Isolating Analytes of Different Analyte Classes

Description

[0001] The present invention relates to methods and devices for isolating different analyte classes from the same sample volume, and more particularly to methods and devices for isolating extracellular vesicles, EV, and / or circulating tumor cells as a first analyte class and cell-free nucleic acids as a second analyte class from the same sample volume of a biological sample. Introduction

[0002] Early diagnosis of diseases is often crucial for patients' chances of recovery, which is why there is an urgent need for high diagnostic sensitivity. Furthermore, specific diagnosis of diseases is important for making correct treatment decisions and minimizing false-positive findings, as these are associated with a significant burden for patients and high additional costs for the healthcare system due to follow-up examinations.

[0003] Thus, in addition to high sensitivity, there is a need for high diagnostic specificity. The sensitivity and specificity of a diagnostic test are defined by determining a common cutoff value that divides test results into positive and negative. Thus, sensitivity and specificity are interrelated and cannot be optimized independently as long as the diagnosis is based on a single analyte.

[0004] A promising way to increase both diagnostic sensitivity and specificity is the analysis of multiple analytes or analyte classes in a patient sample and at a single point in time. However, this usually requires either the division of the patient sample into multiple sub-volumes or the collection of larger sample volumes, which not only incurs costs, e.g. multiple blood tubes, but also increases the burden for the patient. This problem is exacerbated by the fact that for the disease-associated analytes known today from non- or minimally invasive samples, a very large amount of sample is collected anyway, which is stressful for the patient, e.g. 10 mL of whole blood for the analysis of cfDNA (cell-free deoxyribonucleic acid) in plasma. The reason for this is that the concentration of the analytes within the sample matrix is ​​often very low, particularly in the early stages of the disease or.following therapy (monitoring of minimal residual disease, MRD).

[0005] For example, centrifugal microfluidic systems can be used to provide analytes for analysis from a biological sample and / or to analyze analytes. A centrifugal microfluidic system is defined as a system designed to handle liquids by utilizing the centrifugal force generated during rotation. Centrifugal microfluidics deals with the handling of liquids in the picoliter to milliliter range in rotating systems. Such systems are usually disposable polymer cartridges used in or instead of centrifuge rotors with the intention of automating laboratory processes. Standard laboratory processes such as pipetting, centrifuging, mixing, or aliquoting can be implemented in a microfluidic cartridge. For this purpose, the cartridges contain channels for fluid flow and chambers for collecting liquids.Generally, such structures designed to handle fluids can be referred to as fluidic structures. Such cartridges can generally be referred to as fluidic modules.

[0006] The cartridges can be subjected to a predefined sequence of rotation frequencies, the frequency protocol, so that the liquids contained in the cartridges can be moved by centrifugal force. Centrifugal microfluidics is primarily used in laboratory analysis and mobile diagnostics. State of the art

[0007] Methods and devices for isolating analytes from biological samples using centrifugal microfluidic systems are known.

[0008] Chi-Ju Kim et al., "Fully automated, on-site isolation of cfDNA from whole blood for cancer therapy monitoring," Lab Chip, 2018, 18, pages 1321 to 1329, disclose a centrifugal microfluidic chip and an associated device that can be used to fully automatically isolate cfDNA from whole blood in less than 30 minutes. The cfDNA is bound to silica beads, washed, and eluted. The fluidics are based on membrane valves that are opened or closed by a plunger.

[0009] Hyun-Kyung Woo et al., "Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Sample," ACS Nano 2017, 11, pages 1360 to 1370, disclose a fluidic module in the form of a disc with fluidic structures for isolating EVs (extracellular vesicles) using centrifugal microfluidics. The fluidic module contains two nanofilters, which are used to isolate EVs in the size range of 20 nm to 600 nm from biological samples within 30 minutes. Large particles are first removed from the sample using a first filter, and then EVs are isolated using a second filter. The EVs are then washed and eluted. The fluidics system is based on membrane valves that are opened or closed by a plunger.

[0010] There are also methods in which two or more analyte classes are isolated from a single sample.

[0011] EP 3 167 062 B1 discloses a method in which particles, cells, and / or cell fragments or other contaminants are removed from a biological sample by centrifugation or filtration before microvesicles are isolated, purified, and / or enriched. High-quality nucleic acids are then extracted from the microvesicles. In the method described, a biological sample is provided and brought into contact with a capture surface under conditions sufficient to obtain cell-free DNA (deoxyribonucleic acid) and microvesicles from the biological sample on or in the capture surface. The capture surface is brought into contact with a phenol-based lysis reagent while cell-free DNA and the microvesicles are on or in the capture surface, releasing DNA and RNA (ribonucleic acids) from the sample and producing a homogenate. DNA, RNA, or both DNA and RNA are then extracted from the homogenate.

[0012] A similar method for isolating extracellular vesicles and co-isolating cell-free DNA from biofluids is known from US Pat. No. 10,808,240 B2. A biological sample is brought into contact with a solid capture surface under conditions sufficient to retain cell-free DNA and microvesicles from the biological sample on or within the capture surface. The capture surface is brought into contact with a GTC-based elution buffer while cell-free DNA and microvesicles are on or within the capture surface, releasing the DNA and RNA from the sample and creating a homogenate. The DNA, RNA, or both DNA and RNA are extracted from the homogenate.

[0013] EP 2 245 458 B1 also discloses a method in which a homogenate is produced consisting of DNA from extracellular vesicles and cfDNA.

[0014] US 2014 / 030788 A1 discloses devices and systems with fluid channels or fluid chambers and permeable barriers arranged therein, each comprising a plurality of aligned nanostructures defining an outer surface of the barrier and an inner network of cavities. The barriers enable the capture of particles of a first type in a fluid sample or the selective separation or concentration of particles of the first type from particles of a second type. Particles of the first type can be, for example, cancer cells, and particles of the second type can be, for example, microvesicles or nucleic acid. Description of the invention

[0015] It has been recognized that a significant disadvantage of the methods known in the prior art is that after these methods have been carried out, the EV DNA and the cfDNA (and, depending on the method, RNA) are present as a homogenate and cannot be analyzed in detail. The extracellular vesicles are destroyed, meaning that characteristics such as surface markers, size distribution, etc., cannot be further investigated. Extracellular vesicles contain other relevant analytes (e.g., proteins). These are lost in the known methods that produce a homogenate. The amount of cfDNA already represents a biomarker. This amount is distorted by additional EV DNA.

[0016] It was further recognized that analyzing different analyte classes from the same sample volume could provide a solution. Several different analytes can increase diagnostic sensitivity / specificity, but this previously required a large sample volume. Preferably, the methods used for pre-analytical isolation of the different analyte classes should be compatible with each other and, at the same time, capable of being combined in an integrated, automated process chain. This could offer an economically attractive solution, as it would avoid the high costs associated with manual intermediate steps and the need for multiple different laboratory devices for isolation.At present, there is no method available that allows the isolation of multiple analyte classes from an identical sample volume and in a single integrated process chain, since the existing methods would either influence / interfere with each other and / or are based on different principles that cannot be easily combined in one laboratory device.

