Microfluidic method for preparing a sequencing library for nucleic-acid-containing cells, in particular circulating tumour cells, with particles

Abstract

The invention relates to a method (500) for microfluidically preparing a sequencing library for nucleic-acid-containing cells (40), in particular for circulating tumour cells (40), wherein particles (50) are added to a liquid (10) comprising nucleic-acid-containing cells (40), in particular circulating tumour cells (40), wherein the particles (50) in each case comprise at least one cell-binding entity (51), in particular at least one anti-EpCAM antibody, and one or more sequencing entities (52), preferably an oligonucleotide, in particular a primer and / or an enzyme, in particular a polymerase, wherein after the addition (504), preferably a separation (505) of at least some of those cells (40) which have each bound one or more of the particles (50) via the cell-binding entity (51) is carried out for a subsequent creation of a sequencing library.

Description

[0001] Description

[0002] title

[0003] Microfluidic method for preparing a sequencing library for nucleic acid-containing cells, in particular circulating tumor cells, with particles

[0004] State of the art

[0005] CTCs (short for circulating tumor cells) are circulating tumor cells released into the patient's bloodstream from a primary tumor or metastases from a primary tumor. They play a crucial role in tumor metastasis and are therefore of high diagnostic value and predictive of the further progression of the disease. In the simplest case, the number of CTCs per unit volume of blood, for example, per milliliter (ml) of blood, is determined, the so-called CTC count. Traditionally, various enrichment or depletion methods are used to increase the relative number of CTCs compared to the background.The CTCs are then detected by specifically labeling the cells, for example using one or more types of fluorescently labeled antibodies directed against specific tumor markers such as EpCAM (epithelial cell adhesion molecule on the cell surface) and / or HER2 (human endothelial growth factor receptor 2), etc., as described, for example, in D. Lin et al., “Circulating tumor cells: biology and clinical significance,” Signal Transduction and Targeted Therapy, Vol. 6, No. 1, 2021. doi: 10.1038 / s41392-021-00817-8. https: / / www.nature.com / articles / s41392-021 ■ 00817-8.

[0006] The CTC count alone is highly informative, for example, regarding treatment response, and can therefore be used for therapy monitoring, which is particularly useful in the case of adjuvant therapies (measure-control-regulate rather than "flying blind"). An increase in the CTC count is particularly indicative of a lack of response or failure of the therapy used and preferably a reason for a change in therapy.

[0007] The determination of the CTC count can, for example, be carried out using isolation-free methods which, after or parallel to erythrocyte lysis and sedimentation of all nucleated cells into a microstructured silicon substrate, can be specifically stained using various reagents (for example, a selection of fluorescently labeled antibodies with different antigen targets) and thus optically quantified compared to non-CTCs, as described, for example, in the published patent application DE 10 2021 203 897 A1.

[0008] As a basis for therapy changes, the quantified CTCs can be subjected to more detailed follow-up analyses, for example with regard to characteristics such as genetic alterations (mutations, methylation patterns, etc.), which in turn can represent therapeutic targets and therefore provide clues to the therapies to be used. See, for example, Z. Habli et al., “Circulating Tumor Cell Detection Technologies and Clinical Utility: Challenges and Opportunities,” Cancers, Vol. 12, No. 7, p. 1930, 2020. doi: 10.3390 / cancers12071930. https: / / www.mdpi.com / 2072-6694 / 12 / 7 / 1930.

[0009] Disclosure of the invention

[0010] Advantages of the invention

[0011] Against this background, the invention relates to a method for the microfluidic preparation of a sequencing library for nucleic acid-containing cells, in particular for circulating tumor cells. Particles are added to a fluid comprising nucleic acid-containing cells, wherein the particles each comprise at least one cell-binding entity and one or more sequencing entities.

[0012] Sequencing library preparation refers to preparatory measures, particularly the provision of entities such as reagents, for the creation of a sequencing library. Sequencing library preparation, also referred to as library preparation, is a component of next-generation nucleic acid sequencing, a process for determining the nucleotide sequence in a DNA or RNA molecule using next-generation sequencing (NGS) methods.

[0013] The particles can be spherical particles, called "beads," commonly used in microfluidics, such as magnetic or ferromagnetic beads. For example, these particles can be selected so that their diameter is 3 to 1000 times smaller than a typical cell diameter, for example, a diameter between 10 nanometers (nm) and 5 micrometers (pm).

