Combined cell-specific labelling and enrichment of biomarkers

Abstract

The invention relates to a microfluidic method for the combined cell-specific labelling and enrichment of biomarkers in a reaction compartment, comprising the following method steps: a) providing a microfluidic device comprising at least one carrier substrate for receiving a sample fluid, wherein the carrier substrate comprises at least one microcavity (110), and further comprising at least one electrode arranged at or in the microcavity (110) to generate an electric field designed to trap a cell (510) and / or a primer particle (512) in the microcavity (110); b) loading the at least one microcavity (110) of the microfluidic device with a cell (510) and trapping it; c) loading the at least one microcavity (110) of the microfluidic device with a primer particle (512) comprising at least one primer population coupled thereto and trapping the primer particle (512), wherein steps b) and c) can also be carried out in reverse order; d) introducing an amplification mixture into the at least one microcavity (110); e) displacing the amplification mixture above the at least one microcavity by a non-aqueous phase, so that the microcavity (110) represents a closed reaction compartment; f) inducing cell lysing and primer (516, 517) releasing conditions; and, g) binding the at least one primer population (517) to biomarker regions of the DNA (539) and / or RNA (539) of the lysed cell (510a) for cell-specific labelling and amplification of a biomarker target region (530).

Description

[0001] Description

[0002] title

[0003] Combined cell-specific labeling and enrichment of biomarkers

[0004] The present invention relates to a microfluidic method for combined cell-specific labeling and enrichment of biomarkers, as well as to a control device configured to carry out and / or control the steps of the method, according to the preamble of the independent claims. The present invention further relates to a computer program.

[0005] State of the art

[0006] A biomarker is a measurable biological characteristic with prognostic or diagnostic significance. In molecular diagnostics, diseases can be detected based on nucleic acid biomarkers, and appropriate therapies can be initiated. These biomarkers can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) biomarkers.

[0007] The query of targeted, multiple biomarkers is of great value and is used, for example, in infection diagnostics, where different species are queried simultaneously, or in tumor diagnostics, where different mutation sites in the genome are queried simultaneously.

[0008] The number of these so-called biomarker targets varies depending on the assay, from a few dozen to several tens of thousands. The goal is always to specifically enrich these biomarker targets compared to the uninformative, but usually highly concentrated background, in order to make the readout of the biomarker targets simpler and more robust, for example, using quantitative real-time quantitative polymerase chain reaction (qPCR), digital droplet polymerase chain reaction (ddPCR), or parallel sequencing (next-generation sequencing, NGS). Two fundamentally different principles are primarily pursued for the enrichment of nucleic acids: multiplex PCR with specific primers or primer pairs, and hybridization-based positive selection with specific probes.However, these enrichment methods have in common that they involve numerous sequential process steps and can take several hours to a day. This makes them unsuitable for use at the point of care in a lab-on-a-chip environment. Furthermore, several nanograms of nucleic acids are required as starting material, which, for example, requires additional pre-amplification (whole genome amplification, WGA, and / or whole transcriptome amplification, WTA) for low-input sample material. This, in turn, requires additional process steps and time.

[0009] In numerous disciplines of biology, such as immunology, neurobiology, oncology, or stem cell and developmental biology, researchers are interested in analyzing as many nucleated cells (several hundred thousand to millions) of a heterogeneous cell population as possible at the single-cell level in the shortest possible time (hours to a few days). Single-cell analysis can be performed either molecularly, for example, at the DNA, RNA, and / or protein level, or functionally, for example, through cell cultures and / or drug testing.

[0010] Droplet-based, microtub-based, or valve-based microstructured substrates are known for spatially isolating cells. However, none of these systems meets the requirements for highly parallelized enrichment of DNA and / or RNA-encoded biomarkers with a clear molecular labeling of the target amplicons in just a few process steps, for example, to enable point-of-care analyses.

[0011] Disclosure of the invention

[0012] According to the invention, a microfluidic method for combined cell-specific labeling and enrichment of biomarkers in a reaction compartment is provided with the features of the independent patent claim.

[0013] A device with which the method according to the invention can be carried out, as well as a method for operating the same, are described in the as yet unpublished German application with the file number 102022203848.7, with a filing date of April 19, 2022, and a priority date of April 20, 2022. The content of this application is incorporated into the description by reference to this application. It should be noted that it is clearly evident that the features of this application contribute to solving the technical problem underlying the present invention.

[0014] The microfluidic method for combined cell-specific labeling and enrichment of biomarkers in a reaction compartment comprises at least steps a) - g).

[0015] In step a), a microfluidic device as described in the as yet unpublished application with the file number 102022203848.7 is provided. This device comprises at least one carrier substrate for receiving a sample fluid. The carrier substrate has at least one microcavity and furthermore at least one electrode arranged on or in the microcavity to generate an electric field configured to trap a cell, in particular a nucleated cell, and / or a primer particle in the microcavity.