[0017] It is the object of the invention to provide methods and devices that make it possible to isolate analytes of different analyte classes, namely extracellular vesicles and / or circulating tumor cells on the one hand and cell-free nucleic acids on the other hand, from the same sample volume in a way that enables implementation in an integrated process chain.

[0018] This object is achieved by a method according to claim 1 and a centrifugal microfluidic device according to claim 6. Further developments of the invention are defined in the dependent claims.

[0019] Examples of the disclosure provide methods for isolating analytes of a first analyte class, which are extracellular vesicles and / or circulating tumor cells, and analytes of a second analyte class, which are cell-free nucleic acids, from the same sample volume of a biological sample in a centrifugal microfluidic system comprising a fluidic module including a first isolation chamber containing a first isolation structure and a second isolation chamber containing a second isolation structure. The sample volume is directed into the first isolation chamber such that the analytes of the first analyte class are retained by the first isolation structure, while the analytes of the second analyte class are not retained by the first isolation structure and pass through the first isolation structure as part of a residual fluid.The residual liquid is directed into the second isolation chamber so that the analytes of the second analyte class are retained by the second isolation structure. The analytes of the first analyte class are separated by the first isolation structure, and the analytes of the second analyte class are separated by the second isolation structure in order to provide the analytes of the first analyte class and the analytes of the second analyte class separately from one another for subsequent analysis. The first isolation structure is a filter with a pore size in a range of 20 nanometers to 200 nanometers, preferably in a range of 20 nanometers to 45 nanometers. The second isolation structure is a surface for binding analytes of the second analyte class, wherein the surface is formed by particles, and wherein the particles are magnetizable.The method comprises rotating the fluidic module to move the magnetizable particles in the second isolation chamber by means of one or more stationary or movable magnets of a processing device in order to support mixing of the particles with the residual liquid and / or to support retention of the magnetizable particles in the second isolation chamber during a transfer of analytes of the second analyte class into the second collection chamber.

[0020] Examples of the disclosure provide a device with a processing device and fluidics module for isolating analytes of a first analyte class, which are extracellular vesicles and / or circulating tumor cells, and analytes of a second analyte class, which are cell-free nucleic acids, from the same sample volume of a biological sample in a centrifugal microfluidic system. The fluidics module comprises a first isolation chamber containing a first isolation structure, a second isolation chamber containing a second isolation structure, a first fluid line, a second fluid line, a first collection chamber, and a second collection chamber.The first fluid line is designed to direct the sample volume into the first isolation chamber such that the analytes of the first analyte class are retained by the first isolation structure, while the analytes of the second analyte class are not retained by the first isolation structure and pass through the first isolation structure as part of a residual liquid. The second fluid line is designed to direct the residual liquid into the second isolation chamber such that the analytes of the second analyte class are retained by the second isolation structure. The first collection chamber is connected to the first isolation chamber and designed to receive analytes of the first analyte class released from the first isolation structure.The second collection chamber is connected to the second isolation chamber and is configured to receive analytes of the second analyte class dissolved by the second isolation structure, or to receive the second isolation structure after the analytes of the second analyte class have been dissolved therefrom. The first isolation structure is a filter with a pore size in a range of 20 nanometers to 200 nanometers, preferably in a range of 20 nanometers to 45 nanometers. The second isolation structure is a surface for binding analytes of the second analyte class, and the surface is formed by magnetizable particles.The processing device comprises one or more stationary or movable magnets arranged outside the second isolation chamber to assist mixing of magnetizable particles with the filtrate upon rotation of the fluidic module and / or to assist retention of the magnetizable particles in the second isolation chamber upon transfer of analytes of the second analyte class into the second collection chamber.

[0021] According to examples of the disclosure, analytes of different analyte classes are thus obtained from the same sample volume and provided separately from one another for further use, for example, analysis or investigation. Since both analytes are obtained from the same sample volume, with the analytes of the second analyte class being obtained from the residual liquid remaining after the isolation of the analytes of the first analyte class, isolation of both analyte classes from a small sample volume is possible. Furthermore, it has been recognized that it is possible to perform both isolations using a fluidics module in a centrifugal microfluidic system, thus enabling integration into a single process chain. Short description of the drawings

[0022] Embodiments of the invention are explained in more detail below with reference to the accompanying drawings. They show: Fig. 1 a schematic representation of a fluidics module according to an example of the present disclosure; Fig. 2 a schematic representation of components of a fluidics module according to an example of the present disclosure; Fig. 3 a diagram schematically showing a method according to the present disclosure; and Fig. 4 a schematic representation of a fluidic module in the form of a centrifugal microfluidic cartridge according to an example of the present disclosure; Fig. 5A and 5B schematic representations of devices using fluidic modules as described herein. Detailed description

[0023] Examples of the present disclosure are described in detail below with the use of the accompanying drawings. It should be noted that like elements or elements having the same functionality are provided with the same or similar reference numerals, and repeated description of elements provided with the same or similar reference numerals is typically omitted. In particular, like or similar elements may each be provided with reference numerals having the same number with a different or no lowercase letter. Descriptions of elements having the same or similar reference numerals may be interchangeable. In the following description, many details are described in order to provide a more thorough explanation of examples of the disclosure.However, it will be apparent to those skilled in the art that other examples may be implemented without these specific details. Features of the various described examples may be combined with each other, unless features of a corresponding combination are mutually exclusive or such a combination is expressly excluded.

[0024] Before further explaining examples of the present disclosure, definitions of some terms used herein are provided.

[0025] The term isolation refers to processes aimed at removing interfering components of a sample matrix, e.g., inhibitors or falsely analyzable components, and / or increasing the concentration of an analyte in a sample, e.g., by reducing the aqueous portion of a sample. Isolation can be achieved either by enriching the analyte or by depleting non-analyte substances. Synonyms for the term isolation are enrichment, extraction, and purification.

[0026] In this context, a sample refers to a biological material, usually derived from a human and collected using a non-invasive or minimally invasive technique. The sample can be either a liquid sample (e.g., whole blood, blood plasma, blood serum, urine, saliva, tear fluid, cerebrospinal fluid, seminal plasma) or a liquefied sample of solid origin (e.g., stool). This definition also includes samples generated from a human cell sample, such as cell culture supernatant, even if the original cell sample was collected invasively.

[0027] A sample matrix refers to the components of a sample that are not to be analyzed. The matrix can complicate analysis, for example, if the analyte is present in very low concentrations or if components of the matrix interfere with the analysis procedure.