[0014] The liquid may, in particular, comprise a body fluid, preferably blood. The blood may be whole blood. The blood may have been obtained as a sample from a human or animal, particularly from a blood sample taken immediately beforehand. The liquid may also comprise other body fluids, for example, urine, stool, or a smear.

[0015] Nucleic acid-containing cells are understood to be cells of a living being, in particular a mammal, that contain nucleic acids, especially DNA or RNA, especially in a cell nucleus. These nucleic acid-containing cells are preferably circulating tumor cells, abbreviated to CTCs (circulating tumor cells) or CETCs (circulating epithelial tumor cells).

[0016] A cell-binding entity is understood in particular to be an entity or species that can bind to the surface of a cell, preferably specifically to a given cell type, for example, to a cell of epithelial origin. This can in particular be a molecule, in particular a protein, preferably an antibody. The cell-binding entity preferably binds specifically to given nucleic acid-containing cells, which are also referred to below as target cells. For example, the cell-binding entity comprises an anti-EpCAM antibody (https: / / www.antikoerper-online.de / antibody / 349703 / anti- Epithelial+Cell+Adhesion+Molecule+EPCAM+antibody / ), i.e., an antibody that specifically binds to the epithelial cell adhesion molecule (EpCAM for short), or an antibody that binds to another, e.g., mesenchymal, surface marker such as cell surface vimentin (CSV), or an scFv fragment, scFv antibody, Fab fragment, a DARPin, or an aptamer, each of which preferably also binds to EpCAM or another desired surface marker. A "bond" here and in the following refers in particular to a physical bond between two binding partners, here an entity and a cell, whereby the bond can be reversible, for example, via van der Waals forces.

[0017] A sequencing entity is understood to mean, in particular, an entity for creating a sequencing library, preferably an essential component for library preparation, for example an oligonucleotide, in particular a primer, or an enzyme, in particular a polymerase, for use in preparing or carrying out sequencing. A universal selection marker such as biotin is preferably bound to the sequencing entity for selective isolation of the sequencing entities together with the respective particle and the cell bound thereto. The sequencing entity is, in particular, reversibly bound to the respective particle. For this purpose, the sequencing entity can be bound to the particle in particular via an antibody, via a direct bond or via an indirect bond, in particular via the cell-binding entity, for example via an antibody of the cell-binding entity.The sequencing entity may be bound to an antibody, in particular an antibody which is specific for the cell-binding entity, in particular in the case of the cell-binding entity being designed as an antibody.

[0018] The invention advantageously enables simple and reliable preparation for sequencing nucleic acids from the cells contained in a sample using a microfluidic method. By binding the particles, which already contain one or more entities for creating a sequencing library, to the cells, the components required for sequencing are brought into practically immediate spatial proximity, facilitating subsequent sequencing. By indirectly binding the sequencing entities to the cells to be sequenced via the particles, this spatial proximity can advantageously be maintained in a well-defined manner until subsequent sequencing.

[0019] In a step following particle addition, at least some of the nucleic acid-containing cells, each of which has bound one or more of the particles via the cell-binding entity, can be separated for subsequent creation of a sequencing library. The separation of the cells can be understood, in particular, as a removal of the cells bound via the particles from other components of the liquid or sample, in particular an accumulation or concentration of these bound cells in a spatial region of the liquid or a separation or removal of the bound cells from the liquid. Except in special embodiments, complete separation or complete removal of the cells from the liquid is not necessary.The solution according to the invention advantageously allows for selective library preparation, which, when using a cell-specific cell-binding entity, is selective for selected cells, in particular CTCs, and thus does not occur for the usually dominant cellular background (in particular non-CTC cells). The invention thus offers the possibility of selectively genetically examining individual target cells against a high background using the described particles. The invention thus also relates to a particle with at least one cell-binding entity, in particular at least one anti-EpCAM antibody, and one or more sequencing entities, preferably an oligonucleotide, in particular a primer, and / or an enzyme, in particular a polymerase, for sequencing.The invention advantageously further automates essential steps of library preparation, which supports acceleration and scalability of sequencing while reducing the susceptibility to errors by reducing manual activities.

[0020] Furthermore, the invention generally facilitates the separation or isolation of the cells together with the sequencing entities from the sample, which can be achieved, for example, through the use of magnetic forces when using (ferro)magnetic or magnetizable substances as part of the particles, particularly in the case of magnetic or magnetizable beads as described above. Alternatively or additionally, as also described above, the particles can comprise universal selection markers such as biotin for the purposes of separation or isolation, for example by exploiting the streptavidin / biotin interaction. The selection markers can, for example, each be bound to a sequencing entity, in particular to a primer. Alternatively or additionally, selection markers can also be bound directly to the particles or to the cell-binding entity.

