with it. Embodiments of the invention describe a first solution that allows for such measurements. In one example, measurements of weight, density, size, and shape can be performed concurrently, simultaneously, or in combination on samples with dimensions ranging from one micrometer to five thousand micrometers. In addition to the advantage of operating with a single instrument for a large size distribution of the sample, this also allows for solid-state correlations between data obtained on individual particles.

[0062] Embodiments of the fluid apparatus and related methods described in this invention allow for the performance of the above analyses in an economical, accurate, and non-invasive manner. Such fluid methods and apparatus can be used not only to perform measurements of organic and biological targets but also to perform measurements of inorganic samples.

[0063] The following paragraphs describe several exemplary embodiments of the invention. These embodiments are described by way of example, and for ease of understanding, with reference to the analysis of biological materials (such as cells or cell aggregates), wherein biological materials are referred to hereinafter by the general term microparticles. However, it is understood that the methods and apparatus associated with the invention shown herein allow for the measurement of the mass density, weight, size, and shape of non-biological microparticles. In particular, embodiments of the exemplary embodiments described below can also be used to analyze microparticles other than cells or cell aggregates. Therefore, in the context of embodiments of the invention, the term "microparticle" used to refer to cells, cell aggregates, or biological materials can also generally be used to refer to microparticles, residual microparticles from industrial processes, suspended atmospheric dust, pollen, vesicles, oil droplets in aqueous suspensions, and bubbles in liquids.

[0064] Similarly, the term "analytical medium" or the more general term "liquid" is used in the illustrative examples of the invention to refer to a medium compatible with cell cultures. However, the terms "analytical medium" and "liquid" must be interpreted in their most general sense, as will be apparent to those skilled in the art. In particular, depending on the design requirements and properties of the microparticles to be analyzed, the liquid may be a cell culture medium or liquid of another nature, such as an aqueous solution or oil, as will be apparent to those skilled in the art.

[0065] The present invention aims to bridge the gaps in existing technologies that may involve the use of a single technical device and related methods to simultaneously measure particle mass density, weight, size and / or shape, as well as different sizes. Indeed, existing devices can operate partially within narrow size distributions. Furthermore, they do not allow for simultaneous or combined measurements of these parameters. This results in fragmented information that is difficult to correlate.

[0066] Even though the purpose has been described with particular reference to the accompanying drawings, reference numerals are used in the specification and claims to enhance the understanding of the invention, and do not constitute any limitation on the scope of protection claimed.

[0067] The present invention discloses a novel device and related methods of use, which are capable of performing accompanying measurements of the mass density, weight, size and / or shape of biological and non-biological particles (such as microspheres, cells, spheres and organoids) having a size between 1 and 5000 micrometers.

[0068] After describing in detail the various embodiments suitable for obtaining such measurements, a variety of modes of use of the invention will be disclosed. The latter includes adaptations to a basic system, which are particularly relevant to its usefulness in the biomedical field. The invention is applicable to the analysis of particles (420) in gravity-guided motion under static flow conditions. Figure 1 The movement of particles in this way is mainly influenced by their mass density, volume, shape, orientation, and the mass density and viscosity of the surrounding liquid.

[0069] exist Figure 2 The figure shows an illustrative depiction of an embodiment of the present invention.

[0070] refer to Figures 3 to 16 The described embodiments describe Figure 1 Subsequent improvements and developments in the fluid apparatus. Therefore, the various features described below with reference to a single figure can be combined with features described with reference to other figures.

[0071] The fluid device includes a settling chamber 100, a pumping system 200, a detection device 300, and a processor 500. The movement of the particles 420 occurs within a fluid channel with transparent walls, referred to herein as the settling chamber 100. The pumping system 200 includes a flow generation system 230, which, by way of example, can be a peristaltic pump or a pressure control system. Inlet channels 210 and outlet channels 220 can be connected to loading or unloading tanks; alternatively, they can serve as vents. The system includes a detection device 300 connected to the processor 500, which performs the dual function of monitoring the movement of the particles 420 and measuring their shape and size. The dimensions of both the settling chamber 100 and the detection device 300 are configured to allow accurate measurement of the movement, shape, and size of the particles 420, as described in detail below.

[0072] Although Figure 2 The device includes a detection device 300, but the detection device is optional. In an alternative embodiment, the fluid device may be configured without a detection device.

[0073] Specifically, in the context of embodiments of the invention, "measuring the shape and size of a particle" refers to the measurement of any feature of the particle that can be used to represent or approximate the three-dimensional geometry of the particle. In the case of spherical particles, this may be consistent with the measurement of the radius, or in the case of nearly spherical and non-spherical particles, this may be consistent with other geometric features.

[0074] For proper operation of the apparatus, the particles 420 contained in the analytical medium 450 are removed from the tank 400, introduced into the settling chamber 100 through the inlet channel 210, and transported within the settling chamber 100. The transport of the particles within the pumping system 200 occurs through the activation of the flow generation system 230. After the introduction of the particles 420, the flow is stopped, and the particles move within the analytical medium 450 under gravity. Without other flow, and after a brief period due to the acceleration step, the particles will reach their constant velocity, defined here as the final velocity, drift velocity, or settling velocity.

[0075] Once measurements have been taken, inlet channel 210 and outlet channel 220 can be used indiscriminately to recover particulate 420.

[0076] This device can be used to perform relative measurements (where differences between different particles are evaluated) or absolute measurements. In the latter case, knowing the temperature is useful in order to define the mass density and viscosity of the analytical medium 450. In embodiments of the invention, the device is suitable for use within a controlled temperature environment, such as in an incubator. Alternatively, the temperature can be measured during the experiment. In one embodiment of the invention ( Figure 3 The average temperature of the analytical medium 450 is measured by a temperature control device 600, which consists of at least one sensor 610 and is positioned near the sedimentation chamber 100. The processor 500 then adapts the calculated viscosity as a function of the measured temperature.