[0028] Analytes are defined as components to be analyzed. Analytes dissolved in the sample matrix include substances, molecules, and particles, which can be analyzed for number, concentration, biomarker signatures, etc. in an analytical procedure connected to the isolation. Possible analytes in this context include nucleic acids, proteins, peptides, metabolites, secondary metabolites, vitamins, cells (human cells, as well as fungi, bacteria, or mycoplasmas), exosomes, and viruses.

[0029] The term analyte classes refers to those analytes that can be isolated using a single technical procedure. In this context, DNA and RNA in their various forms are considered one analyte class, namely cell-free nucleic acids (cfNA). The heterogeneous subtypes of extracellular vesicles are also considered one analyte class, namely extracellular vesicles (EV). This analyte class can furthermore or alternatively include circulating tumor cells. It should be noted that depending on the nature of the analyte of interest, for example, small EV versus large EV, the parameters of the isolation procedure can vary, for example, varying pore sizes during filtration. Other analyte classes according to this definition can be proteins, cells, and cell fragments.

[0030] The term cell-free nucleic acids (cfNA) encompasses various forms of DNA and RNA that occur outside of cells in the human body. Currently, this includes, among others, circulating cell-free DNA (ccfDNA) of both nuclear and mitochondrial origin, circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cellular DNA, circulating tumor RNA (ctRNA), microRNA (miRNA), and non-coding RNA (ncRNA).

[0031] The term extracellular vesicles (EVs) refers to particles released by cells. They represent a heterogeneous population of various subtypes, which currently include exosomes, microvesicles, ectosomes, and apoptotic bodies. The size of the particles varies depending on the subtype, between a few tens of nm and a few 1 µm. Various analytes, such as proteins, nucleic acids, lipids, and metabolites, are found both inside the EVs and, in some cases, on their surfaces.

[0032] Proteins are macromolecules consisting of amino acids linked by peptide bonds. They perform a wide variety of functions in the human body and are involved in a multitude of disease-associated processes. The analysis of cell-free proteins in a sample matrix, such as blood serum, can therefore be used for diagnostic purposes, either alone or in conjunction with other analytes.

[0033] The term "cells" or "cell fragments" refers to all human blood cells (erythrocytes, leukocytes, and platelets), as well as other circulating (e.g., in the blood) or transported (e.g., in stool) cells. Examples of circulating cells in the blood include circulating tumor cells (CTCs) and tumor-associated platelets (TEPs).

[0034] A fluidic module is understood here to be a module, for example a cartridge, that has microfluidic structures designed to enable liquid handling as described herein. A centrifugal microfluidic fluidic module (cartridge) is understood to be a corresponding module that can be subjected to rotation, for example in the form of a fluidic module that can be inserted into a rotating body or a rotating body.

[0035] Examples of the invention can be applied in particular in the field of centrifugal microfluidics, which involves the processing of liquids in the picoliter to milliliter range. Accordingly, the fluidic structures can have suitable dimensions in the micrometer range for handling corresponding liquid volumes.

[0036] When the term radial is used herein, it means radial with respect to the center of rotation around which the fluidic module or the rotating body is rotatable. In the centrifugal field, a radial direction away from the center of rotation is radially decreasing, and a radial direction towards the center of rotation is radially increasing. A fluid channel whose beginning is closer to the center of rotation than its end is thus radially decreasing, while a fluid channel whose beginning is further from the center of rotation than its end is radially increasing. A channel with a radially increasing section therefore has directional components that increase radially or run radially inwards. It is clear that such a channel does not have to run exactly along a radial line, but can run at an angle to the radial line or be curved.

[0037] Unless otherwise stated herein, room temperature (20°C) shall be assumed with regard to temperature-dependent quantities.

[0038] Generally, examples of the present disclosure relate to methods and devices for purifying multiple analytes from a sample volume. Examples relate to a method for isolating multiple different classes of analytes, such as cell-free nucleic acids and extracellular vesicles (EVs), from a single sample volume, where the sample typically originates from a human and is collected using a non-invasive or minimally invasive technique, such as blood, urine, or stool. Examples allow this to be done without dividing the sample volume into multiple subvolumes, since such a division is associated with a loss of absolute analyte quantity per analysis, which in turn results in a loss of achievable sensitivity.Furthermore, the isolation of different analyte classes together into a single isolate should be avoided, as this would prevent the structurally identical components of the various analytes, for example, cell-free nucleic acids and EV-associated nucleic acids, from being analyzed separately from one another. A centrifugal-microfluidic process chain was identified as a suitable method for avoiding sample partitioning. This process enables the isolation of multiple classes of analytes from the same sample volume, with the individual isolation methods being compatible with each other and not interfering with each other. In contrast to previous methods, which either require partitioning the sample prior to isolation or multiple serial isolation methods, the methods described here enable the automated isolation of multiple analytes in a single, integrated process chain.The products of the process are several isolates, each containing only a single defined class of analytes.

[0039] Examples of the disclosure thus provide methods for isolating two or more analyte classes from a single sample volume in a combined, integrated process in a centrifugal microfluidic cartridge, as well as fluidic modules in the form of rotating bodies and devices designed to carry out such methods. The goal is to save sample material and time and minimize manual and instrumentation effort, while simultaneously isolating as many analytes as possible in high purity for subsequent analyses.

[0040] Fig. 1schematically shows an example of a fluidic module 10 according to the present disclosure, which is designed to carry out a method for isolating two analyte classes, as described herein. The fluidic module 10 can, for example, be a rotational body rotatable about a rotation center R or a fluidic module insertable into such a rotational body. The fluidic module 10 has fluidic structures that have a first isolation chamber 12 and a second isolation chamber 14. The first isolation chamber 12 contains a first isolation structure 16. The second isolation chamber 14 contains a second isolation structure 18. A sample volume is directed into the first isolation chamber 12, as indicated by an arrow 20 in Fig. 1For this purpose, the fluidic structures comprise a first fluid line 22. Analytes of the first analyte class are retained by the first isolation structure 16, while analytes of the second analyte class are not retained by the first isolation structure 16, but pass through the first isolation structure 16 as part of a residual liquid. The residual liquid is guided into the second isolation chamber 14 through a second fluid line 24, which connects the first isolation chamber 12 to the second isolation chamber 14, as indicated by an arrow 26 in Fig. 1 The analytes of the second analyte class are retained by the second isolation structure 18.