[0021] According to a particularly preferred development of the invention, the separation comprises sedimentation of the affected cells, i.e., the cells associated with the particles, in cavities in a surface of an analysis substrate. The cavities are formed as recesses on the surface for accommodating individual cells. For example, the analysis substrate is formed as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1. The cavities or microcavities can have widths and depths in the single- to double-digit micrometer range for accommodating at least one cell, for example for accommodating a maximum of five, preferably a maximum of three cells, in particular CTCs of a typical size of approximately 10 pm in diameter.At least some of the cavities can have a preferably individual cell barcode sequence, preferably in the form of a randomized nucleotide sequence, wherein the cell barcode sequence is preferably reversibly bound to an inner wall of the cavity. These cell barcode sequences can advantageously be incorporated into a replicated nucleic acid segment during processing of cells accommodated in the cavities for individualized identification.

[0022] For separation or sedimentation, the liquid can be placed above the surface after particle addition for a specified sedimentation time, for example, for a period of up to 40, up to 30, or up to 20 minutes, depending in particular on the amount of liquid and the geometric conditions of the wells and the analysis substrate, as well as the usable volume above the analysis substrate. This further development thus has the advantage that the separation of the cells can take place in well-defined local areas, namely in the wells of the analysis substrate, in which subsequent analysis, in particular further steps of library preparation and sequencing, can then also take place.Due to the different properties of cells and particles, particularly with regard to their volume and mass, the cells sediment significantly faster than the unbound particles (for example, 100 times faster for a particle radius 10 times smaller, since the sedimentation rate scales with the square of the particle radius according to the Stokes equation). Therefore, the majority of all unbound particles effectively remain suspended within the limited sedimentation time of, for example, 20 minutes and do not settle into the cavities at the bottom. This further development therefore also has the advantage that, depending on the selected sedimentation time, hardly any particles not bound to cells are found in the cavities.In contrast, sequencing entities bound to the particles are advantageously located almost exclusively in cavities that also contain cells specifically bound to the particles, so that library preparation and subsequent sequencing are performed only for the desired target cells. The invention also relates to a microfluidic kit comprising particles according to the invention and such an analysis substrate.

[0023] In a particularly advantageous embodiment, the particles comprise detection molecules, especially fluorophores. This allows for easy localization of the particles, especially in the respective cavities for counting or analyzing the sedimented cells.

[0024] According to an advantageous embodiment of the invention, the particles are added during or after a lysis of other cells, also called background cells, in the liquid. Background cells are understood to mean, in particular, cells that are not intended for further analysis, in particular sequencing, and in particular not target cells. This has the advantage that such potentially interfering cells are damaged or even destroyed and, preferably, the binding of the particles to the predetermined nucleic acid-containing cells, in particular the target cells, can occur more efficiently or more quickly. The background cells can be, in particular, red blood cells, which are lysed via the specific lysis. Lysis of the background cells, in particular the red

[0025] Blood cells, also advantageously contributes to preventing these cells from sedimenting into the wells of an analysis substrate used.

[0026] After the addition and preferably before the end of the sedimentation time, at least a portion of the liquid can be moved through a size-based filter, particularly in the device, for a separation of cells with bound particles from unbound particles.

[0027] After the sedimentation time has elapsed, a portion of the liquid located above the surface, including any remaining particles, is preferably removed. Furthermore, preferably subsequently, the surface can be rinsed and / or the cavities decanted, particularly to further remove remaining particles or sample residues, with a majority of the sedimented cells remaining in the cavities.

[0028] Subsequently, as described above, a count of the sedimented cells, in particular the sedimented CTCs (CTC count), can optionally be carried out, preferably a fluorescence-based readout upon excitation of the fluorophores preferably bound to the particles, as described above.

[0029] Preferably after rinsing or decanting, and preferably after an optional counting, further reagents are added for library preparation, which preferably takes place in the wells. Subsequently, a sealing fluid, particularly an oil, can be added to cover the surface of the analysis substrate and the wells, effectively fluidically separating the individual wells from each other, thus forming closed, individual reaction compartments.

[0030] Subsequently, at least one step of the preparation of the sequencing library, in particular an amplification reaction for the replication of nucleic acid segments, can be carried out in at least some of the wells into which cells with particles bound to them have been taken up.