[0077] In another embodiment, when operating conditions require a specific temperature value (e.g., 37°C for live biological samples), the temperature control device 600 is configured to adjust the temperature of the liquid in the sedimentation chamber based on information provided by the processor 500 (such as the current temperature value, particle type, liquid viscosity, current liquid viscosity value, etc.). In one specific embodiment, the temperature control device 600 manages the temperature adjustment unit 620, which allows a specific value to be maintained during analysis. For example, the temperature control device 600 may include several heaters (620a and 620b) and sensors (610a and 610b) of the pumping system 200, located at various locations in the fluid device, and particularly near the sedimentation chamber 100.

[0078] From a physics perspective, and to maximize the accuracy of the obtained data, particle movement can be considered when the particle reaches its final velocity, rather than during the transition period. The relative velocity v(t) of the particle with respect to the analytical medium can be obtained by solving the dynamic equations in the equations of motion:

[0079] F = m p a=(ρ p -ρl V p g-kv

[0080] v(t) = v d +(v0-v d )e -t / τ

[0081] Where τ is the transition period of particle dynamics:

[0082]

[0083] Where m p It is the mass of the particle, ρ p ρ is the particle mass density, a is the particle acceleration, and ρ is the particle acceleration. l It is the mass density of the analytical medium, V p It is the particle volume, v d v0 is the initial particle velocity, r is the particle radius, k is the coefficient of friction, η is the viscosity of the analytical medium, and g is the gravitational acceleration.

[0084] To assess the magnitude of this transition period, calculations for its measurement in aqueous solution are presented below, considering biological microparticles of varying sizes and types. The aqueous solution has a viscosity of approximately 1 mPa·s, and the average mass density of the biological microparticles is 1020 fg / μm. 3 In the case of particles with diameters between 1 μm and 2000 μm, the magnitude of τ varies from 60 ns (for a diameter of 1 μm) to 250 ms (for a diameter of 2000 μm). Within this interval, the particle reaches its final velocity in a very short time. Since this time span is negligible, the particle's acceleration has no effect on the measurement, and the particle dynamics can therefore be considered as uniform linear motion.

[0085] Other technical embodiments of the present invention allow for the measurement of larger particles, particularly those with diameters between 2 and 5 millimeters. For such embodiments, the transition time must be considered, as it can reach 1.5 seconds in the case of a 5-millimeter diameter sample.

[0086] For example, in one embodiment of the invention, the particles are brought to a predetermined distance above the working area of ​​the detection device 300 in order to exclude the transition period from the measurement.

[0087] In another embodiment of the invention, the working area of ​​the detection device 300 is large enough, and the sedimentation chamber 100 is long enough, to allow the detection of the full dynamic motion of the particles, including acceleration and final velocity. Data collected during the transition period of particle motion may not be considered for the calculation of the final velocity.

[0088] In one embodiment of the invention, the theory used for measurement is based on a detailed explanation of Stokes' law, wherein:

[0089]

[0090] and

[0091]

[0092] W p =ρ p V p (Equation 3)

[0093] Among them, W p It is the weight of the particles.

[0094] To demonstrate the reliability of this invention, its specific implementation method was used. Figure 2 This was used to measure the mass density and size of polystyrene microspheres between 20 and 90 micrometers.

[0095] In this embodiment, the detection system 300 consists of an optical microscope system with 4X magnification. The precipitation chamber 100 is obtained within a transparent polydimethylsiloxane (PDMS) chip covalently adhered to a glass slide. The precipitation chamber 100 has a length of 6 cm and a cross-section of 1 x 1 mm. Particles are positioned in the upper part of the precipitation chamber 100 by a flow generation system 230, which in this embodiment consists of a peristaltic pump. Before the sample reaches the precipitation chamber 100, it is transported through a pumping system 200, in this case, a polytetrafluoroethylene (PTFE) tube. The processor 500 consists of a computer and associated software capable of processing images collected by the microscope unit. From these images, a two-dimensional projection of the particle shape and its final velocity are inferred. The latter is calculated via linear regression, which is obtained from the change in the position of the particle's centroid as a function of time. Figure 4 Then, processor 500 calculates the mass density and diameter of the particles using the previously shown mathematical model. This process is repeated several times for each particle to obtain statistically significant results.

[0096] This specific embodiment was used to measure microparticles with known density and diameter to verify the invention. Specifically, microspheres from three batches of polystyrene (PS) were selected. Polysciences (USA) produced microspheres with diameters of 20, 50, and 90 micrometers. Product flakes provided by the seller indicated an average mass density of 1.050 ± 10 fg / µm³ for PS spheres. For each batch of microspheres, seven different units were analyzed, and each unit was measured seven times to increase statistical significance. Figure 5 The average density values ​​of seven different microspheres analyzed are shown, along with the corresponding standard deviations extrapolated from seven replicates for each batch. Consistent with experimental expectations, the standard deviations of the measurements performed on all microspheres are comparable, demonstrating the accuracy and reliability of this implementation method.

[0097] Furthermore, the density values ​​obtained for PS microspheres were found to be significantly more accurate than the average values ​​claimed by the manufacturer. Figure 3 a). In particular, it can be noted that the mean standard deviation has a higher accuracy than the commercial value ( Figure 6a The magnitude is even lower than that of the theoretical final velocity (Equation 1). By comparing the theoretical final velocity (Equation 1) and the experimental final velocity (Equation 2), we can see that the final velocity is on a lower order of magnitude. Figure 6b This allows for further confirmation of the accuracy of the results.