[0041] The analytes of the first analyte class are separated by the first isolation structure 16 and the analytes of the second analyte class are separated by the second isolation structure 18. This is indicated by arrows 28 and 30 in Fig. 1shown. As a result, the analytes of the first analyte class and the analytes of the second analyte class are provided separately from one another for subsequent analysis. Separating 28 the analytes of the first analyte class from the first isolation structure can, for example, comprise directing the analytes of the first analyte class into a first collection chamber 32. For this purpose, the first collection chamber 32 can be connected to the first isolation chamber 12 via a fluid line. Separating 30 the analytes of the second analyte class from the second isolation structure 18 can comprise directing the analytes of the second analyte class into a second collection chamber, which is connected to the first collection chamber via a fluid line. The first collection chamber 32, the second collection chamber 34, and fluid lines connecting them to the first isolation chamber 12 and the second isolation chamber 14, respectively, can be part of the fluidic structures of the fluidic module 10.The first collection chamber 32 is thus designed to receive analytes of the first analyte class dissolved by the first insulation structure 16, and the second collection chamber 34 is designed to receive analytes of the second analyte class dissolved by the second insulation structure 18.

[0042] In examples of the described method, the order in which the steps of separating the analytes of the first analyte class from the first isolation structure and directing the residual liquid into the second isolation chamber take place is not important. In examples, the residual liquid can first be directed into the second isolation chamber before the analytes of the first analyte class are separated from the first isolation structure. In examples of the present disclosure, the first isolation structure is configured to perform filtration based on size differences. In examples, the first isolation structure 16 is a filter with a pore size in a range of 20 nanometers to 200 nanometers, preferably in a range of 20 nanometers to 45 nanometers. Thus, the first isolation structure enables the isolation of analytes of the first analyte class, namely extracellular vesicles and / or circulating tumor cells.Alternatively, platelets could also be isolated by such filtration.

[0043] In examples, the first isolation structure comprises capture structures designed to bind analytes of the first analyte class. Such capture structures can be, for example, antibodies or aptamers. Alternatively, affinity-based methods can be used, for example, peptides on magnetizable particles that bind components of the EV membrane. In examples, the first isolation structure can be designed to cause polymer precipitation. In this case, a polymer is added, which forms a grid in which the EVs or circulating tumor cells (CTCs) are captured. Subsequently, sedimentation can be performed to separate the grid, i.e., to separate the analytes of the first analyte class from the first isolation structure.

[0044] In examples, separating the analytes of the first analyte class from the first isolation structure may include washing the analytes retained on the first isolation structure using a washing solution, eluting the analytes from the first isolation structure using an elution solution, and transferring the analytes released from the first isolation structure to a first collection chamber. Thus, examples enable isolating and separating the analytes of the first analyte class in a conventional manner.

[0045] In examples, the first isolation structure has a volumetrically defined chamber geometry of the first isolation chamber for the sedimentation of analytes of the first analyte class. The geometrically defined chamber geometry can, for example, be provided by a channel that opens into the first isolation chamber at a specific radial height. Rotation can cause sedimentation in the first isolation chamber, through which the analytes of the first analyte class reach the chamber region that lies radially outside the channel opening into the chamber. The residual liquid can then be guided into the second isolation chamber by withdrawing the supernatant via the channel opening into the first isolation chamber at the specific radial height. In this case, the first isolation structure is formed by the lower part of the first isolation chamber.In examples, EVs can be sedimented in a polymer forming the first isolation structure, after which the supernatant can be removed, a dissolving buffer can be added to redissolve the polymer, and then the solution containing the EVs can be transferred. In such a case, the channel for withdrawing the supernatant can be located at the radially outer end of the first isolation chamber to withdraw the residual liquid, leaving the polymer containing the analytes of the first analyte class in the first isolation chamber.

[0046] In examples, the isolation structures, in particular the second isolation structure, can be a surface for binding analytes. The surface can be formed by particles, columns, a membrane, or a surface of the fluidic module. Such an isolation structure can effect solid-phase isolation (bind, wash, elute) due to controlled binding conditions to a surface, for example, isolation of cell-free nucleic acids or isolation of proteins. The surface can be part of the fluidic module, the cartridge, for example, a channel or a chamber, or can be a body introduced into the fluidic module or a plurality of bodies introduced into the fluidic module, for example, a column / membrane or beads / nanoparticles.

[0047] In examples, at least the second isolation structure has a surface for binding analytes of the second analyte class, which surface is formed by particles, for example beads, wherein the particles are magnetizable, wherein the method comprises rotating the fluidics module in order to move the magnetizable particles in the second isolation chamber by means of one or more stationary or movable magnets of a processing device in order to support mixing of the particles with the liquid in the respective chamber, for example with the residual liquid. In examples, the magnets can serve not only for mixing, but can also be designed to retain particles in the second isolation chamber when the elution liquid with the analyte is transferred into the collection chamber.In examples, one or more magnets may be arranged stationary or movable outside the second isolation chamber to assist in retaining the magnetizable particles in the second isolation chamber during the transfer of the analytes of the second analyte class into the second collection chamber.

[0048] Examples of the present disclosure provide a centrifugal microfluidic device comprising a fluidic module as described herein and a processing device configured to impart rotation to the fluidic module. The processing device may comprise one or more stationary or movable magnets that, upon actuation of the fluidic module, i.e., upon rotation thereof, move magnetizable particles in the second isolation chamber to assist in mixing the particles in the residual liquid.

[0049] In examples of the methods disclosed herein, isolating the analytes of the second analyte class comprises mixing the sample volume with a binding buffer, contacting the resulting mixture with a surface to bind the analytes of the second analyte class to the surface, washing the surface with the bound analytes using a wash buffer, eluting the analytes from the surface using an elution buffer, and transferring the analytes released from the second isolation structure to a second collection chamber. In examples, a comparable method can be used to isolate the analytes of the first analyte class from the sample volume, wherein the released analytes can be transferred to a first collection chamber. Examples thus enable isolation of the analytes of the first and / or second analyte class using common bind-wash-elute methods.

[0050] In examples of the present disclosure, analytes can be isolated from the sample volume by centrifugation. Such centrifugation can be used to further separate the sample based on density differences before, between, or after filtration, for example, for blood-plasma separation or for platelet isolation. Examples of the present disclosure are designed to extract analytes of two or more analyte classes from the sample volume. If three or more analytes are to be isolated, the fluidic structures are designed accordingly and comprise at least one further isolation chamber with at least one further isolation structure and at least one further collection chamber. The various analyte classes can then be removed from the various collection chambers of the fluidic module, the microfluidic cartridge, for analysis.

[0051] In examples, the fluidic module comprises at least one filter fluidically connected to the inlet side of the first isolation chamber, which filter is permeable to the analytes of the first analyte class and the analytes of the second analyte class, and which is configured to filter cells, cell fragments, particles, and contaminants from the sample volume. Accordingly, examples of the method comprise passing the sample volume through a corresponding upstream filter prior to passing the sample volume into the first isolation chamber. Thus, examples enable preparation of the sample volume for the subsequent isolation of the analytes of the first analyte class and the second analyte class from the sample volume.