[0031] The method according to the invention can preferably be carried out with a microfluidic cartridge, also called a lab-on-a-chip (LoC) cartridge. The invention thus also relates to a microfluidic cartridge comprising particles according to the invention or the microfluidic kit according to the invention, i.e., the particles according to the invention and an analysis substrate with cavities as described above. Brief Description of the Drawings

[0032] Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description

[0033] It shows

[0034] Figure 1 is a flowchart of an embodiment of the method according to the invention and

[0035] Figure 2 shows an embodiment of the device according to the invention,

[0036] Figures 3a-d show cross-sectional views through a chamber of the device at different times during the process and

[0037] Figures 4, 5 Embodiments of the particles according to the invention

[0038] Embodiments of the invention

[0039] The method according to the invention, the particles according to the invention and thus also the microfluidic kit according to the invention as well as the device according to the invention can be used particularly advantageously for the sequencing of CTCs in the context of the analysis of CTCs with next generation sequencing methods (NGS), because the mutation profile of CTCs can be comprehensively analyzed molecular genetically using NGS, in particular by reading out base sequences of individual nucleic acids in the form of amplicons, i.e. products of a PCR or other, for example, isothermal amplification.

[0040] An overall process for nucleic acid analysis using NGS can preferably comprise four parts: (i) nucleic acid isolation, (ii) sample preparation, in particular including the creation of a sequencing library (library preparation), (iii) sequencing, and finally (iv) bioinformatics analysis. The greatest challenge in the NGS process is the creation of the sequencing library, as this is associated with a high laboratory expenditure of time and is very diverse depending on the question (e.g., whole genome sequencing, transcriptome sequencing, targeted panel sequencing, etc.). The classic method for untargeted, whole genome shotgun (WGS) libraries (for whole genome sequencing) consists of four steps: (i) DNA fragmentation, (ii) end repair, (iii) adapter ligation with barcodes, and (iv) PCR enrichment. In a classic method for targeted panel sequencing, for example,A fifth step must be introduced to enrich the interesting gene regions to be sequenced compared to the non-interesting entire genome (usually via high multiplex PCR or pull-out using specific probes) (v). For transcriptome sequencing, for example, a sixth step is added to transcribe the RNA into DNA, the so-called cDNA synthesis (vi). Automation of these processes is currently only available to a limited extent, so the creation of a sequencing library consists of numerous steps that are carried out manually or with liquid handlers in the laboratory, as described, for example, in JF Hess et al., “Library preparation for next generation sequencing: A review of automation strategies,” Biotechnology advances, Early Access, doi: 10.1016 / j.biotechadv.2020.107537.In order to extend CTC count analysis to NGS, a highly scalable sample preparation method must be used that allows the necessary processing steps to be performed quickly and in significantly fewer reaction steps. It can be applied directly on the chip specifically for individual target cells, so that only the nucleic acids of CTCs are processed from the high background of non-CTCs. Furthermore, an isothermal reaction, which runs in a one-pot approach, is advantageous (saves time, reagents, and reaction chambers). A further challenge is the small amount of starting material for the nucleic acids. With the classic method, for a small number of cells, the entire genetic material must first be pre-amplified before the actual library preparation can begin, as described, for example, in S. Panelli, G. Damiani, L. Espen, G. Micheli, and V.Sgaramella, "Towards the analysis of the genomes of single cells: further characterization of the multiple displacement amplification," Gene, Early Access, doi: 10.1016 / j.gene.2006.01.032. The following exemplary embodiment is set in the context of nucleic acid analysis of nucleic acid-containing nucleated cells, in particular CTCs, using NGS, without limiting the application of the invention thereto.

[0041] Figure 2 schematically shows an embodiment of a device 100, which is used, for example, for the method 500, the method steps of which are shown as a flow chart in Figure 1. Preferably, the device 100 can be based on a microfluidic cartridge described in the published patent application DE 10 2021 203 897 A1.

[0042] According to a first step 501 of the method 500, a blood sample, for example comprising 100-200 microliters (μl) of whole blood from a particularly immediately preceding blood draw from a human or animal, is provided and, in a second step, introduced into an input chamber 101 of the device 100, which can then preferably be closed. For example, a number or concentration of CTCs is to be determined for this sample. Before introduction, a substance, for example plasmin, can be added to the sample to unmask the target cells, in particular CTCs, i.e., to expose structures on the surface of the target cells that can be detected, for example, using staining reagents.