[0016] For example, approximately 30,000 individually actuatable microcavities or reaction compartments per square centimeter can be integrated into this microfluidic device. These microcavities have, for example, a square base area and a cavity width of approximately 50 μm. The microcavities are arranged, for example, in a regular matrix. The microcavities can capture or repel individual cells and / or particles. Individual cells can sediment into a microcavity in a controlled manner. Furthermore, irrelevant cells can be removed from the microcavities, while relevant cells can be retained by using an electric field to create a dielectrophoresis cage (D EP cage) in the microcavity. This cage can be opened, closed, or switched off depending on the current strength, for example.

[0017] The DEP cages can be implemented using active components, such as transistors or memory elements. These are integrated within individual electrodes using Complementary Metal Oxide Semiconductor (CMOS) technology.

[0018] In step b), at least one microcavity of the microfluidic device is loaded with a cell, in particular a nucleated cell, and the cell is trapped within the microcavity. For this purpose, the DEP cage is closed by a voltage change. Other cells located outside the DEP cage can no longer enter it, while the cell inside the DEP cage is stably trapped. This ensures that only a single cell is located within the microcavity.

[0019] In step c), at least one microcavity of the microfluidic device is loaded with a primer particle (primer bead), and the primer particle is trapped in the microcavity. The primer particle comprises, for example, at least one primer population coupled to it. As soon as the primer particle has settled into the cell-filled microcavity, the DEP cage is closed by a voltage change. Additional primer particles located outside the DEP cage can no longer enter it, while the cell and the primer particle are stably trapped within the DEP cage. This ensures that only a single primer particle is present within the microcavity.

[0020] Steps b), c) and d) can also be carried out in any reversed order; in particular, steps b) and c) can be carried out in the reverse order.

[0021] In step d), an amplification mixture is introduced into the at least one microcavity. For this purpose, the components of the amplification mixture diffuse into the at least one microcavity.

[0022] The amplification mixture comprises a buffer compatible with its components, a polymerase, in particular an SD polymerase, such as Bst, Bst2.0, Bst3.0, EquiPhi, Phi29, Vent exo, and / or others. Furthermore, the amplification mixture optionally comprises a reverse transcriptase (RT), e.g., RTx, SSIV, and / or others. Furthermore, the amplification mixture comprises deoxyribonucleoside triphosphates (dNTPs) and / or analogues thereof. The dNTPs can be additionally modified. Furthermore, the amplification mixture can also contain components that positively influence the amplification reaction and / or cell lysis and / or primer release, for example. Such components include additives such as polyethylene glycol (PEG), dithiothreitol (DTT), detergents, or enzymes such as restriction enzymes or nicking enzymes.

[0023] In step e), the amplification mixture above the at least one microcavity is displaced by a non-aqueous phase, in particular by an oil phase or air, so that the microcavity is closed by the non-aqueous phase in the region of its opening and thus represents a closed reaction compartment. If the microfluidic device comprises multiple microcavities, all microcavities represent mutually isolated and closed reaction compartments, each of which contains a cell and a primer particle containing the amplification mixture.

[0024] In step f), conditions are created that lead to cell lysis and release of the primers from the primer particle. Such conditions are created, for example, by increasing the temperature, for example, to above 70°C for more than one minute, and / or by electroporation and / or by ultrasound, and / or by other conditions or reagents conducive to this reaction, for example, by introducing them into the amplification mixture.

[0025] In step g), the at least one primer population binds to biomarker regions of the DNA and / or RNA which were released from the cell during cell lysis, so that an amplification of the biomarker target region occurs and the biomarker target amplificate is cell-specifically labeled.

[0026] For amplification, DNA double strands and / or secondary and / or tertiary structures of RNA are denatured, for example, by applying appropriate temperatures, so that the DNA and / or RNA are present as single strands. In a further step, the primers anneal to the DNA and / or RNA, thereby initiating amplification of the corresponding DNA and / or RNA.

[0027] The advantage here is that the method according to the invention is an automated, lab-on-chip-compatible process with which DNA and / or RNA-encoded biomarkers can be enriched in a highly parallelized manner, and the biomarker target amplificate can be unambiguously assigned to its original cell via molecular cell barcode. The biomarker target amplificates are thus available for downstream detection methods, such as NGS analyses, within a few minutes to hours, making the method suitable for point-of-care analyses.

[0028] No additional equipment is necessary for this.

[0029] Another major advantage is that only a few process steps are required. For example, prior pre-amplification / WGA / WTA or prior pooling of several pre-sorted single cells is no longer necessary. This leads to a significant reduction in the required process time, reagents, costs, and work steps.

[0030] The process ensures unique labeling of each individual cell, thus achieving nearly 100% mating efficiency and nearly 100% loading efficiency. This means that each microcavity can be loaded with exactly one cell and exactly one primer particle. Furthermore, multiple process steps can be performed sequentially, as the microcavities can be loaded and unloaded multiple times.

[0031] The simultaneous enrichment of the relevant DNA and / or RNA biomarker target regions can be achieved with a degree of parallelization of, for example, 30,000 microcavities per cm 2 take place.

[0032] Another advantage is that the sample input volume is theoretically unlimited, allowing various samples to be input, such as blood or saliva. Furthermore, the method allows for downstream analysis of only those cells that are relevant, for example.

[0033] Another advantage is that the primer particle that has entered the microcavity is trapped in the microcavity together with the cell and no further cells or primer particles can enter the microcavity.

