Sample substrate, in particular for LC-MS analysis, liquid sample analysis device and method for preparing liquid sample

Abstract

A sample substrate (100) suitable for preparing a sample for LC-MS analysis, comprising: a reaction substrate arrangement (10, 20) having a plurality of containers (12, 22), each container having at least one accommodation space configured to accommodate a liquid sample and a container connection (15) with a container opening (13); and a plurality of collection cartridges (40), each collection cartridge having a cartridge connection (42) with a cartridge opening (43) and configured to contain a liquid sample from one container (12, 22). Each container connection (15) is matched by a form fit to each cartridge connection (42) such that they are adapted to connect the reaction substrate device (10, 20) and the plurality of collection cartridges (40) to provide direct liquid communication of the container (12) and the collection cartridges (40) via the openings (13, 43) of the container and collection cartridges. Furthermore, a liquid sample analysis device comprising the sample substrate (100) and a method of performing LC-MS sample analysis using the sample substrate (100) and an LC-MS device are described.

Description

Technical Field

[0001] The present invention relates to a sample substrate, a liquid sample analysis device, and a method for preparing a liquid sample, which are particularly used for LC-MS (liquid chromatography-mass spectrometry) analysis. The application of the present invention can be used in the field of processing liquid samples, especially biological samples, such as proteomics research. Background Art

[0002] In this specification, reference is made to the following prior art related to the background art of the present invention, particularly related to the preparation of cell samples for mass spectrometry:

[0003] [1] US 2019 / 0209592 A1;

[0004] [2] H. Specht et al. "Single-cell mass-spectrometry quantifies the emergence of macrophage heterogeneity" doi: http: / / dx.doi.org / 10.1101 / 665307H;

[0005] [3] Y. Zhu et al. "Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells" in "NATURE COMMUNICATIONS" (2018) 9:882, DOI: 10.1038 / s41467-018-03367-w;

[0006] [4] Z. Y. Li et al. "Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis" in "Anal. Chem." 2018, 90, 5430−5438Z;

[0007] [5] US 2019 / 0250130 A1;

[0008] [6] EP 3 964 290 A1; and

[0009] [7] EP 4 075 145 A1。

[0010] As is well known, people are making increasing efforts to study the proteome, especially to analyze the entire set of proteins contained in biological materials such as biological cells or cell aggregates or cell components. Various antibody-based techniques and mass spectrometry-based techniques can be used to analyze the proteome, such as mass cytometry (CyTOF), matrix-assisted laser desorption / ionization (MALDI), single-cell proteomics mass spectrometry (SCoPE-MS), antibody-based techniques, or liquid chromatography-mass spectrometry (LC-MS). The latter has an advantage in the ability to detect a large number of proteins in each cell with high specificity.

[0011] LC-MS generally includes the following processes. For example, biological cells are separated using a fluorescence-activated cell sorter (FACS) and subjected to cell lysis to provide the proteins contained in the cells. The proteins are enzymatically digested into peptide fragments. In the case of analyzing a single biological cell, these fragments are usually labeled with a labeling molecule used as a mass reporter, such as a tandem mass tag (TMT) label, and then optionally subjected to label quenching (removing unbound labeling molecules). After optionally pooling the labeled samples, the peptide fragments are separated by liquid chromatography (LC). The peptide fragments are introduced into a tandem mass spectrometer and identified using bioinformatics techniques by peptide fingerprinting or tandem mass spectrometry. The steps from cell separation to pooled sample preparation can be challenging in terms of parallel processing of a large number of samples contained in a small volume of liquid, maintaining the assignment of peptide fragments to certain samples, and avoiding cross-contamination between different samples.

[0012] For example, the current techniques for sample preparation are described in [1] to [4], which use the same conventional workflow for cell separation, lysis, digestion, TMT labeling, and sample collection, but differ in the details of the sample substrate and the reagents applied for lysis and digestion. According to [1] and [2], lysis and digestion are performed in a 384-well microplate, and the labeled samples are collected into a single glass HPLC (High Performance Liquid Chromatography) insert. According to [3], a homemade patterned slide is used for sample preparation. In [4], it is proposed to use an oil layer to reduce the evaporation of small liquid samples, where a layer stack is used to create a sample container, and a self-supporting oil film is spanned over each sample container. In [6], a sample preparation device for preparing a liquid sample for sample analysis is disclosed, in which a carrier plate device having a reaction site array is combined with a collection device having a collection container. Each collection container is adapted to collect a liquid sample from at least two reaction sites and provide the liquid sample for sample analysis. In [7], it is described that the collection device is adjusted to be directly positioned in the turntable of an automatic sampling device (such as an automatic sampling device for liquid chromatography).