[0052] In examples of the present disclosure, preparing the biological sample comprises one or more of the following processes: Performing a blood-plasma separation of the biological sample to obtain plasma, from which the sample volume is obtained. Performing a blood-plasma separation of a coagulated biological sample to obtain serum, from which the sample volume is obtained. Performing platelet isolation of the plasma to obtain platelet-poor plasma, from which the sample volume is obtained. Performing platelet isolation of the plasma to obtain platelet-rich plasma, from which the sample volume is obtained. Performing an enzymatic digestion to break down proteins and protein aggregates in the biological sample to obtain the sample volume. Performing liquefaction and / or homogenization of the biological sample to obtain the sample volume.

[0053] In examples, the fluidics module comprises fluidic structures configured to perform one or more of the aforementioned processes.

[0054] Platelet isolation can be performed, for example, by sedimentation, with the sediment corresponding to platelet-rich plasma and the supernatant to platelet-poor plasma. Depending on whether the sample volume is withdrawn via a channel at a radially lower end or a radially lateral end of the sedimentation chamber, platelet-poor or platelet-rich plasma can be obtained. Furthermore, platelet isolation can be achieved by filtration if the fluidic module has a filter with a pore size designed to retain the platelets. The filtrate would then be platelet-poor, and the retentate, or eluate subsequently released from the filter, would be platelet-rich. The size of platelets is approximately 1 to 4 µm. For example, it is known to use filters with an appropriate pore size, for example, a pore size of 600 nanometers, to retain platelets.

[0055] In examples of the present disclosure, the first isolation structure is configured to retain EVs and / or CTCs, wherein separating the analytes from the first isolation structure comprises separating the EVs and / or CTCs from the isolation structure in whole or in their constituent parts. In examples, EVs / CTCs can be separated from the isolation structure while simultaneously retaining the cfDNA in the flow-through by one of the following methods: - filtration; - methods in which the analytes are captured ("capture"), for example, using antibodies, aptamers, or affinity-based methods such as peptides on magnetizable particles that bind components of the EV membrane; - polymer precipitation, where a polymer is added that forms a grid in which the EVs / CTCs are captured, followed by sedimentation to separate the grid; - sedimentation to isolate CTCs from the sample volume.

[0056] Generally, in examples of the method described herein, a sample with a volume of a few microliters to several milliliters can be transferred automatically or manually into the fluidic module (the microfluidic cartridge). The fluidic module is then further processed automatically in a device. In this process, analytes of several analyte classes are successively isolated from the sample in a single process using various methods. For this purpose, the device can be designed to subject the fluidic module to rotations that move the various fluids through the fluidic structures, as described herein. In examples, the fluidic module can be subjected to a corresponding frequency protocol for this purpose, so that the fluids contained in the cartridges can be moved by centrifugal force.The fluidic structures can also be designed to support the handling of the liquids through additional processes, such as hydrodynamic processes. Instead of splitting the sample for each isolation procedure, it is automatically passed on via microfluidic connecting channels, ensuring the respective analytes are isolated from the entire sample volume.

[0057] Fig. 2schematically shows components of a fluidics module according to an example of the present disclosure. The components include a first filter structure 40, which represents a first isolation structure, a first collection structure 42, which represents a first collection chamber, a separation structure 44, which represents a second isolation structure, and a second collection structure 46, which represents a second collection chamber. A first fluid line 47 leads to the first filter structure 40, a second fluid line 48 connects the filter structure 40 to the separation structure 44, a third fluid line 50 connects the first collection structure 42 to the filter structure 40, and a fourth fluid line 52 connects the second collection structure 46 to the separation structure 44. As in Fig. 2As shown, in examples, a further filter structure 54 can optionally be arranged upstream, which has a pore size that allows extracellular vesicles, circulating tumor cells, and cell-free nucleic acids to pass through. The first filter structure 40 is configured to retain extracellular vesicles and allow cell-free nucleic acids to pass through. In examples, the first filter structure can be configured to retain circulating tumor cells. In alternative examples, the first filter structure can be replaced by an isolation structure configured to retain circulating tumor cells by sedimentation.

[0058] Referring to Fig. 3 A possible implementation of a process for purifying multiple analytes from a single sample volume is explained below. Fig. 3To the right of a dividing line T, the steps are shown that actually relate to the method for isolating two different analyte classes from a sample volume, while to the left of the dividing line T, a method for preparing the sample volume is shown. Examples of fluidic modules of the present disclosure have fluidic structures to Fig. 3 However, the procedure does not have to include all of the Fig. 3 shown processes, whereby in particular the process steps shown to the left of the dividing line T can be regarded as optional in whole or in part.

[0059] A blood sample is transferred into the fluidics module, 60. A blood-plasma separation is performed to obtain plasma, 62, whereby cells and cellular fragments are separated, 64. Subsequently, a platelet separation can be performed to separate platelets from the plasma, 66. Subsequently, a protein digestion by proteinase K (PK) can be carried out for protein digestion, 68. This results in PK-treated plasma, 70. The plasma thus obtained can now be subjected to coarse filtration with a large pore size, for example, through a filter structure 54 as shown in Fig. 2 This filtration, which is shown in Fig. 3 designated as Filtration 1, a retentate 72 and a filtrate 74 are obtained. The filtrate 74 represents the sample volume of the biological sample from which EV and then NA are isolated. The primary separation in the example shown is carried out by means of a filtration, which is Fig. 3referred to as filtration 2. In this process, the optionally previously prepared sample is pressed through one or more filter membranes using centrifugal forces. The filter membranes can be made of different materials and have different pore sizes, for example, between 20 nm and 10 µm. Possible materials for the membranes are polyester (PES), recycled material (RC), anodized aluminum oxide (AAO), or TEPC (fabric-equivalent material). If multiple filter membranes are used, the sample is pressed successively through the various filter membranes, first filter membranes with a larger pore diameter and subsequently filter membranes with a smaller pore diameter. For example, the upstream filtration 1 can separate matrix components, for example, cells, cell fragments, or protein aggregates, with a large diameter, whereas the EV and cfNA pass through the filter, for example, the further filter structure 54 in Fig. 2The final filter, which represents the first isolation structure, has a pore size such that smaller contaminants, such as individual proteins, and the cfNA pass through the membrane, but the EV are retained. This filtration is in Fig. 3 referred to as filtration 2. This filtration 2 produces a retentate 76 and a filtrate 78. The filtrate 78 represents the residual liquid that is passed to the second isolation structure.