[0034] Further advantageous embodiments emerge from the subclaims.

[0035] In an advantageous embodiment, different populations of primers are reversibly coupled to the primer particle. The primer particle comprises, for example, a polystyrene, in particular in the form of a polystyrene bead. The different primer populations are, in particular, biomarker-specific forward primers (Specific Forward Primers, SFP), biomarker-specific reverse primers (Specific Reverse Primers, SRP), and / or random primers (Random Primers, RP).

[0036] The primer populations are coupled to the primer particle, for example, via base pairings that can be broken at specific temperatures, or via specific sequences or other chemical modifications in the base composition that can be broken, for example, by enzymes or light of a specific wavelength. This can be, for example, a cleavage site for a restriction enzyme or the base analogue dUTP for uracil-DNA glycosylase (UDG).

[0037] In a further advantageous embodiment, the SFP and SRP each comprise a selection marker coupled to a universal sequence, a unique cell barcode sequence and specific target sequences flanking the biomarker target regions in forward and reverse orientation.

[0038] Each primer particle contains a clonal unique cell barcode sequence (BCi- n) with a length of > 8 bases. The selection marker is, for example, biotin at the 5' end of the SFP or SRP. In a particularly advantageous embodiment, the selection marker is coupled to an internal, e.g., modified base of the universal sequence, whereby the universal sequence is extended toward the 5' end by a single-strand break sequence (nicking site), which induces a single-strand break via a corresponding nicking enzyme.

[0039] The selection marker enables subsequent positive selection of the biomarker target amplifications. The universal sequence is required, for example, for later amplification of a sequencing library or a known sequence with a function of choice, such as a restriction site, particularly for releasing the biomarker target amplification, for example from a capture molecule. Another advantage is that, by labeling the individual biomarker target amplifications with a cell barcode, they can be clearly assigned to the individual cells. Further analyses of a large number of cells, for example molecular genetic analyses using NGS, in which relevant biomarkers can be assigned to the individual cells, for example from isolated cells from tissue biopsies, can be of great benefit, for example, in the diagnostics and therapy monitoring of tumor patients.

[0040] Because the cell and the primer particle are trapped in the microcavity and no other cells or primer particles can enter the microcavity, unique single-cell labeling is possible, as each primer particle is equipped with a unique, molecular cell barcode.

[0041] In a further advantageous embodiment, several, in particular 2-4, SFPs and SRPs with different specific forward and reverse target sequences flanking the biomarker target region are used for each biomarker target region. These can also be used, for example, in different concentrations.

[0042] The advantage here is that, for example, with an increasing number of SFPs or SRPs per biomarker target region, not only the amplification rate but also the chances of binding of the SFPs or SRPs to the target region increase, or in another example, by adjusting the concentration of the SFPs or SRPs of individual, for example more difficult to amplify, biomarker target regions, a more uniform amplification can be achieved compared to other biomarker target regions.

[0043] Furthermore, in one embodiment, it is advantageous if SFP and SRP further comprise a unique molecular identifier (UMI) in the form of a randomized sequence. The UMI can be located, for example, between the universal sequence and the cell barcode sequence, or between the cell barcode sequence and the forward or reverse target sequence. The UMI is preferably >6 bases long.

[0044] In an advantageous embodiment, the primer particles are designed such that the SFP and SRP are already reversibly bound to the primer particles via the selection marker. In the case of a reversible connection, the SFP and SRP are, for example, via antibodies or terminal chemical modifications, such as biotin coupling. Alternatively, the SFP and SRP can also be reversibly bound to the primer particles via aptamers, temperature-stable groups, pH-sensitive groups, or light-cleavable groups such as o-nitrobenzyl.

[0045] In this embodiment, the random primers are added with the amplification mixture for amplification, particularly for ssMDA (described below). The advantage here is that the target amplification products are produced directly on the primer particle and can be captured by DEP forces after removal of the non-aqueous phase and purified directly in the microcavity. Thus, in this embodiment, no capture molecules are necessary, since the amplification of the biomarker target region occurs via the SFP and SRP bound to the primer particle. A selection marker can be omitted here.

[0046] A further advantage is that the biomarker target amplificates, immobilized on the primer particles held in the DEP cage, can be further processed in the microcavity if required, for example by adding additional primer particles or liquid reagents.

[0047] In a particularly advantageous embodiment, the universal sequence of the SFP and SRP is selected such that a direct linkage point in the form of a specific sequence is introduced for further amplification, in particular for the completion of a sequencing library.

[0048] The advantage here is that additional amplification or preparation of the purified biomarker target amplicons is possible using other sequencing technologies, for example from another manufacturer.

[0049] The necessary asymmetric design can be implemented, for example, such that all SFPs receive a first universal sequence and all SRPs receive a second universal sequence that differs from the first universal sequence. This way, only one highly standardized PCR, such as an index PCR, is required before the sequencing library can be sequenced. Flexible use of the universal sequences ensures that the method is compatible with downstream, preferred analytical methods, such as various NGS technologies for single-cell analysis.