[0043] In the input chamber 101 or in another chamber 110 of the device, hereinafter referred to as the processing chamber 110, the sample can be mixed in a second step 502 with further liquids 80 or substances 80, which can be stored in a pre-storage chamber 120 of the device 100. The liquids or substances are preferably substances for lysing erythrocytes, for example in the form of a commercially available erythrocyte lysis buffer (in particular containing ammonium chloride for minimal lysis effect on nucleated cells). The sample thus obtained with lysed erythrocytes is hereinafter also referred to as blood lysate. The substances 80 can also comprise staining reagents as color markers, in particular for CTCs, for example in the form of a staining solution.The device 130 also comprises a chamber 130 (hereinafter also referred to as substrate chamber) shown schematically in vertical cross-section in Figure 3a, wherein the chamber 130 has an analysis substrate 135 with recesses 137 on a surface 136 of the analysis substrate 135 for receiving and analyzing, in particular counting, biological cells. The analysis substrate 135 can, for example, be arranged on the bottom 138 of the chamber 130, embedded in the bottom 138, or connected to the chamber 130 in a recess in the bottom 138. Figure 3b shows a plan view of the chamber 130 and of the analysis substrate 135 with, for example, circularly defined cavities 137. The recesses 137 can preferably be formed as microcavities 137. The analysis substrate 135 can, for example, be designed as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1.For example, the chamber has dimensions of 13.45 x 13.45 x 0.64 mm with a volume of 100 pL, and the analysis substrate 135 has dimensions of 12.5 x 12.5 x 0.38 mm. The recesses 137 have, for example, widths or diameters and depths in the single- to double-digit micrometer range for each accommodating at least one cell, in particular a nucleated, preferably human cell, in particular CTC. Preferably, the cavities are designed to accommodate a maximum of three cells of a predetermined size, preferably three of the aforementioned cells, in particular CTCs. These microcavities 137 can be created in the silicon substrate using methods of semiconductor technology or microsystem technology, for example by plasma deep etching processes, in particular using the "Bosch-DRIE" process.The substrate chamber 130 has a first inlet 131 and a second inlet 132, wherein the two inlets 131, 132 are arranged in different walls of the chamber 130, in particular in opposite walls, so that the analysis substrate 135 is preferably located between the two inlets 131, 132.

[0044] In a third step 503, the blood lysate 10 is placed as a liquid phase above the surface of the analysis substrate 135 in the so-called headspace 133, for example, through the first inlet 131, so that at least some of the cells, in this example CTCs, can sediment into the recesses of the analysis substrate 135. In a fourth step 504, particles 50 in the form of microfluidic beads are added. As explained above, these particles 50 can, for example, have a diameter between 10 nanometers and 5 micrometers, such as the commercially available MACS® Beads or Dynabeads™. The particles 50 can preferably be stored upstream in the device, for example, in the pre-storage chamber 120 or in another chamber, for example, directly in the substrate chamber 130.As shown schematically in Figure 4a, a particle 50 comprises on its surface both at least one, preferably several, cell-marking species 51 (for example, an anti-EpCAM antibody, for example from mouse or goat, alternatively, for example, one or more scFvs or aptamers) that specifically mark only the CTCs, as well as one or more essential components 52 of the downstream library preparation (for example, primers, polymerase, ...) as entities for creating the sequencing library (hereinafter also referred to as sequencing entities 52), which are immobilized on the surface. The cell-marking species 51 acts as a cell-binding entity 51 for binding the respective particle to the cell 40.As shown in Figure 4b, preferably several particles 50 bind to a target cell 40, preferably at least some particles 50 each via multiple bonds, wherein the target cell 40 has at least one, preferably several, binding sites 41 for the specific binding of the cell-binding entity 51, in particular EpCAM in the case of CTCs as target cells 40. Furthermore, a universal selection marker 53, in particular biotin, which is preferably bound via one of the sequencing entities 52, in particular to a primer, enables the isolation of the finished library from the reaction mixture. This fourth step 504 can also be performed earlier, in particular parallel to the selective erythrocyte lysis in the second step 502 or before or during the placement of the blood lysate 10 in the headspace 133 in the third step 503.