[0060] To further deplete the matrix components in the retentate 76, washing steps can be performed using aqueous solutions, wash buffers. The purified EVs can then be separated from the filter for further analysis using an elution buffer and transferred as EV eluate 80 into a collection chamber of the fluidics module. The filter flow, i.e., the filtrate 78, contains the remaining sample matrix as well as the cfNA. This can then be concentrated and purified using additional methods. Various methods are available for this on centrifugal microfluidic cartridges, the fluidics module, such as cfNA solid-phase isolation on beads. In such a solid-phase isolation, cfNA is bound to beads, then washed using a wash buffer, eluted using an elution buffer, and transferred as cfNA eluate 82 into a collection chamber of the fluidics module.Thus, analytes of the first analyte class and analytes of the second analyte class are provided separately for subsequent analysis.

[0061] In examples where the second isolation structure can be moved out of the second isolation chamber, after the analytes of the second analyte class have been separated from the second isolation structure, the second isolation structure can be moved out of the second isolation chamber while the analytes of the second analyte class remain in the second isolation chamber. For example, in the above example, the solid-phase matrix in the form of beads could be transferred out of the isolation chamber after elution, while the eluate remains in the isolation chamber.

[0062] As already explained above, a blood-plasma separation can optionally be performed upstream of blood samples by utilizing centrifugal forces in a centrifugal microfluidic system. This can further reduce the manual effort required for sample preparation and, at the same time, increase the reproducibility of the entire analytical process. In contrast to manual blood-plasma separation, the plasma supernatant can be removed from whole blood after centrifugation using a centrifugal-pneumatic operation, thereby virtually eliminating re-sedimentation effects. Another goal of an integrated, upstream blood-plasma separation is the automatically achieved isolation of cells and cell fragments, which can be considered a further class of analytes.In addition to the analysis of healthy cells, the analysis of disease-associated cells, such as circulating tumor cells and CTCs, can be particularly relevant.

[0063] In some examples, the sedimentation of circulating tumor cells does not represent a pretreatment of the biological sample, but rather an isolation of analytes of the second analyte class. In other examples, the isolation of circulating tumor cells represents the isolation of analytes of a third analyte class, which precedes the isolation of EV as an analyte of a first analyte class and the isolation of cfNA as an analyte of a second analyte class.

[0064] Analogous to blood-plasma separation, the separation of plasma into platelet-rich plasma (PRP) and platelet-poor plasma (PPP) can be achieved by applying the inherent centrifugal force and by removing the supernatant one or more times. The resulting PRP can be taken from the cartridge as an isolate for the analysis of platelets and used, for example, for the analysis of tumor-educated platelets (TEP). The resulting PPP, however, can be used in the further process of successive isolation of the analyte classes EV and cfNA. If cell-free DNA of mitochondrial origin (cf-mtDNA) is to be analyzed, such upstream platelet isolation can be essential to prevent or minimize contamination of the cell-free mitochondrial DNA with mitochondrial DNA from lysed platelets (mtDNA).TEPs can be considered as analytes of a third analyte class, which are removed from the sample volume before the analytes of the first and second analyte classes.

[0065] Optionally, and depending on the matrix, liquefaction and / or homogenization of the biological sample, for example, in the case of sputum or stool samples, or enzymatic digestion with protease (e.g., proteinase K) can be performed prior to filtration. This process breaks down proteins and protein aggregates that make analyte isolation difficult, for example, by clogging the filter membranes or binding nucleic acids. The enzymes can be removed by washing steps, for example, the Fig. 3 indicated wash buffer, or a solid phase isolation (bind-wash-elute) are used to separate the analytes from the analytes so that they do not hinder further analyses.

[0066] Fig. 4schematically shows a fluidic module 10 in the form of a rotating body 10 rotatable about a rotation center R, which has fluidic structures designed to perform automated, combined isolation of cfNA and EV from a single sample volume in the form of a human plasma sample. Fluidic structures formed in the fluidic module include a sample inlet chamber 83, a prefiltration chamber 84, a filtration chamber 85 for EV isolation, a chamber 86 for collecting the filtrate from the filtration chamber 85, a collection chamber 87 for removing the EV isolate, a chamber 88 for solid-phase isolation of cfNA, a collection chamber 89 for washing liquids from the solid-phase isolation, and a collection chamber 90 for removing the cfNA isolate. As in Fig. 4As shown, the respective chambers are connected to each other by fluid lines. The fluid lines are configured to enable a transfer of liquid between the respective chambers by utilizing centrifugal force and possibly other effects, such as hydrodynamic effects. The filtration chamber 85 for EV isolation represents a first isolation chamber as described herein, and the chamber 88 represents a second isolation chamber as described herein.

[0067] A filter membrane can separate an inlet-side section of the fluid chamber, in which the filter membrane is arranged, from an outlet-side section of this fluid chamber. In examples, the inlet-side section of the fluid chamber and the outlet-side section of the fluid chamber are arranged one above the other in the direction of a rotation axis passing through the center of rotation.

[0068] During operation, the plasma sample is introduced into sample inlet chamber 83 and then sequentially filtered through the filter membranes arranged in chambers 84 and 85. The filter membrane arranged in chamber 84 has larger pore diameters than the filter membrane arranged in chamber 85, so that the analytes of the first and second analyte classes can pass through, but larger matrix components are retained. The second filter membrane in chamber 85 is designed so that cfNA can pass through, but the EV are retained. This allows both analytes to be separated from the sample without loss. The cfNA filtrate is collected in chamber 86 and from there transferred to chamber 88 for solid-phase isolation. Alternatively, the filtrate could also be transferred directly to the solid-phase isolation chamber.The EVs retained on the second filter membrane in chamber 85 are subsequently washed with aqueous buffer solutions, which can be added manually or automatically to chamber 83 to remove matrix components that can pass through the filters. The washing solutions can then be collected in chamber 86 after the cfNA filtrate has been transferred to chamber 88. After washing the EVs, they are transferred from the filter in chamber 85 to the collection chamber 87, from which they can be removed manually or automatically. The filtrate containing the cfNAs is subjected to solid-phase isolation in chamber 88 by contacting it with a surface. In examples, solid-phase isolation is carried out using the bind-wash-elute method using magnetic or magnetizable particles with which the filtrate is contacted, which bind the NA via a silica surface.Matrix components, such as proteins, can be removed in one or more washing steps using solvent-containing liquids. The washing solutions are collected in chamber 89. After washing, the cfNA are eluted from the magnetizable particles using an aqueous elution buffer and then transferred to chamber 90 for removal.