[0050] In an advantageous embodiment, the amplification mixture promotes cell lysis. This can be achieved, for example, by a reagent that lyses the cells and releases nucleic acids, but does not inhibit the subsequent reaction or even promotes it, such as with a detergent or by changing the osmolarity.

[0051] The advantage here is that no separate process steps for cell lysis and mixing with the amplification mixture are required, and the respective reagents can also be stored premixed. This leads to time, reagent, and cost savings. Furthermore, the reduced steps minimize dilution effects and losses that would otherwise result from repeated rinsing. This results in increased efficiency.

[0052] In an alternative or additional embodiment, cell lysis occurs after introduction of the amplification mixture by brief heating, for example to >50 °C for >1 min. In a further alternative or additional embodiment, cell lysis occurs by increasing the pressure within the microcavities, for example by applying an overpressure.

[0053] In a particularly advantageous embodiment, the amplification is an isothermal amplification, in particular a semi-specific multiple displacement amplification (ssMDA). This method is also described in the as yet unpublished German application with the file number 102022211087.0.

[0054] The ssMDA offers the option of simultaneously enriching DNA and RNA from minimal amounts both genomically and transcriptomically and of preferentially amplifying and enriching individual biomarker-relevant regions, i.e. the biomarker target regions.

[0055] This is achieved by using, in addition to random primers, for example, RP6 with the sequence NNNNNN or RP9 with the sequence NNNNNNNNN, where N stands for one of the bases A, C, T, or G, specific forward and reverse primers for amplifying the biomarker target regions in ssMDA. These primers anneal to the DNA or RNA up to 3 kilobases (kb) upstream of the biomarker target region, and reverse primers anneal to the DNA or RNA up to 3 kb downstream of the biomarker target region. The SFPs and SRPs are additionally equipped with a selection marker, for example, biotin. A subsequent purification of the reaction, for example with a suitable capture molecule such as streptavidin, allows a direct enrichment of the specific biomarker target amplicons compared to the background amplicon produced in parallel.

[0056] Particularly preferred in ssMDA are single-strand break sites, where the SFP and SRP have a single-strand break cleavage site. This single-strand break site provides an additional amplification starting point for a polymerase after the single-strand break is generated by a suitably added single-strand break-inducing endonuclease (nicking enzyme). This allows biomarker target amplification to continue at these sites without further primer hybridization, thus increasing processivity.

[0057] During amplification, a single-strand break-inducing endonuclease is added to the polymerase. The endonuclease is preferably selected from the group consisting of Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.Bsml, Nt.BsmAI, Nt.BspQI, Nb.BsrDI, Nb.BssSI, Nt.BstNBI, Nb.BtsI, and Nt.CviPII.

[0058] The primers used in ssMDA are preferably primers that have at least one modification selected from the group consisting of LNA (Locked Nucleic Acid), MGB (Minor Groove Binder), C-5 propynyl deoxycytidine, C-5 propynyl deoxyuridine, aminoethyl phenoxazine deoxycytidine, 5-methyl deoxycytidine, 2-amino deoxyadenosine, trimethoxystilbene, pyrene, and spermine. ZNA primers (Zip Nucleic Acids) are particularly preferred. These are spermine-modified primers. These have the advantage of forming particularly temperature-stable hybridized nucleic acids with single-stranded nucleic acids (DNA and / or RNA) and allowing higher hybridization temperatures to be used without requiring sequence extension of the primers. In addition, they reduce self-hybridization of the primers when appropriate spermine loading is used.This is particularly advantageous when the primers used are short and the isothermal amplification is carried out at temperatures of at least 45°C.

[0059] The ssMDA occurs isothermally, for example, at temperatures between 40-72°C for more than 10 minutes.

[0060] For ssMDA, an SD polymerase is preferably used. The SD polymerase can, for example, be an enzyme mutant of the DNA polymerase of Bacillus subtilis phage Phi29, especially Equi Phi29, or of Thermococcus litoralis, especially Vent (exo-). The SD polymerase Vent (exo-) has the advantage of being stable and processive up to temperatures of 100°C. Therefore, it can be added before denaturing the DNA and / or RNA without being damaged by the high denaturation temperature. However, these SD polymerases lack RNA processivity.

[0061] The SD polymerase is preferably an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus. These SD polymerases exhibit not only processivity towards DNA but also processivity towards RNA. Particularly preferred enzyme mutants of the DNA polymerase of Bacillus stearothermophilus are selected from the group consisting of Bst, Bst 2.0, and Bst 3.0. Among these enzyme mutants, Bst 3.0 exhibits the highest RNA processivity, which is why Bst 3.0 is particularly preferred.

[0062] SD polymerase is a DNA polymerase suitable for isothermal amplification reactions and is already known for its high strand displacement activity in amplification reactions.

[0063] The use of SD polymerase has several advantages. It enables isothermal amplification. Furthermore, some SD polymerases tolerate high temperatures during isothermal amplification, which increases the reaction rate to such an extent that a significant reduction in reaction time is possible. Finally, some SD polymerases can use RNA as a template for amplification in addition to DNA.