[0045] The particles 50 bound to the target cells 40, in particular beads, sediment together with the target cells in the fifth step 505 within a predetermined period of time for the sedimentation (sedimentation time), which is for example 20 minutes, into the cavities located on the surface of the microstructured analysis substrate and are thus separated from the liquid 10 (the blood lysate) in the headspace 133 above the analysis substrate 135. Due to the different properties of cells and particles, the cells sediment significantly faster than the unbound particles (e.g. 100x with a 10x smaller radius of the particles, in particular beads, since the sedimentation speed scales according to the Stokes equation in the square of the particle radius), which is why the majority of all unbound particles remain in suspension within the limited sedimentation time and do not sediment into the cavities located at the bottom.The sedimentation and thus separation of the bound cells 40 can be assisted and, in particular, accelerated by a magnet 134 if the particles 50 comprise (ferro)magnetic material or are magnetizable, whereby it must be taken into account that both bound and unbound particles 50 are accelerated by the magnetic force. In particular, the particles can be magnetic beads. The magnet 134 can, for example, be arranged below the analysis substrate 135, as shown in Figure 3a. The magnet 134 can be a permanent magnet, which can be movably arranged, for example, to set different magnetic field strengths in the headspace 133 and in the cavities 137, or an electromagnet for adjusting the magnetic field strength.

[0046] After the sedimentation time has elapsed, the blood lysate is displaced from the headspace 133 and from the substrate chamber 130 in a sixth step 506, for example, by a gas phase or a wash buffer, for example, through the second inlet 132 or back through the first inlet 131. In the process, the beads remaining in the area above the analysis substrate are removed. Individual unbound beads that have sedimented into a well without a target cell are washed out of the cavities during the subsequent rinsing / washing due to their significantly smaller size than cells, while the cells are retained by the structure of the cavities. Thus, residues of the blood lysate 10 remain in the recesses 137 or microcavities 137 together with the sedimented cells 40 due to the adhesion, capillary, and gravitational forces acting there.An optional advantageous embodiment of the invention provides that the device 100 has an additional size-based filter 139, for example a silica filter, which phase-separates the unbound particles 50 from the cells 40 by retaining the cells 40. The unbound particles are removed from the chamber 130, for example, via the second inlet 132 and are preferably subsequently flushed in the opposite direction through the filter 139 in order to detach the retained cells 40 from the filter 139. This filter 139 is preferably used after the particles 50 have bound to the target cells 40 or, in the case of non-target cells, have not bound, and before sedimentation, so that no free beads are left in suspension on the analysis substrate.This results in various possible states of a cavity 137: (i) empty, (ii) occupied by non-target cells, (iii) occupied by target cell-particle complex(es), and (iv) occupied by both target cell-particle complex(es) and non-target cells. Due to the significantly smaller diameter of the particles compared to the target cells 40, many particles can bind to the cell surface (in the case of CTCs: surface area of ​​an average CTC: 300 pm). 2 , particle / bead diameter, for example, 1 pm). This serves, on the one hand, to amplify the signal. In a positive event, many particles 50 bind to a target cell 40. On the other hand, it provides internal process control. False-positive events can be identified because in these cases only a few particles bind nonspecifically.

[0047] In a preferred seventh step 507 of the method 500, a second liquid phase, in particular a buffer, is placed above the upper side 136 of the sedimentation substrate 135, so that the residues of the blood lysate from the recesses 137 can diffuse into the second liquid phase and are thus removed from the recesses 137 or at least reduced in quantity, which can also be referred to as microfluidic decanting. In other words, such diffuse mixing effectively replaces the remaining blood lysate in the recesses 137 by strongly diluting it with the second liquid phase. When using a transparent second liquid phase, transparency now also prevails in the recesses 137 and in the headspace 133, which also enables undisturbed optical analysis, in particular counting, of the sedimented target cells 40.The second liquid phase is preferably, as preferably all liquid phases, moved, in particular pumped, through the first inlet 131 into the chamber 130.

[0048] The second liquid phase or, alternatively, a further liquid phase introduced into the chamber after removal of the second liquid phase can preferably also contain reagents, in the present example in particular further, preferably all still missing reagents for the library preparation, in particular reagents for nucleic acid amplification as part of the library preparation. The introduction of this phase 20 with these reagents 60 is shown in Figure 3c, wherein the reagents 60 are shown significantly enlarged compared to the cavities 137 and the target cells 40 for better illustration. The reagents 60 can also be stored upstream in the device 130, for example likewise in a pre-storage chamber, in particular for mixing as required with the preferably also upstream second or further phase.According to a particular embodiment, two or more of these reagents may also already be mixed with each other and / or pre-mixed with the second or further liquid phase.