[0098] Specifically, Figure 2 The same configuration shown was used for validation analysis of PS microspheres and can be applied to the analysis of large biological samples, such as spheres or organoids.

[0099] The relevance of these applications involves the possibility of using the methods described herein, for example, to monitor the growth stages of spheroids. Indeed, being able to pinpoint the moment when a spheroid colony reaches the operator's desired or expected level of maturity is a crucial aspect of continuing biological experiments. In fact, the spheroid formation process undergoes compaction over time, beginning with the aggregation of decomposed cells and reaching a compact aggregate stage. This is particularly important in the biomedical field, where the biological materials typically used for analysis are not standardized, leading to less reliable results. Simultaneous measurement of the packing density of a sample, along with its weight, shape, and size, allows for the determination of optimal conditions for the growth of standard spheroids. Furthermore, spheroids can be generated from different types of cells or combinations thereof, so the time required to reach maturity and the mass density characteristics of the maturation period differ for each type of spheroid. Determining the mass density values ​​during the spheroid formation process is important for a key application area involving industrial drug screening processes. In this context, the efficacy of pharmacological therapies can be evaluated by studying drug absorption within the spheroids themselves. Indeed, drug penetration is known to be highly correlated with the compactness level of the aggregates, which in turn can be correlated with their mass density. Therefore, in terms of mass density, the accurate assessment of the structure of the spheres can significantly improve the reproducibility of these results. This embodiment of the invention aims to improve this aspect of the research, which is currently one of the main limitations representing the application of 3D biological models for drug discovery and development.

[0100] However, spheroids are not the only particles used in the biomedical field. The diverse properties of particles are also reflected in the wide range of sizes they encompass, from single cells to organoids. Given researchers' focus on the entire particle range, the possibility of simultaneously measuring their mass density, weight, size, and shape using a single instrument will have considerable implications for the scientific community.

[0101] In this regard, the methods and / or technical embodiments described in the following paragraphs are employed to expand the size range of particles that can be analyzed.

[0102] For example, in an adaptation of the invention, high-magnification optics are required for analyzing micron-sized particles, and sufficient resolution is needed to detect their size and shape. Another adaptation involves the fact that, typically, a one-millimeter particle tends to fall at a higher rate than a one-micrometer particle. Therefore, the sedimentation chamber 100 must be long enough to allow for the measurement of its final velocity. Thus, the proper selection of the specific detection device 300 and the sedimentation chamber 100 can relate to expanding the operational scope of the invention.

[0103] In the example of the above embodiment, the detection device 300 comprises an image acquisition system that acquires a series of frames of the particles 420 as they move within the sedimentation chamber 100. These images are processed to obtain the size, shape, and position of the particle centroid relative to time. Figure 4 As shown, a linear regression algorithm is used to calculate the final velocity. This specific implementation requires collecting a sufficient number of images of moving particles to obtain the correct number of points, which are then used by the regression algorithm. For example, under the condition of spherical particles, the acceptable number of points can be 5, while under the condition of non-spherical particles, the acceptable number of points can be more, due to the additional uncertainty in identifying the centroid of the particles. Specifically, the number of points that the device can obtain during the measurement depends on various experimental and technical factors. These include the final velocity of the particles, the length of the portion of the sedimentation chamber observable by the identification system, the frame rate, and the optical resolution. These factors can be combined in a formula that includes the number of analytical points detected under specific technical and experimental conditions, as shown herein:

[0104]

[0105] In a particular embodiment of the invention, the acceptable number of analysis points is fixed at a specific value, such as 10. Conversely, in another embodiment of the invention, such an acceptable number of analysis points is variable and adjusted by an optimization algorithm. For example, the algorithm may adjust the ideal number of analysis points based on the fit quality of particle position data as a function of time.

[0106] Furthermore, to improve the accuracy of these measurements, the apparatus allows particles to be centered in the settling chamber before analysis is performed. For example, in one embodiment of the invention, the apparatus uses a particle centering system by varying and / or narrowing the geometry of the settling chamber 100. This operating principle is based on the use of a specific geometry of the settling chamber that allows laminar flow with a non-zero horizontal component to be obtained therein. Under such conditions, with proper control of the pumping system 200, particles can be transported to the region of the settling channel where the flow has a zero horizontal component. Hereinafter, and more generally, in the context of the invention, vertical refers to a direction parallel to the gravity vector, while horizontal refers to any vector perpendicular to the vertical direction.

[0107] For example, if the settling chamber is vertically arranged, and if a particle is located inside the settling chamber where the streamline has a non-zero horizontal component, then the particle is also affected by the lateral thrust caused by the horizontal component of the flow. In a specific embodiment of the invention, the settling chamber 100 is vertically oriented and consists of two parts with different horizontal cross-sections, such that the channel (100a) with the wider cross-section is located below the channel (100b) with the narrower cross-section, as... Figure 7 As shown in A. Furthermore, in this particular example, the two sections (100a and 100b) of the sedimentation chamber (100) are coaxially connected by a channel section having an inclined cross-section (100c). The time required for particle centering can vary depending on the value of the inclination angle of the wall of section 100c, the percentage narrowing of the cross-section of the smaller portion of the sedimentation channel (100b) relative to the larger portion (100a), or the relationship between the areas of the two cross-sections. Figure 7 A). In any case, the specific values ​​of these parameters are not necessary for the operation of the centering system. In another embodiment of the invention, the channel portion having an inclined cross-section (100c) is replaced by a discontinuous change in cross-section or a wall at 90° relative to the vertical axis.