[0064] Even if the SD polymerase is capable of processing RNA in addition to DNA, it is preferable to use at least one additional reverse transcriptase for ssMDA. This is particularly true for RTx or SSIV. In principle, however, any reverse transcriptase that processes under similar reaction conditions to the SD polymerase used is conceivable. This specifically supports the amplification of RNA, allowing the required amplification time to be shortened even further. If an SD polymerase is used that lacks RNA processivity, then the use of a reverse transcriptase is necessary to process RNA in addition to DNA.

[0065] For amplification using ssMDA, the DNA and / or RNA to be amplified is denatured, particularly by heating to a suitable temperature. In the case of thermophilic polymerases, such as Vent (exo) polymerase, denaturation preferably occurs at a temperature in the range of 75°C to 98°C, and most preferably at a temperature in the range of 80°C to 95°C. Heating is preferably carried out for a period of more than 10 seconds, particularly preferably for a period in the range of 30 seconds to 120 seconds.

[0066] In the case of non-thermophilic SD polymerases, such as Bst3.0, temperatures of 65-85°C are preferably used, and temperatures of 72-80°C are particularly preferred.

[0067] The primers for hybridizing the single-stranded DNA and / or RNA can preferably be brought into contact with the DNA and / or RNA prior to denaturation, particularly preferably by dissolving them in the amplification mixture. Hybridization of the primers to the single strands of DNA and / or RNA then preferably takes place at a temperature in the range of 4°C to 65°C for a period of time that is preferably more than 10 seconds.

[0068] SSMD is preferably performed at a constant temperature ranging from 40°C to 72°C. A preferred amplification time is between 10 and 120 minutes. The higher the temperature during amplification, the faster the amplification process, and the shorter the amplification time can usually be advantageously selected.

[0069] The advantage of ssMDA is that DNA and RNA biomarkers can be simultaneously amplified and enriched from single cells in the same reaction compartment, which represents a huge improvement and simplification of biomarker enrichment, also with regard to the effort required, the duration of the experimental procedure, the number of work steps and the associated costs.

[0070] In an alternative embodiment to ssMDA, amplification is performed using reverse transcriptase-PCR. Here, for example, only one SFP and one SRP are used per biomarker target region. The amplification mixture in this embodiment comprises, for example, a Taq polymerase and a reverse transcriptase.

[0071] Furthermore, in one embodiment, it is advantageous if, in order to transfer an amplification reaction from the at least one microcavity and / or to combine several amplification reactions from different microcavities, the non-aqueous phase above the microcavities, which had previously closed them in the region of their opening, is displaced. The displacement can be carried out, for example, with water, an elution buffer, or a detergent-containing buffer. The amplification reactions can be transferred via the opening into a microfluidically connected system, and the individual amplification reactions can be combined if necessary. In the context of the present invention, an amplification reaction is understood to mean the reaction solution present in the reaction compartment after amplification.

[0072] In an alternative advantageous embodiment for transferring an amplification reaction from the at least one microcavity and / or for combining multiple amplification reactions from different microcavities, the at least one microcavity has a further opening in the wall, in particular one directed downwards. Such a further opening is closed, for example, by an actuatable membrane during the reaction. After the reaction has ended, the further opening is opened or the actuatable membrane is removed in order to transfer the amplification reaction, in particular downwards, into a microfluidically connected system and, if necessary, to combine the individual amplification reactions.

[0073] The microfluidically connected system is, for example, a microfluidically connected reaction chamber with a volume of, for example, 20 μl. In this chamber, the biomarker target amplifications can be further enriched and purified. For further enrichment, the biomarker target amplifications are amplified again with the universal sequence if necessary and read, for example, using NGS. Using the cell barcode sequence retrieved during readout, the biomarkers can be clearly assigned to the individual cells.

[0074] In an advantageous embodiment, the biomarker target amplification is carried out using capture molecules, in particular capture molecules immobilized on magnetic beads. The capture molecules, in particular streptavidin, bind, for example, to the selection marker of the SFP and SRP, in particular biotin. For this purpose, appropriate binding conditions are established for the capture molecules and the biomarker target amplifications containing the selection marker, and a "bind-wash-elute" procedure, which can be carried out according to known protocols, is applied.

[0075] Alternatively, instead of the classic elution, a restriction enzyme can also be used to release the biomarker target amplifications, for example from the capture molecule. A prerequisite for this is that corresponding cleavage sequences are incorporated into the universal sequence in the SFP and SRP. In one embodiment of the invention, it is advantageous if the protelomerase TelN or a mutant enzyme is used for elution of the biomarker target amplifications from the capture molecule. This enzyme covalently links the two single-stranded molecules of the DNA double helix (sense and antisense strands) at the cleavage site, leaving behind a hairpin structure. Such a hairpin structure is required, for example, for the production of NGS libraries, for example, for the sequencing library of the sequencing technology from Pacific Biosciences.The advantage here is that no further steps for adapter ligation are required.

[0076] Furthermore, further enrichment of the biomarker target amplifications can be achieved using isothermal rolling circle amplification (RCA) without the need for additional sample preparation steps for the amplification.