[0049] Furthermore, depending on the application, the second liquid phase or a further liquid phase can comprise further reagents 70 (also shown relatively greatly enlarged in Figure 3c) for treating or analyzing the sedimented cells, for example for fixation (e.g., 4% formaldehyde and / or others) and permeabilization (e.g., 0.5% Triton X-100 and / or others) and / or one or more different markers for labeling the captured cells 40, for example, fluorescently labeled antibodies for intracellular or surface antigens such as cytokeratin, vimentin, CD45, or others. Alternatively or additionally, cell-binding entities 51 immobilized on the particles 50 could additionally carry molecules 54 for detecting the labeled cells (as shown by way of example for a cell-binding entity 51 in Figure 4a), so that a distinction between target cells and non-target cells is possible.These could, in particular, be fluorophores 54 for optical detection or redox labels 54 for electrical detection. Another possibility is the use of intrinsically colored particles 50, such as the commercially available Fluorsebrite® BB Carboxylate Microspheres 0.5 pm. Thus, in an eighth step 508, a fluorescence-based count of the cells 40 collected in the wells 137 can now be performed, in particular the CTC count described above, to obtain a measure of the number of CTCs contained in the sample.

[0050] As discussed above, such quantification of the target cells present, especially CTCs, provides significant information, particularly regarding treatment response, and can be advantageously used for therapy monitoring. Additionally or alternatively, library preparation can be performed, as described below, to supplement the information provided by the target cell / CTC count using the orthogonal NGS method.

[0051] In a ninth step 509, which is thus alternative to or subsequent to the eighth step 508, a sealing fluid 30, in particular oil, for example a mineral oil, is added to the chamber 130, which, as shown in Figure 3d, can fill the entire headspace 133 and, furthermore, the entire chamber 130. The sealing fluid 30 serves to cover the surface 136 of the analysis substrate 135 and thus the cavities 137, whereby the individual cavities 137 are effectively fluidically separated from one another and can now be considered as closed, individual reaction compartments, as shown in Figure 3d (where the reagents 60, 70 accommodated in the cavities 137, in particular for the library preparation, are not shown).Since only the cavities containing the target cell-particle complex contain all the necessary reagents of the nucleic acid amplification mixture, the library preparation performed in the subsequent tenth step 510 can only take place in such cavities. This means that only the genetic information of the target cells can be amplified. For this purpose, the device 100 or an analysis device that processes the device 100 can have a heating element 150, for example a Peltier element or a resistance heater, for setting a temperature, in particular a temperature that can be changed over time, in the analysis substrate 135 and in particular in the cavities 137 for carrying out a two- or three-step PCR or an isothermal amplification reaction, and preferably for lysing the target cells to release the nucleic acids to be amplified.The heating element 150 can, for example, border on the analysis substrate via a wall, in particular the floor or a side wall of the chamber 130, or directly, as shown in Figure 3d. As already explained above, the cavities 137 are preferably shaped such that a maximum of three target cells are found in one cavity. Therefore, a condition (iv) with a maximum of two non-CTCs cannot be completely ruled out, and amplification could also occur here. However, a ratio of two non-CTCs to one CTC is tolerable by the method, so that this scenario still delivers meaningful results. By appropriately designing the analysis substrate, in particular with regard to the size and number of cavities, as well as by the concentration of target cells in the sample, multiple occupancy of cavities can be ruled out, which can be modeled using Poisson statistics.

[0052] After the library preparation, in particular after the amplification reaction, the products of the library preparation can be combined in an eleventh step 511. For this purpose, the sealing fluid 30 can first be removed. Alternatively, the cavities 137 can have further openings, in particular at the bottom of the cavities 137, which can preferably be closed by a movable membrane as needed, in particular during processing in the cavities.

[0053] Figure 5 shows an exemplary embodiment of a further embodiment of the particle 50 according to the invention. In contrast to the particle 50 shown in Figure 4a, one or preferably several sequencing entities 52 are each bound to a cell-binding entity 51, as shown by way of example in Figure 5. For this purpose, the cell-binding entity 51 has at least one or more binding sites for the sequencing entities 52. The cell-binding entity 51 can preferably also be linked to a detection label 54 (for example a fluorophore or redox label) in this embodiment, as shown by way of example for an entity 51 in Figure 5. For example, the cell-binding entity 51 is an antibody, in particular a primary anti-EpCAM antibody from a mouse, goat or another animal.Instead of direct binding of the one or more sequencing entities 52 to the respective cell-binding entity 51, the sequencing entity 52, as shown in Figure 5b, can also have an antibody 55 for binding to the antibody of the cell-binding entity 51, for example, an anti-animal secondary antibody, for example, an anti-mouse or anti-goat secondary antibody, for example from the goat or mouse, which specifically binds only to the cell-binding primary antibody. The primary antibody has at least one, but ideally several, binding sites for the secondary antibody, thereby enabling signal amplification and thus improved performance of the downstream library preparation.