[0108] exist Figure 7 A specific example of the centering system is shown in Figure A to illustrate its operation. In this specific example, the widest portion of the settling chamber 100 has a square cross-section (100a) with a side length of 2 mm, while the narrowest portion of the settling chamber 100 has a square cross-section (100b) with a side length of 1 mm. These two portions are connected by a narrowing section (100c), which in this case is a gradual transition at an angle of 45° relative to the longitudinal axis of the settling chamber. The measurements given in this example relating to the square cross-section of the settling chamber are indicative. Therefore, the relationship between the cross-sections of the two portions of the settling chamber and the inclination of the narrowing section are not limited to those derived from such measurements. The dimensions given in this example are indicative, and these specific values ​​are optional.

[0109] In a particular application of the centering device, the fluid system is regulated in a pulse manner. In one example, at time t1 (420t1), particle 420 is at... Figure 7 The position described in B. Once the pumping system 200 has been activated, the particles are transported along the streamlines imposed by the geometry until they reach the position at time t2 (420t2). Figure 7Positioning is shown in B. The flow is then interrupted, allowing the particles to begin falling under the balance of gravity and hydrostatic pressure. After this, at time t3 (420t3), the particles will be in a more central position relative to the settling chamber 100. Further activation of the pumping system 200 allows this process to be repeated until the particles 420 are centered.

[0110] In another application of the centering system, the particles 420 are held within the narrowed portion 100c by a specially modulated flow inside the settling chamber 100 to counteract the effects of gravity, such as... Figure 7 As shown in C. Therefore, the lateral component caused by the streamlines generated by the change in channel geometry serves as a lateral thrust, thereby propelling the particle from time t. a The lateral position at (420ta) moves to the point at time t. b A more central position at (420tb).

[0111] For both solutions shown, the pumping system 200 can be operated manually or automatically.

[0112] In one embodiment of the invention, the detection device 300 comprises an optical device including a microscope system mounted on a movable support. The resolution of the optical device is suitable for acquiring images of all particles of interest, achieving a level of detail sufficient to handle their size and shape. The movable support is guided by a processor 500 to follow the movement of the particles within the sedimentation chamber 100, allowing the observed fall length to be long enough to measure sufficient points for reliable handling of the final velocity of the particles 420. Similarly, in another embodiment, the movable support is adapted to move the sedimentation chamber 100 such that the particles 420 remain within the operating range of the detection device 300.

[0113] In another embodiment, the detection device 300 includes an optical system capable of supporting various magnifications. This is achieved, for example, via a mechanically operated optical axis. The processor 500 selects the correct magnification for the specific particle being inspected and is also able to select an appropriate balance between resolution (used to measure the particle's geometry) and the size of the field of view. The latter must be large enough to allow for the final speed at which the particles are processed.

[0114] In another embodiment, the correct magnification for measuring a specific particle can be selected without the aid of moving parts. In this embodiment, the precipitation chamber includes multiple flow channels, and the detection device monitors these different flow channels, each with a different magnification. Before measurement, the detection device identifies the particle, and the processor manages its introduction into the appropriate flow channel. Furthermore, the architecture of the flow channels can be implemented in different configurations to meet the needs of particle diversity. In fact, the size, cross-section, and geometry can vary, with particular attention to the channel length. Thus, larger particles are inserted into longer channels and monitored at lower resolution for a larger field of view. Conversely, smaller particles are inserted into shorter channels and monitored at higher resolution for a smaller field of view.

[0115] In a variant of this specific embodiment, the mechanism for introducing particles into the sedimentation chamber 100 is a fluid multiplexer. In another variant of this embodiment ( Figure 8 The sedimentation chamber includes several flow channels (e.g., 110a, 110b, and 110c) connected to independent flow regulators (e.g., on / off valves 271a, 271b, and 271c). All flow channels are connected in parallel within the fluid device. In this configuration, the detection device 300 identifies the particle in a specific optically accessible channel 150 before it reaches the branch and selects a flow channel into which the particle is introduced via control of these valves. In another variation of this embodiment, the operator manually selects the flow channel for measurement by operating a selection valve positioned on the inlet channel 210.

[0116] A different strategy that allows for the measurement of the movement of particularly large particles lies in its different positioning method, wherein the detection device 300 is able to identify the passage of particle 420 through different target locations in the sedimentation chamber 100. The distances between these target locations and the total distance covered by this positioning system can be adjusted to accurately determine the final velocity of the particle. The final velocity of the particle is then determined by dividing the distance traveled by the elapsed time.

[0117] In one technical embodiment of this apparatus and related method, the detection device 300 includes several image recognition systems specifically for the target location of the sedimentation chamber 100. In addition to aiding in the measurement of the final velocity, the latter also collects information about the particle shape and size. In another technical embodiment, the detection device 300 includes a series of sensors (e.g., ...) located at the target locations (e.g., 115a, 115b, and 115c) in the sedimentation chamber 100. Figure 9 315a, 315b and 315c in the above), and a separate image recognition system 320 for collecting shape and size information of particles 420.

[0118] Another strategy that allows for the measurement of the movement of particularly large particles lies in the application of mass density and viscosity to the analytical medium. For example, in one embodiment of the invention, the system is adapted to carry an analytical medium 450 having a specific mass density for the type of particles 420 being analyzed, and specifically corresponding to the average mass density of the particle swarm (e.g., 1035 fg / µm³ in the case of SW620 spherical particles). Therefore, the total force acting on the particles, i.e., the sum of gravity and hydrostatic pressure, is directed downwards or upwards depending on the particle's mass density. Thus, during a measurement step without flow, particles with higher density in the analytical medium tend to move downwards, while those with lower density tend to move upwards.