[0069] Compared to the state of the art in serial individual procedures, in which, for example, extracellular vesicles are isolated by filtration in a first apparatus, followed by manual transfer and subsequent isolation of nucleic acids by solid-phase isolation in a second apparatus, the combination method presented here enables the isolation of various analytes not only in a single apparatus, a centrifugal microfluidic processing device, but also in a single consumable, namely a fluidics module in the form of a centrifugal microfluidic cartridge. In addition to advantages such as ease of handling and lower material and personnel costs, this integrated process chain is expected to have a beneficial effect on the reproducibility of the isolation.This aspect is of utmost relevance, as pre-analytical variations are considered a major hurdle in the clinical translation of circulating analytes. In contrast to the state of the art in combination methods, the presented method enables the isolation of different analyte classes from a single sample volume without negatively affecting the respective analytes. Specifically, the method described here allows the analyte class of extracellular vesicles to be separated from the analyte class of nucleic acids and each to be isolated from the sample matrix without significantly destroying, damaging, or minimizing either analyte class. The resulting high-quality isolates of the respective analyte classes enable accurate and reproducible analyses at the single and multi-analyte level to increase sensitivity and specificity without an undesirable increase in sample volume.

[0070] Referring to the Figures 5A and 5B Examples of centrifugal microfluidic systems that use or comprise a fluidic module as described herein will now be described. In other words, the fluidic module can be used in the systems in the Figures 5A and 5B any of the fluidic modules described herein.

[0071] Fig. 5Ashows a device with a fluidic module 110 in the form of a rotating body, which has a substrate 112 and a cover 114. The substrate 112 and the cover 114 can be circular in plan view, with a central opening through which the rotating body 110 can be attached to a rotating part 118 of a drive device 120 via a conventional fastening device 116. The rotating part 118 is rotatably mounted on a stationary part 122 of the drive device 120. The drive device 120 can be, for example, a conventional centrifuge, which can have an adjustable rotational speed, or a CD or DVD drive. A control device 124 can be provided, which is designed to control the drive device 120 in order to subject the rotating body 110 to a rotation or to rotations at different rotational frequencies.The control device 124 can, as will be apparent to those skilled in the art, be implemented, for example, by an appropriately programmed computing device or a user-specific integrated circuit. The control device 124 can further be configured to control the drive device 120 in response to manual inputs from a user in order to effect the required rotations of the rotating body. In any case, the control device 124 can be configured to control the drive device 120 in order to impart the required rotation to the rotating body in order to implement embodiments of the invention as described herein. A conventional centrifuge with only one direction of rotation can be used as the drive device 120.

[0072] The rotating body 110 has the required fluidic structures. The required fluidic structures can be formed by cavities and channels in the cover 114, the substrate 112, or in the substrate 112 and the cover 114. In embodiments, for example, fluidic structures can be depicted in the substrate 112, while fill openings and vent openings are formed in the cover 114. In embodiments, the structured substrate (including fill openings and vent openings) is arranged on top and the cover is arranged on the bottom.

[0073] With an alternative in Fig. 5BIn the embodiment shown, fluidic modules 132 are inserted into a rotor 130 and, together with the rotor 130, form the rotating body 110. The fluidic modules 132 can each have a substrate and a cover, in which corresponding fluidic structures can be formed. The rotating body 110 formed by the rotor 130 and the fluidic modules 132 can, in turn, be subjected to rotation by a drive device 120, which is controlled by the control device 124.

[0074] In the Figures 5A and 5B The center of rotation around which the fluidic module or the rotation body can rotate is again denoted by R.

[0075] The Figures 5A and 5BThe device shown represents a processing device designed to impart the rotations required for carrying out the methods described herein to the fluidic module. The processing device may comprise one or more stationary or movable magnets 140 designed to promote mixing of particles with the residual liquid in the second isolation chamber. For this purpose, the magnets 140 may be mounted at a suitable location on the stationary part of the drive device 120, which passes through the second isolation chamber, so that its magnetic field can act on magnetizable particles in the second isolation chamber.

[0076] In embodiments of the invention, the fluidic module or the rotating body comprising the fluidic structures can be formed from any suitable material, for example a plastic such as PMMA (polymethyl methacrylate), PC (polycarbonate), PVC (polyvinyl chloride) or PDMS (polydimethylsiloxane), glass or the like. The rotating body 110 can be considered a centrifugal microfluidic platform. In preferred embodiments, the fluidic module or the rotating body can be formed from a thermoplastic such as PP (polypropylene), PC, COP (cyclic olefin polymer), COC (cyclo olefin copolymer) or PS (polystyrene).

[0077] The fluidic modules described herein thus enable multiple analyte classes to be isolated from an identical sample volume and removed from the process as separate isolates. Previously, to generate multiple isolates each containing only one analyte class, either the sample was fractionated into multiple subvolumes, resulting in a loss of sensitivity, or a larger sample volume was fractionated to achieve the same sensitivity. However, this is not feasible in practice due to patient burden or limited sample resources. There is no known technology in centrifugal microfluidics that offers all the principles required to isolate different analyte classes (centrifugation, supernatant removal, single- and multi-stage filtration, and solid-phase isolation) and enables their combination in integrated, automated process chains.In particular, it was surprisingly discovered that the individual steps of the process chain presented here, namely EV filtration and cfNA solid-phase isolation, as well as various optional steps, do not negatively influence each other to any significant degree. For example, it was surprisingly discovered that EV purification by filtration does not result in a significant loss of cfNA due to absorption at the filter surface or in the filter volume.

[0078] Examples of the present disclosure provide a rotating body in which one or more series-connected filter structures are integrated for filtering, through which fluid can flow using centrifugal force and whose filter pores have a diameter between 20 nm and 200 nm, and in which a collecting structure for the flow is provided. The collecting structure can contain an isolation structure for mixing the flow with a buffer and subsequently bringing the buffer-flow mixture into contact with an (active) surface in order to bind nucleic acids to this surface. Furthermore, the rotating body can have an integrated further fluidic structure for collecting the buffer-flow mixture from the separation structure in the further fluidic structure and also for collecting at least one washing buffer that washes the (active) surface. The (active) surface is formed from magnetizable particles.A magnet is integrated into the device to drive the rotation. This magnet can be movable and can assist in mixing the beads with the liquid. In some examples, the active surface can be a silica membrane.

[0079] In examples, the second insulation structure, as described herein, may be a separation structure as described in DE 10 2018 219 091 A1. In examples, the first and second insulation structures may be formed by insulation structures as are known per se and described above.

[0080] It goes without saying that chambers described herein, such as isolation chambers and fluid chambers, may each have multiple chamber sections or may be formed by multiple chambers. Chambers, such as fluid chambers or isolation chambers, that are connected via a fluid line do not have to be directly connected via the fluid line; rather, additional fluid chambers may be arranged between them.

[0081] Although features of the invention have been described in each case with reference to device features or method features, it is obvious to those skilled in the art that corresponding features can also be part of a method or device. Thus, the device can be configured to perform corresponding method steps, and the respective functionality of the device can represent corresponding method steps.