[0077] In an advantageous embodiment, the cells are circulating tumor cells (CTCs). Molecular genetic single-cell analysis of these CTCs is a highly sensitive method, for example, in the therapy monitoring of cancer patients, to collect early information on the course of therapy (prognostic) and on the therapy adjustment (diagnostic). Here, the rare CTCs (e.g., 10 1 up to 10 2 per mL of blood; individually or a few as a pool) from a high background of “healthy” blood cells (>10 6Leukocytes, >10 9 Erythrocytes per mL of blood) are isolated. According to conventional methods, the genetic material of these individual, isolated cells must be pre-amplified to generate sufficient input for downstream analyses.

[0078] The method according to the invention eliminates process steps for pre-amplification of the DNA and / or RNA of the CTCs, as well as previous steps for combining several pre-sorted cells, which significantly shortens the process time, requires fewer work steps, requires fewer reagents and thus also results in lower costs.

[0079] Cells used in the method according to the invention can be pre-stained cells, such as EpCAM-positive CTCs. These carry the surface antigen EpCAM (epithelial cell adhesion molecule). However, all cells can also be examined without prior staining, such as all individual cells detached from the cell network of a tissue.

[0080] Alternatively, the cells can also be microbial cells or single-cell cells. The invention further relates to a control device configured to execute and / or control the steps of the method according to the invention in corresponding units, in particular in a microfluidic cartridge.

[0081] The invention furthermore relates to a computer program configured to carry out and / or control the steps of the microfluidic method, as well as to a machine-readable storage medium on which the computer program is stored.

[0082] Short description of the drawing

[0083] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0084] Fig. 1: the schematic representation of an embodiment of a method according to the invention for the combined cell-specific labeling and enrichment of biomarkers in a reaction compartment, and

[0085] Fig. 2: The schematic representation of a biomarker-specific forward or

[0086] Reverse primers.

[0087] Embodiments of the invention

[0088] Figure 1 shows the method according to the invention for the combined cell-specific labeling and enrichment of biomarkers in a reaction compartment in an embodiment, which is described using an isothermal ssMDA.

[0089] A device with which the method according to the invention can be carried out is described in the as yet unpublished German application with the file number 102022203848.7. This device has a carrier substrate for receiving a sample liquid. The carrier substrate has at least one microcavity 110 and furthermore at least one electrode arranged on or in the microcavity 110 to generate an electric field designed to capture a cell 510, in particular a nucleated cell 510, and / or a primer particle 512 and / or another particle in the microcavity 110. Figure 1 of the present invention shows only one microcavity 110 of this device.

[0090] In a preceding step 0 (not shown in Figure 1), a first sample liquid containing cells 510 is introduced into the microfluidic device or placed onto the carrier substrate. The cells 510 sediment towards the microcavities. As soon as a cell 510 approaches the microcavity 110, the DEP cage is switched to an open state or switched off, allowing this cell 510 to enter the microcavity. As soon as the cell 510 has entered, the DEP cage is closed again, allowing no further cells to enter and allowing a maximum of one cell per microcavity. Once all cells have sedimented and, for example, all other microcavities are also loaded with one cell each, the sample liquid is rinsed away. The DEP cage remains closed, retaining the cell 510 until further notice.

[0091] In step S1 of Figure 1, a cell 510 with a cell nucleus 511, here, for example, a CTC, is imaged in the microcavity 110. A second sample fluid containing primer particles 512 is then introduced into the microfluidic device or deposited onto the carrier substrate. This displaces, for example, the first sample fluid located above the carrier substrate, which comprises the cells 510.

[0092] The primer particles 512 comprise, for example, a particle 513 made of polystyrene, with different primer populations reversibly coupled to them, here, for example, biomarker-specific forward primer (SFP) 517 and biomarker-specific reverse primer (SRP) 517 with a selection marker 514 and random primer (RP) 516. In Figure 1, the SFP 517 and SRP 517 are visually indistinguishable from one another, but it should be noted that they are different populations of primers.

[0093] In step S2, the DEP cage is transferred to an open or switched-off state so that a primer particle 512 can settle into the microcavity 110. As soon as a primer particle 512 has settled into the microcavity 110, the DEP cage is transferred to a closed state by a voltage change, and the primer particle 512 is trapped therein with the cell 510. In this state, no further primer particles 512 can enter the microcavity 110, ensuring that, in addition to the single cell 510, only a single primer particle 512 is present in the microcavity 110. The dashed line with the reference number 520 is intended to visually illustrate that the DEP cage is closed after being loaded with the primer particle 512. In an alternative embodiment not shown in Figure 1, the microcavity 110 can also be first loaded with a primer particle 512 and then with a cell 510.

[0094] In a further alternative embodiment, not shown in Figure 1, the primer particles can also carry only the SFP and SRP and the RP are introduced via the amplification mixture in step S3.

[0095] In a step S3, an amplification mixture is then introduced into the microfluidic device or applied to the carrier substrate. Either the second liquid containing the unused primer particles 512 is first removed from the carrier substrate and the amplification mixture is subsequently applied to the carrier substrate, or the liquid containing the primer particles 512 is directly mixed with the amplification mixture on the carrier substrate. The amplification mixture comprises a buffer containing SD polymerase 525 and deoxyribonucleoside triphosphates (dNTPs), as well as optionally a reverse transcriptase and / or nicking enzyme. For ease of understanding, only polymerase 525 is shown in Figure 1.