[0119] In this specific embodiment, the processor 500 and associated processing algorithms are adapted to calculate the final velocity of the falling or rising particles. Furthermore, the flow generation system 230 is capable of promoting flow within the sedimentation chamber 100 to both utilize and counteract the gravity vector. This allows the sample to be moved in the relative direction with respect to its final velocity, thus allowing for repeatable measurements. Similarly, in another embodiment of the invention, the system is adapted to contain a high-viscosity analytical medium 450, which reduces the final velocity of the particles.

[0120] In another embodiment of the invention, the device is adapted to change the analytical medium 450, wherein one or more analytical media 450 have different mass densities. The architecture of the device in this embodiment is similar to... Figure 2 The architecture described herein is the same, wherein the pumping system 200 is adapted to select a new analytical medium to be introduced into the system. In this case, the detection device 300 measures the final particle velocity and activates the pumping system 300 to modify the analytical medium so that the final particle velocity decreases. This process can be repeated until the final particle velocity is low enough to be easily detected and measured.

[0121] In the foregoing description of embodiments and some implementations of the invention, methods and related tools for performing combined measurements of the mass density, weight, size, and / or shape of a large group of spherical microparticles have been described. However, many biological samples used in the biomedical field (including the samples described above) may deviate from sphericity. In particular, for spheroids and organoids, sphericity depends primarily on the local variability of composition and / or cellular activity during the formation and maturation of the aggregate itself. Individual cells can have slight variations compared to a spherical shape due to, for example, the internal structure of the cytoskeleton, non-uniform external pressure of the extracellular matrix, or the presence of rigid cellular structures. Therefore, in the following paragraphs, variations of the invention are disclosed that take into account cases where the three-dimensional shape of the microparticles deviates from sphericity.

[0122] For spherical particles, many detection methods and algorithms can be used to calculate the friction coefficient related to their radius. Unlike spherical particles, the case of non-spherical particles is more complex and requires generalization of the previously discussed theories.

[0123] Specifically, Equations 1 and 2 are generalized as follows:

[0124]

[0125] and

[0126]

[0127] In this form, the coefficient of friction k and the volume V p Both depend on the particle shape and orientation. Numerous papers exist on theories that describe the geometry of an object to approximate the coefficient of friction, k. Such geometry could be ellipticity, or the ratio between the surface area of ​​a sphere (which has the same volume as the particle being examined) and the actual surface area of ​​the particle itself. Furthermore, if the complete 3D shape of the particle is known, the coefficient of friction, k, can theoretically be calculated through computational fluid dynamics simulations.

[0128] Therefore, the term "radius" as used in the context of embodiments of the invention (including the foregoing paragraphs) is intended to refer to the geometric radius in the case of spherical particles. However, the term radius should be understood as "effective radius" and can also be used to represent a one-dimensional geometric feature representing a nearly spherical or non-spherical particle. For example, the term radius can be understood as the average distance from a surface point to the centroid, or even twice the ratio of the two-dimensional area to the perimeter, or the Feret radius (perimeter divided by twice π).

[0129] Therefore, as previously written, the terms “shape” and “size” as used in the context of embodiments of the invention are intended to be any geometric description of the particles that can be used to represent or approximate their two-dimensional or three-dimensional geometry. For example, in the case of spherical particles, they can be understood as a quantity of radius, or in the case of nearly spherical and non-spherical particles, they can be understood as a quantity of other geometric features.

[0130] Furthermore, accordingly, the term "mass density" or simply "density" as used in the context of embodiments of the invention should be understood as the ratio of mass to volume in the case of spherical particles. However, these terms can also be used to refer to "effective mass density," which, in addition to the previously described definition of density, includes variations depending on shape factors such as ellipticity and surface roughness in the case of nearly spherical and non-spherical particles.

[0131] Similarly, the term "weight" as used in the context of embodiments of the invention should be understood as gravitational mass in the case of spherical particles. In the case of non-spherical or nearly spherical particles, it is intended to be understood as "effective weight." Similar to the description given for the term "mass density," the term "effective weight" includes variations depending on shape factors such as ellipticity and surface roughness.

[0132] As previously described in various embodiments of the invention, performing different measurements on the same microparticles allows for improved reliability of the results. In the case of spherical microparticles, the obtained statistical distribution is exclusively related to the measurement uncertainty. Conversely, for nearly spherical and non-spherical microparticles, the experimental distribution of the final velocity measurements can be used to describe the size and shape of the microparticles. In a variant of this embodiment, the standard deviation of the final velocity can be used as a parameter for determining the ellipticity of the microparticles. This information is relevant, for example, for the generation and selection of uniform spheres, to be used as a model for drug development. In various variants of this embodiment, the asymmetry of the data distribution obtained from these measurements can be related to the geometric symmetry factor, the kurtosis index can be related to the heterogeneity of the shape, and the multimodal distribution can be related to the distribution of the two-dimensional microparticle projection.

[0133] In another embodiment of the invention, the particle image is processed to obtain its three-dimensional reconstruction, for example, using a tomographic imaging algorithm. This reconstruction can then be processed by a computational tool for calculating the coefficient of friction. This method improves the measurement of the absolute value of the mass density of nearly spherical and non-spherical particles.

[0134] In particular, different technical implementation methods can be used to reconstruct the 3D shape of the particles or improve their morphological characteristics.

[0135] In one specific embodiment of the present invention, the detection device 300 includes a dual-camera system (…). Figure 10 The perpendicular orientation of these cameras relative to the sedimentation chamber 100 ensures that images of the particles are acquired from two different angles. These images are then processed by the processor 500 to obtain a 3D model.

[0136] Conversely, if a single image acquisition system is used, different optical, fluid, or mechanical strategies can be employed. For example, in another embodiment of the invention, the fluid device includes two optical mirrors 113 and 114 located near the rear wall of the sedimentation chamber 100 and having a predetermined tilt relative to the device 300. Figure 11 The front-facing camera maintains a focal plane to include reflected images of particles 430 and 440 obtained from mirrors 113 and 114, respectively. This implementation allows images to be collected simultaneously from different spatial perspectives of the particles and used for their 3D reconstruction.