Claims

Claims 1 . Method (500) for the microfluidic preparation of a sequencing library for nucleic acid-containing cells (40), in particular for circulating tumor cells (40), wherein particles (50) are added to a liquid (10) comprising nucleic acid-containing cells (40), in particular circulating tumor cells (40), wherein the particles (50) each comprise at least one cell-binding entity (51), in particular at least one anti-EpCAM antibody, and one or more sequencing entities (52), preferably an oligonucleotide, in particular a primer, and / or an enzyme, in particular a polymerase, wherein after the addition (504) preferably a secretion (505) of at least some of those cells (40) which have each bound one or more of the particles (50) via the cell-binding entity (51) takes place for a subsequent creation of a sequencing library.

2. The method (500) according to claim 1, wherein the separation (505) comprises a sedimentation (505) of the cells (40) in cavities (137) in a surface of an analysis substrate (135) during a sedimentation time 3. Method (500) according to claim 1 or 2, wherein the addition (504) takes place during or after a lysis (503) of background cells, in particular red blood cells 4. Method (500) according to one of the preceding claims, wherein in at least some of the particles (50) at least one sequencing entity (52) is bound to the cell-binding entity (51) of the respective particle (50), for example via an antibody of the cell-binding entity (51) and / or via an antibody (55) associated with the sequencing entity (52).

5. Method (500) according to one of the preceding claims, wherein the particles (50) comprise detection molecules (54), in particular fluorophores, and / or preferably universal selection markers (53), in particular biotin (53), bound to the sequencing entities (52), in particular primers (52).

6. The method (500) according to any one of the preceding claims, wherein at least some of the sequencing entities (51) are bound to the cell-binding entities (51) and wherein preferably the respective sequencing entity (52) or the cell-binding entity (51) has a bound detection molecule (54) 7. The method (500) according to any one of the preceding claims, wherein at least some of the sequencing entities (52) each comprise an entity (51), in particular an antibody, for binding to the cell-marking / cell-binding species.

8. The method (500) according to any one of claims 2 to 7, wherein after the addition (504) and preferably before the end of the sedimentation time (505), at least a portion of the liquid is moved through a size-based filter (139) for a separation of cells (40) with bound particles (50) from unbound particles (50).

9. Method (500) according to one of claims 2 to 8, wherein after the addition (504) and after the expiry of the sedimentation time (505), a removal (506) of a part of the liquid (10) located above the surface (136) with particles (50) remaining therein and preferably a counting (508) of the cells (40) sedimented in the cavities takes place.

10. The method (500) according to claim 9, wherein after the removal (506) and preferably before the counting (508) a decantation (507) of the cavities (137) takes place, in particular for the further removal of remaining particles, wherein a majority of the sedimented cells (40) remain in the cavities (137).

11. Method (500) according to one of the preceding claims, wherein after the removal (506) and preferably after the decantation (507) an addition of further reagents for a library preparation, preferably followed by an addition (509) of a sealing fluid (30) to cover the cavities (137).

12. The method (500) according to any one of claims 2 to 11, wherein a sealing fluid (30) is added to cover the surface (136) of the analysis substrate (135) and the cavities (137), and preferably a step of creating the sequencing library, in particular an amplification reaction for replicating nucleic acid segments, is carried out in at least some of the cavities (137) into which cells (40) with particles (50) bound thereto have been taken up.

13. The method (500) according to any one of the preceding claims, wherein at least some of the cavities (137) comprise a preferably individual cell barcode sequence, preferably in the form of a randomized nucleotide sequence, wherein the cell barcode sequence is preferably reversibly bound to an inner wall of the cavity (137).

14. Particles (50) for the microfluidic preparation of a sequencing library, in particular for a method according to one of the preceding claims, with at least one cell-binding entity (51), in particular at least one anti-EpCAM antibody, and one or more sequencing entities (52), preferably an oligonucleotide, in particular a primer, and / or an enzyme, in particular a polymerase, for sequencing.

15. A microfluidic kit comprising a plurality of particles (50) according to claim 14 and an analysis substrate (135) with cavities (137) for receiving at least one nucleic acid-containing cell with at least one attached particle (50).

16. Microfluidic device (100), in particular microfluidic cartridge (100) comprising particles (50) according to claim 14 or a kit according to claim 15.

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