[0096] In step S3, the diffusion-based distribution of the components or polymerase 525 of the amplification mixture into the microcavity 110 is shown. Here, the DEP cage is closed, which is illustrated by the dashed line with reference numeral 520, since the components of the amplification mixture do not represent entities with dielectric properties and can therefore pass through the DEP cage diffusion-based even in the closed state.

[0097] The microcavity 110 now contains the cell 510, the primer particle 512 and the SD polymerase 525 as well as other components of the amplification mixture.

[0098] In a step S4, the amplification mixture above the at least one microcavity 110 is displaced by a non-aqueous phase 526, for example, an oil phase or air, so that the microcavity 110 is closed in the region of its opening by the non-aqueous phase 526 and thus represents a closed reaction compartment in which the cell 510 and the primer particle 512 with the amplification mixture are enclosed. Conditions are now created that lead to lysis of the cell 510 and to the release of the primers 516, 517 from the particle 513.

[0099] S4 shows a lysed cell 510a from which the cell components, for example, RNAs 539, have been released, as well as a lysed cell nucleus 511a, which has released the DNA 539 located therein. Such conditions are created, for example, by increasing the temperature to, for example, above 70°C for, for example, more than one minute and / or by electroporation and / or by ultrasound.

[0100] During the temperature-induced lysis of the cell 510, denaturation of the DNA double strands and / or the RNA secondary structures and / or tertiary structures occurs simultaneously, particularly preferably at temperatures between 72°C and 80°C, for example for more than one minute.

[0101] In a further step S5, the amplification reaction is carried out using ssMDA. The random primers 516 bind randomly to regions of the DNA 539 and / or RNA 539, allowing an isothermal, strand-displacement-based amplification of the total DNA 539 and / or RNA 539 using the polymerases 525. This produces total DNA amplification products 539 and / or total RNA amplification products 539. Furthermore, the specific forward primers 517 and the specific reverse primers 517 bind upstream and downstream of the biomarker target regions 530 of the DNA 539 and / or RNA 539, resulting in amplification of the biomarker target region 530, which is cell-specifically labeled by the selection marker 514, for example, biotin. For amplification, polymerases 525 bind to the SFP and SRP, which provide the starting point for amplification to the SD polymerases 525. This creates biomarker target amplification proteins 542.

[0102] For example, the SFP and SRP carry additional nicking sequences at the 5' end that extend the universal sequence. By adding a corresponding nicking enzyme to the amplification mixture in step S3, an additional, disproportionate enhancement of the biomarker target amplification can be induced, with the biomarker target amplicons 542 continuing to carry the selection marker 514.

[0103] The amplification of RNA can, for example, be further enhanced by reverse transcriptase.

[0104] In step S6, it is indicated that in order to combine multiple amplification reactions from different microcavities 110, the non-aqueous phase 526 above the microcavities 110, which had previously closed them in the region of their opening, is displaced. The displacement can be carried out, for example, with an elution buffer or a detergent-containing buffer. The amplification reactions can be transferred via the opening into a microfluidically connected system, for example, a microfluidically connected reaction chamber, and the individual amplification reactions can be combined.

[0105] Here, the biomarker target amplifications 542 can, for example, be further enriched and purified. In a step S7, the amplified biomarker target amplifications 542 are purified using capture molecules 543, which are immobilized, for example, on magnetic particles (beads). The capture molecules 543, for example, streptavidin, bind to the selection marker 514 of the SFP 517 and SRP 517, in particular biotin. Thus, the biomarker target amplifications 542 can be separated from the remaining reaction components, such as the total DNA amplifications 539 and / or total RNA amplifications 539. Subsequently, the biomarker target amplificates 542 are eluted from the capture molecules 543, for example by classical elution methods or by a restriction enzyme.

[0106] Figure 2 shows an example of a biomarker-specific forward primer (SFP) 517 and a biomarker-specific reverse primer (SRP) 517.

[0107] The SFP or SRP comprises, starting at the 5' end, a selection marker 514 coupled to a universal sequence 501, followed by a unique cell barcode sequence 503 and a specific biomarker target sequence 506.

[0108] By means of the specific biomarker target sequence 506, the SFP 517 binds to the DNA 539 and / or RNA 539 in a forward-facing manner up to 3 kb upstream of the biomarker target region 530, and the SRP 517 binds to the DNA 539 and / or RNA 539 in a reverse-facing manner up to 3 kb downstream of the biomarker target region 530.

[0109] The unique cell barcode sequence 503 is clonally contained in each SFP 517 and SRP 517 of a primer particle 512, for example, with a length of 8 or more bases. The selection marker 514 is, for example, biotin at the 5' end of the SFP 517 or SRP 517. Using the selection marker 514, a subsequent positive selection of the amplified biomarker target amplifications 542 is possible. The universal sequence 501 is required, for example, for a subsequent amplification of a sequencing library or a known sequence with a function of choice, such as a restriction site, in particular for releasing the biomarker target amplification 542, for example, from a capture molecule 543.