[0137] In another embodiment of the invention, the device is adapted to acquire images of particle 420 stacked together. In this embodiment, the detection device 300 comprises at least one camera, wherein the focal plane is adjusted to allow scanning of the particles by acquiring multiple images. These collected images can be processed by a processor 500 through the application of image filters (such as deconvolution) to perform 3D reconstruction of the particles. Other embodiments, such as other image stacking techniques, holographic microscopy, or light sheet microscopy, can also be implemented.

[0138] In another embodiment of the present invention, the detection device 300 consists of an image acquisition system that orbits around the sedimentation chamber 100, such as... Figure 12 As shown in the illustration. In a specific embodiment, the precipitation chamber 100 is cylindrical. This allows images to be acquired from different particle angles, which are then processed by tomographic reconstruction for 3D reconstruction of their morphology. In this specific embodiment, reference markers are used near the precipitation chamber 100 to reduce uncertainty in the position of the measured particle 420, which is introduced by the rotation of the detection device 300. The same strategy can be used to minimize the uncertainty introduced in any other embodiment that includes a moving part of the detection system 300. In a variant of this embodiment, the rotating detection device 300 is adapted to move vertically and follow the movement of the sample in relation to the final velocity of the sample. The above embodiments and methods employ known techniques that have been rearranged to obtain 3D reconstruction of the particles. Of course, other existing solutions not shown herein can also be combined with embodiments of the present invention.

[0139] The methods, techniques, and main modes of use of the invention have been described in the preceding paragraphs. However, different modes of use of the invention will be shown herein to demonstrate its further advantages.

[0140] For example, some alternative examples would involve: combined measurements of microparticle swarms introduced for high-throughput analysis (which results in an increase in the amount of data); time-varying and extended analysis of microparticles related to applications such as drug testing; and microparticle classification related to medical applications or the standardization of biological models.

[0141] High-throughput analysis derived from studies of large numbers of individual particles or populations is of paramount importance in biology. For example, information about the heterogeneity of the examined population allows for an understanding of the specific behavior of subpopulations within a biological sample. Indeed, this behavior often varies significantly from the mean of the entire population and becomes a crucial factor in discriminative analysis. Currently, instruments for measuring the mass density, weight, size, and shape of each particle within a population are not commercially available.

[0142] This invention addresses this shortcoming in the prior art by providing a solution that also allows for the extrapolation of data needed for statistical analysis of particle swarms through the overall implementation of the fluid device. The particle data received by the processor 500 from the detection system 300 relates to a large number of particles and includes at least one of the velocity, shape, position, and size of more than one particle. Specific embodiments utilize… Figure 2 The same scheme described herein, wherein the detection system 300 comprises a microscope system connected to the processor 500. This embodiment allows for the simultaneous measurement of the mass density, weight, size, and shape of each particle in the swarm. Similar to the main method of use described above, particles 420 contained in the analytical medium 450 are collected by the tank 400 and introduced through the inlet channel 210 of the pumping system 200. The flow generation system 230 is activated to guide the particles into the sedimentation chamber 100, then the flow is stopped, and analysis is performed while the particles fall due to gravity. In this specific mode of use, the detection device 300 is adapted to simultaneously monitor each particle 420 present inside the sedimentation chamber 100 during measurement. The final velocity and size of each individual component of the swarm are calculated by indexing each particle 420 monitored inside the sedimentation chamber 100. Thus, after several repeated measurements, the distribution of the mass density, weight, size, and shape of the swarm can be extrapolated while still maintaining information about each particle.

[0143] In another embodiment, a greater amount of data is obtained through the specific geometry of the sedimentation chamber 100. While the sedimentation chamber 200, or the flow channel 110 included therein, is straight and without curves in the apparatus shown in the figures, the sedimentation chamber 200 can alternatively have a curved geometry. In this embodiment, the sedimentation chamber may, for example, comprise a single channel with a serpentine architecture, allowing the detection device 300 to monitor several vertical sections in which analysis occurs. The high throughput applied to embodiments of the invention allows for the extrapolation of important data, such as the statistical distribution of analyzed particle populations. For example, a normal (or Gaussian) distribution provides information about the homogeneity of the population. Analysis of the standard deviation of this distribution can therefore be correlated with defining the quality of the sample. This is important, for example, in the case of cell databases, where the cell database can demonstrate the quality of available samples. Conversely, a bimodal or multimodal distribution can identify the presence of different subpopulations or categories. For example, it can provide information about different cell lines present in heterogeneous populations, or, in the case of homogeneous populations, it can distinguish cells at different stages of their life cycle. In addition, the case of homogeneous groups subjected to pharmacological treatment was also considered, where differences in statistical distributions from normal to bimodal or multimodal could distinguish, for example, drug penetration and / or its efficacy.

[0144] Another aspect of the invention aims to avoid wasting analytical media through a closed-loop fluid system by means of a device capable of keeping particles suspended inside the precipitation chamber. This is particularly important for applications using expensive analytical media, such as during the lengthy pre-market characterization phase of a drug.

[0145] Another embodiment of the invention relates to the simultaneous culture and analysis of single cells or cell clusters over time. Such aspects are of great interest in the biomedical and clinical fields. For example, monitoring changes in the physical properties of a sample during drug treatment is useful in pharmacology applications such as personalized medicine, oncology, and adjunctive medical therapies.

[0146] This implementation comprises an apparatus and a corresponding method for measuring the mass density, weight, size, and / or shape of particles over a desired time period (up to several weeks). Furthermore, the method is non-destructive, and the specific particles selected for analysis can be appropriately recovered after the analysis for further research.