[0082] In the foregoing Detailed Description, various features have been grouped together in examples in order to streamline the disclosure. This manner of disclosure should not be interpreted as intending that the claimed examples include more features than are expressly recited in each claim. Rather, as the following claims reflect, the subject matter may lie in fewer than all of the features of a single disclosed example. Accordingly, the following claims are hereby incorporated into the Detailed Description, with each claim being capable of standing as its own separate example.While each claim may stand as its own separate example, it should be noted that although dependent claims in the claims refer to a specific combination with one or more other claims, other examples also include a combination of dependent claims with the subject matter of any other dependent claim or a combination of any feature with other dependent or independent claims. Such combinations are intended to be encompassed unless it is stated that a specific combination is not intended. Furthermore, it is intended to encompass a combination of features of a claim with any other independent claim, even if that claim is not directly dependent on the independent claim.

[0083] The examples described above are merely illustrative of the principles of the present disclosure. It is understood that modifications and variations of the arrangements and details described will be apparent to those skilled in the art. Therefore, it is intended that the disclosure be limited only by the appended claims and not by the specific details set forth for the purpose of describing and explaining the examples.

Claims

1. A method for isolating analytes of a first analyte class, which are extracellular vesicles and / or circulating tumor cells, and analytes of a second analyte class, which are cell-free nucleic acids, from the same sample volume of a biological sample in a centrifugal microfluidic system, comprising a fluidics module (10, 110, 132) having a first isolation chamber (12, 85) containing a first isolation structure (16, 40) and a second isolation chamber (14, 88) containing a second isolation structure (18, 44), comprising: guiding the sample volume into the first isolation chamber (12, 85) so that the analytes of the first analyte class are retained by the first isolation structure (16, 40), while the analytes of the second analyte class are not retained by the first isolation structure (16, 40) and pass through the first isolation structure (16, 40) as part of a residual liquid; guiding the residual liquid into the second isolation chamber (14, 88) so that the analytes of the second analyte class are retained by the second isolation structure (18, 44); and separating the analytes of the first analyte class from the first isolation structure (16, 40) and separating the analytes of the second analyte class from the second isolation structure (18, 44) to provide the analytes of the first analyte class and the analytes of the second analyte class separately from each other for subsequent analysis, wherein the first isolation structure (16, 40) is a filter having a pore size in a range from 20 nanometers to 200 nanometers, preferably in a range from 20 nanometers to 45 nanometers, wherein the second isolation structure (18, 44) is a surface for binding analytes of the second analyte class, wherein the surface is formed by particles, and wherein the particles are magnetizable, the method comprising rotating the fluidics module (10, 110, 132) to, by means of one or more stationary or movable magnets (140) of a processing apparatus, move the magnetizable particles in the second isolation chamber (14, 88) to assist mixing of the particles with the residual liquid, and / or support retaining the magnetizable particles in the second isolation chamber (14, 88) during transfer of analytes of the second analyte class into the second collection chamber.

2. The method according to claim 1, comprising washing the analytes of the first analyte class retained at the first isolation structure (16, 40) by means of a washing solution, eluting the analytes from the first isolation structure (16, 40) by means of an elution solution and transferring the analytes detached from the first isolation structure (16, 40) into a first collection chamber (32, 42, 87).

3. The method according to any one of claims 1 or 2, comprising mixing the sample volume with a binding buffer, bringing the resulting mixture into contact with the surface to bind the analytes of the second analyte class to the surface, washing the surface with the bound analytes using a wash buffer, eluting the analytes from the surface using an elution buffer, and transferring the analytes detached from the second isolation structure (18, 44) to a second collection chamber (34, 46, 90).

4. The method according to any one of claims 1 to 3, comprising, prior to guiding the sample volume into the first isolation chamber (12, 85), guiding the sample volume through an upstream filter (54) which is permeable to the analytes of the first analyte class and the analytes of the second analyte class in order to filter cells, cell fragments, particles and impurities from the sample volume.

5. The method according to any one of claims 1 to 4, comprising preparing the biological sample, wherein preparing comprises one or more of the following processes: performing blood-plasma separation of the biological sample to obtain plasma from which the sample volume is recovered; performing blood-plasma separation of a coagulated biological sample to obtain serum from which the sample volume is recovered; performing thrombocyte isolation of the plasma to obtain thrombocyte-poor plasma from which the sample volume is recovered; performing thrombocyte isolation of the plasma to obtain thrombocyte-rich plasma from which the sample volume is recovered; performing enzymatic digestion to decompose proteins and protein aggregates in the biological sample to recover the sample volume; and performing liquefaction and / or homogenization of the biological sample to recover the sample volume.

6. A centrifugal microfluidic device comprising a fluidics module (10, 110, 132) for isolating analytes of a first analyte class, which are extracellular vesicles and / or circulating tumor cells, and analytes of a second analyte class, which are cell-free nucleic acids, from the same sample volume of a biological sample in a centrifugal microfluidic system, and a processing apparatus configured to subject the fluidics module to rotation, the fluidics module comprising: a first isolation chamber (12, 85) containing a first isolation structure (16, 40); a second isolation chamber (14, 88) containing a second isolation structure (18, 44); a first fluid line (22, 47) for guiding the sample volume into the first isolation chamber (12, 85) so that the analytes of the first analyte class are retained by the first isolation structure (16, 40), while the analytes of the second analyte class are not retained by the first isolation structure (16, 40) and pass through the first isolation structure (16, 40) as part of a residual liquid; a second fluid line (24, 48) for guiding the residual liquid into the second isolation chamber (14, 88) so that the analytes of the second analyte class are retained by the second isolation structure (18, 44); a first collection chamber (32, 42, 87) connected to the first isolation chamber (12, 85) for receiving analytes of the first analyte class detached from the first isolation structure (16, 40); and a second collection chamber (34, 46, 90) connected to the second isolation chamber (14, 88) for receiving analytes of the second analyte class detached from the second isolation structure (18, 44) or for receiving the second isolation structure from which the analytes of the second analyte class have been detached, wherein the first isolation structure (16, 40) is a filter having a pore size in a range from 20 nanometers to 200 nanometers, preferably in a range from 20 nanometers to 45 nanometers, wherein the second isolation structure (18, 44) is a surface for binding analytes of the second analyte class, and wherein the surface is formed by magnetizable particles, wherein the processing apparatus comprises one or more stationary or movable magnets which are arranged outside the second isolation chamber (14, 88) in order to support mixing of magnetizable particles with the filtrate during rotation of the fluidics module (10, 110, 132) and / or in order to support retention of the magnetizable particles in the second isolation chamber (14, 88) during transfer of analytes of the second analyte class into the second collection chamber.

7. The centrifugal microfluidic device according to claim 6, wherein the fluidics module (10, 110, 132) comprises at least one filter (54) fluidically connected to the input side of the first isolation chamber (12, 85), which is permeable to the analytes of the first analyte class and the analytes of the second analyte class and which is configured to filter cells, cell fragments, particles and impurities from the sample volume.