[0110] Optionally, and not shown in Figure 2, the SFP 517 or SRP 517 can have a unique molecular identifier (UMI) in the form of a randomized sequence. The UMI can, for example, be located between the universal sequence 501 and the cell barcode sequence 503 or between the cell barcode sequence 503 and the specific biomarker target sequence 506. The UMI is, in particular, 6 or more bases long. Also optionally, and not shown in Figure 2, the SFP 517 or SRP 517 can be extended by a nicking sequence at the 5' end of the universal sequence 501. The selection marker is thus no longer located terminally on the SFP or SRP, but internally at the 5' end of the universal sequence.

Claims

Claims 1. A microfluidic method for combined cell-specific labeling and enrichment of biomarkers in a reaction compartment, comprising the following method steps: a) providing a microfluidic device, comprising at least one carrier substrate for receiving a sample liquid, wherein the carrier substrate has at least one microcavity (110), and further comprising at least one electrode arranged on or in the microcavity (110) to generate an electric field designed to capture a cell (510) and / or a primer particle (512) in the microcavity (110), b) loading the at least one microcavity (110) of the microfluidic device with a cell (510) and capturing the latter, c) loading the at least one microcavity (110) of the microfluidic device with a primer particle (512) comprising at least one primer population coupled thereto and capturing the Primer particle (512),wherein steps b) and c) can also be carried out in reverse order d) introducing an amplification mixture into the at least one microcavity (110) e) displacing the amplification mixture above the at least one microcavity (110) by a non-aqueous phase (526) so that the microcavity (110) represents a closed reaction compartment f) bringing about cell-lysing and primer (516, 517) releasing conditions g) binding the at least one primer population to biomarker regions of the DNA (539) and / or RNA (539) of the lysed cell (510a) for cell-specific labeling and amplification of a biomarker target region (530), 2. Microfluidic method according to claim 1, wherein different populations of primers are reversibly coupled to the primer particle (512), which is in particular a polystyrene primer particle (512), in particular biomarker-specific forward primers (517), biomarker-specific reverse primers (517)and / or random primers (516). The microfluidic method according to claim 2, wherein the biomarker-specific forward (517) and reverse primers (517) each comprise a selection marker (514) coupled to a universal sequence, a unique cell barcode sequence, and, correspondingly, a specific forward or reverse target sequence for the biomarker target region (530). The microfluidic method according to claim 3, wherein, for each biomarker target region (530), several, in particular 2-4, biomarker-specific forward (517) and reverse primers (517) with different flanking specific forward or reverse target sequences (506) are used. Microfluidic method according to any one of claims 2-4, wherein the biomarker-specific forward (517) and reverse primers (517) further comprise a unique molecular identifier (UMI) in the form of a randomized sequence.The microfluidic method according to any one of claims 3-5, wherein the primer particles (512) are configured such that the biomarker-specific forward (517) and reverse primers (517) are bound to the primer particles (512) via the selection marker (514) or via a reversible compound. The microfluidic method according to any one of claims 3-6, wherein the universal sequence (501) of the biomarker-specific forward (517) and reverse primers (517) is selected such that a direct target for further amplification is introduced, in particular for the completion of a sequencing library.The microfluidic method according to any one of claims 3-7, wherein the universal sequence (501) of the biomarker-specific forward (517) and reverse primers (517), in particular at the 5' end, comprises a single-strand break sequence via which a single-strand break can be induced by means of a single-strand break-inducing enzyme, and / or wherein the selection marker is coupled to an internal, non-terminal base of the universal sequence. The microfluidic method according to any one of the preceding claims, wherein the amplification mixture promotes cell lysis and / or a single-strand break. inducing enzyme. Microfluidic method according to one of the preceding claims, wherein the amplification is an isothermal amplification, in particular a semi-specific multiple displacement amplification (ssMDA). Microfluidic method according to one of the preceding claims, wherein, in order to transfer an amplification reaction from the at least one microcavity (110) and / or to combine several amplification reactions of different microcavities (110), the non-aqueous phase is displaced or the at least one microcavity (110) has a further opening in the wall, in particular one directed downwards. Microfluidic method according to one of the preceding claims, wherein a purification of biomarker target amplificates (542) takes place by means of capture molecules (543), in particular by means of capture molecules (543) immobilized on magnetic beads.The microfluidic method according to claim 12, wherein, during elution of the biomarker target amplificates (542) from the capture molecule (543), the protelomerase TelN or an enzyme mutant thereof is used, which covalently links the two single-stranded molecules of the DNA double helix at the interface, thereby leaving a hairpin structure, in particular for carrying out isothermal rolling circle amplification (RCA) for further enrichment of the biomarker target amplificates (542). The microfluidic method according to one of the preceding claims, wherein the cells (510), in particular pre-stained, are circulating tumor cells (CTCs). A control device configured to execute and / or control the steps of a method according to one of the preceding claims 1-14 in corresponding units, in particular in a microfluidic cartridge.Computer program configured to execute and / or control the steps of the microfluidic method according to any one of claims 1-14.

17. A machine-readable storage medium on which the computer program according to claim 16 is stored.

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