[0147] Although Figure 2 and Figure 3 In the fluid apparatus shown, the pumping system 200 is linear, but the pumping system 200 may also include one or more liquid recirculation systems, and one or more flow generation systems for liquid circulation within the pumping system 200 and the settling chamber. Figure 13 In the specific embodiment shown, the pumping system 200 includes another recirculation channel 250 and a secondary pumping system, referred to herein as recirculation device 240. Figure 13 The scheme for introducing particulate matter into the fluid device is similar to that previously described. Particulate matter 420 contained in analytical medium 450 is extracted from tank 400 and introduced into pumping system 200 through inlet channel 210. Flow generation system 230 is activated to introduce particulate matter into settling chamber 100, which is monitored by detection device 300. Pumping system 200 also includes recirculation channel 250 and recirculation device 240. Figure 13 The recirculation channel 250 is circulatedly connected to the sedimentation chamber 100, and the recirculation device 240 is activated by the processor 500 to retain particles within the sedimentation chamber 100. This is achieved by generating a controlled circulation flow based on particle data obtained from the detection system 300. The recirculation channel 250 allows for the recirculation of the analytical medium, which has the advantage of minimizing its consumption.

[0148] One embodiment of the present invention allows for the measurement of particle mass density, weight, size, and shape before and after replacing the primary analytical medium 450 with one or more different analytical media 451. In this embodiment, the pumping system 200 further includes one or more secondary channels fluidly connected to the settling chamber 100 for introducing liquid and / or particles into or removing them from one or more additional tanks. The secondary channels may be directly connected to the settling chamber 100, or they may be connected via inlet channels or via a secondary fluid loop. A specific implementation of this embodiment employs... Figure 2 A variation of the fluid apparatus is shown. Particles 420 are introduced into and kept suspended within a first analytical medium 450 in a sedimentation chamber 100 via a pumping system 200. In this embodiment, the pumping system 200 includes a secondary channel 260 for buffer exchange. Figure 14 The system connects to a second tank 401 containing new analytical medium 451. This system allows new analytical medium 451 to be introduced into the sedimentation chamber 100 via a secondary channel 260 for buffer replacement. During primary analytical medium 450 replacement, microparticles are retained inside the sedimentation chamber 100. After several repetitions, this process results in a complete exchange of analytical medium between both the sedimentation chamber 100 and the recirculation channel 250. In a specific variation of this embodiment, a selection valve 270 is located at the junction between the secondary channel 260 for buffer exchange and the inlet channel 210. In another variation of this embodiment, the system is adapted to draw from the tank 401 containing new analytical medium 451 via the same inlet channel 210, introducing microparticles through the inlet channel 210 via either manual or automatic tank replacement 400. In yet another variation of this embodiment, the pumping system 200 includes a secondary channel 260 for buffer exchange. Figure 14 ), recirculation channel 250 and recirculation system 240 ( Figure 13This implementation allows for measurements of particle mass density, weight, size, and / or shape to be performed over time periods, even days or weeks, during which changes in the liquid containing the particles can be maintained and analyzed. Combinations of these usage modes can be beneficial for various applications. For example, particles can be processed over long periods while frequent changes in the analytical medium are performed. Thus, during each replacement, a second liquid with characteristics different from the previous one can be introduced, for example, a drug concentration with little change, for analysis of its toxicity. Conversely, the intensity of the treatment can be greater, introducing a second analytical medium with characteristics significantly different from the first analytical medium, and then monitoring the particle's response to this change. In one embodiment of the invention, the same particles are analyzed while being subjected to a series of analytical media with varying ionic strengths. With this implementation, the effect of ionic strength on particle 420 can be measured. In some usage methods, the use of the correct analytical medium is a critical aspect of the technical implementation of this embodiment: if the particles have a semi-permeable membrane (e.g., if they are single cells or spheres), then an isotonic analytical medium (e.g., 0.9% w / v PBS for cells) allows for the measurement of the native particle mass density. In different embodiments of the present invention, the test medium is not isotonic with the particles, and the effect of ionic strength on the particles can be measured.

[0149] In various embodiments of the invention, the series of analytical media used to process the microparticles 420 may contain pharmaceutical or other bioactive compounds that can affect their size, volume, mass density, and / or weight. In this case, the system measures the effect of the compound on cells, spheroids, or organoids. Potential results for monitoring bulk density and live sample size are shown in… Figure 15 The diagram illustrates this. Here, the particles are considered to have a spherical geometry, and their size is plotted as diameter. The example shown aims to identify the maturation stages of the spheres and the time required for them to interact with the drug. In this illustrative graph, the particles grow in both diameter and mass density until time T1 is reached. After this, a stabilization of the diameter is observed, although the density continues to increase. This indicates a compaction stage until time T2, where the density also reaches a plateau, where the spheres are in ideal maturation conditions to change the analytical medium. Time T3 represents the moment relevant to drug introduction, and subsequent trends show its impact. This process provides the possibility of observing and comparing changes in density and diameter over time and gathering valuable details about the interaction between the particles and the drug.

[0150] One variation of this embodiment involves the combination of cultures and long-term microbial analysis with solutions used to modify the analytical medium. Indeed, another important aspect of scientific research involves the possibility of monitoring samples during changes in the environment surrounding the sample (such as pH, ionic strength, changes in growth factors in the medium), or during specific treatments using pharmacological reagents and / or chemicals.

[0151] Another mode of use of embodiments of the present invention allows for the classification of microparticles, i.e., the organization and classification of microparticle subpopulations. This operation is important in the biomedical field because it allows for the collection of microparticle subpopulations based on specific properties or geometric features. Cell classification is commonly used in medical, pharmacological, and scientific research. Its applications include, for example, regenerative medicine and personalized medicine, as well as anticancer therapies or virology. Current cell classification methods select samples based on factors such as the presence of protein markers or specific biochemical interactions. However, there is currently no technology capable of selecting and classifying a wide range of samples based on mass density, weight, size, and shape. Therefore, another embodiment of the present invention relates to a method and related apparatus for the selective classification of microparticles based on accompanying measurements of these properties.

[0152] In one specific embodiment of this implementation, the settling chamber 100 includes a branch that connects it to the recovery channel 120, which in turn fluidly connects to a specific portion of the pumping system 200, referred herein as the secondary channel 280 for sorting. Figure 16 The inlet channel 210 and secondary channel 280 used for sorting are both controlled by flow regulators (e.g., valves 270 and 290). In this embodiment, when valve 290 is closed and valve 270 remains open, particles 420 are introduced into the settling chamber 100 through inlet channel 210. Once the particle measurements have been completed as described in the previous embodiments, the results are evaluated based on mass density, weight, size, and shape values ​​for sample sorting. The selected particles are then collected by activating pumping system 200 while valve 270 is closed and valve 290 remains open, allowing the particles to pass through recovery channel 120 and secondary channel 280 for sorting, ultimately reaching tank 700. Figure 16 ).

[0153] In one variation of this embodiment, particles are exclusively selected and classified based on their mass density. This relates to reducing the non-homogeneity of scientific testing, thereby allowing, for example, the selection and collection of spherical subgroups with similar compaction. In another variation of this embodiment, several measurement parameters can be combined to select the collected particles. For example, characteristics such as the standard deviation of the measured diameter and final velocity can be used to classify particles according to their sphericity. This specific application can be related to the diffusion of a test drug across different batches of homogeneous spherical particles and can provide information about the time it takes for the drug to reach the cell nucleus. This same application can also provide information about the concentration levels of the drug required to effectively cross multiple cell layers, as well as for conducting toxicity studies.

[0154] In another variation of this embodiment, the parameters and limits used for classification may be defined by the user or extracted from a database; or they may be automatically defined by an unsupervised algorithm that can classify similar particle populations within a group.

[0155] Another aspect of the invention relates to the importance of shifting from manual to automated processes, particularly when moving from laboratory use to large-scale medical and industrial applications. Due to the small number of hardware components, the present invention is conceived to be easily automated using existing devices. Such components can also be easily miniaturized using existing technology.

Claims

1. A fluid device for measuring at least one of particle mass density and weight, wherein the fluid device comprises: A settling chamber, the settling chamber being fluidly connected to an inlet channel, wherein the inlet channel is configured to be submerged in a liquid; A pumping system connected to the settling chamber, wherein the pumping system is adapted to control the liquid flow in the settling chamber; Processor, the processor being configured to: Obtain particle data related to particles in at least one region of the precipitation chamber, and Calculate at least one of the particle mass density and weight based on the received data; The sedimentation chamber includes a flow channel with a variable internal cross-section to allow the pumping system to induce a flow with a non-zero horizontal component within the sedimentation chamber.

2. The fluid device according to claim 1, further comprising at least one detection device configured to acquire the particle data and provide the particle data to the processor.

3. The fluid apparatus of claim 1 or 2, wherein the processor is configured to control the pumping system based on at least a portion of the received particle data.

4. The fluid device according to claim 1 or 2, wherein the particle data includes at least one of particle velocity, shape, position, and size.

5. The fluid apparatus according to claim 1 or 2, further comprising a temperature control device configured to provide the processor with a temperature measurement of the liquid in the settling chamber; The processor is configured to calculate at least one of the particle mass density and weight, even based on the temperature measurement.

6. The fluid apparatus of claim 5, wherein the temperature control device is further configured to adjust the temperature of the liquid in the settling chamber based on at least one of information provided by the processor and a predetermined temperature value.

7. The fluid device of claim 2, further comprising a movable support member adapted to accommodate at least a portion of the at least one detection device. The processor is also configured to guide the movable support based on at least a portion of the received particle data.

8. The fluid apparatus according to claim 1 or 2, wherein the sedimentation chamber comprises: Flow channels, which are connected in parallel to each other and connected to the inlet channel, wherein each flow channel is also connected to a flow regulator. The processor is configured as Receive input data related to particles in the inlet channel, and The flow regulator is controlled based on the input data.

9. The fluid apparatus according to claim 1 or 2, wherein the pumping system includes a recirculation channel connected in parallel to the settling chamber. The recirculation channel includes a recirculation device, and The processor is configured to control the recirculation device to recirculate the liquid in the sedimentation chamber.

10. The fluid apparatus of claim 1 or 2, wherein the pumping system further comprises a secondary channel fluidly connected to the settling chamber. The processor is configured to selectively control the flow in the secondary channel to introduce liquid into the sedimentation chamber and / or drain liquid from the sedimentation chamber.

11. A method for measuring at least one of particle mass density and weight, the method comprising: The particles to be analyzed are introduced into the sedimentation chamber of the fluid apparatus through an inlet channel that is submerged in liquid; Collecting particle data, wherein the particle data is related to particles in at least one region of the sedimentation chamber, wherein the particles move in a stagnant liquid within the sedimentation chamber, and Calculate at least one of the particle mass density and weight based on the received data; The sedimentation chamber includes a flow channel with a variable internal cross-section to allow the induction of a flow with a non-zero horizontal component within the sedimentation chamber.

12. The method of claim 11, further comprising: Particles are selected based on one of the following: mass density, weight, size, and shape. as well as Selected particles are collected in a predetermined container based on at least one of the particle mass density, weight, size, and shape.

Images