[0013] Using conventional substrates such as 384-well plates, patterned slides, layer stacks, the sample preparation device in [6], or the collection device in [7] has the following drawbacks. First, 384-well plates are suitable for handling relatively large volumes in the range up to 100 µl. In addition, the substrate requires complex lid handling to minimize evaporation. As another drawback, transferring the labeled fragments from the wells to a glass HPLC insert or any other common collection container is time-consuming, including the risk of introducing contaminants and is error-prone. For example, according to [1], time-consuming pipetting one by one is used for the transfer task. In addition, in particular, patterned slides are dedicated devices and are not suitable for all steps of the process chain, including additional previous sample handling steps such as cell separation. The layer stack used in [4] has a complex and expensive structure. Although [6] and [7] introduce some improvements in sample pooling and transferring the sample to an automatic sampling device for liquid chromatography, there are still limitations in LC-MS analysis. Generally, conventional techniques are not sufficient to adapt to the automation of sample preparation, resulting in limitations in terms of time-consuming processes involving human interaction and the risk of contamination.

[0014] Therefore, a significant particular drawback of conventional techniques is that the prepared samples must be manually transferred to sample cartridges to perform LC-MS sample analysis. For example, commercial Evotips ® (see, for example, [5]) must be manually loaded with samples. This means that samples prepared by any sample preparation method are loaded into Evotips ®The steps thereon always include time-consuming pipetting steps. Additionally, the sample comes into contact with the walls of the pipette tip, which may lead to adsorption losses of some peptides in the sample to be analyzed.

[0015] The above problems occur not only in the preparation of samples for mass spectrometry analysis but also in other tasks of preparing liquid samples for sample analysis by applying reagents to multiple samples and processing the samples. Summary of the Invention

[0016] Object of the Invention

[0017] The object of the present invention is to provide an improved sample substrate, a liquid sample analysis device, and a method for preparing a liquid sample, especially for LC-MS, which can avoid the disadvantages or limitations of conventional techniques. In particular, the sample preparation should have the ability to facilitate multiplexing of sample preparation and analysis, reduce operation time, handle reduced sample volumes in the low nanoliter (nl) range, minimize or avoid evaporation problems, and / or facilitate sample pooling. According to a further aspect, the sample substrate should be configured to be as user-friendly as possible and / or suitable for the entire process chain of sample preparation, especially LC-MS analysis, such that, for example, the sample preparation can be automated. Overview of the Invention

[0019] The above object is solved respectively by a sample substrate, a liquid sample analysis device, and a sample preparation method including the features of the independent claims. The features of the preferred embodiments and applications of the present invention are defined in the dependent claims.

[0020] According to a first general aspect of the present invention, the above object is solved by a sample substrate adapted to prepare a sample for LC-MS analysis (e.g., proteomic mass spectrometry). The sample substrate includes a reaction substrate device having a plurality of containers (e.g., wells or a more complex array of containers or receiving spaces). Each container (among the plurality of containers) has at least one receiving space (or sub-container, sub-well) configured to receive a liquid sample, preferably a sample droplet. Each container also has a container connection portion with a container opening.

[0021] The sample substrate further includes a plurality of collection cartridges. Each collection cartridge (among the plurality of collection cartridges) has a cartridge connection portion with a cartridge opening. Each collection cartridge is configured to receive a liquid sample from one container. Each collection cartridge preferably has the shape of a pipette tip and / or a cone and / or a post, particularly having a single opening (i.e., the cartridge opening) at the end face. Preferably, the collection cartridges are commercial Evotips ® (e.g., see [5]) or cartridges having a shape matching that of Evotips ® Evotips ®It is a disposable trap column. Since combining elution with liquid chromatography can eliminate several sample processing steps and reduce injection cycle overhead, it can speed up sample loading and significantly reduce residues, thus simplifying the workflow.

[0022] Preferably, the collection cartridge is an integral part. In particular, the cartridge connection is preferably an integral part of the rest of the collection cartridge. Alternatively, the collection cartridge is made of multiple parts, such as a cartridge body and a cartridge connection fixed to the cartridge body.

[0023] Preferably, the collection cartridge (also known as stage tips) is a pointed container, particularly preferably made of an inert polymer material, such as C18 material, to obtain the advantage of desalting / cleaning protein digestion samples, for example, before injection into a chromatographic separation column. It is known that inert polymer materials generally have polymer non-polar capping and a high carbon content.

[0024] According to the present invention, each container connection mates with each cartridge connection by form fit such that they are adapted to (particularly, liquid-tightly) connect the reaction substrate device and a plurality of collection cartridges, thereby providing direct liquid communication between the container and the collection cartridges via the openings of the reaction substrate device and the plurality of collection cartridges. Preferably, each container connection and each cartridge connection are adapted to be directly connected to each other. Preferably, the contact area where the container connection and the cartridge connection contact each other has a round, particularly circular or oval cross-sectional shape.

[0025] In particular, the sample substrate enables a one-to-one connection between the container and the collection cartridge. In other words, the sample substrate is adapted to effect a (single) connection between (exactly) one container and (exactly) one collection cartridge, particularly via the corresponding container connection and cartridge connection. This connection provides direct liquid communication between (exactly) one container and (exactly) one collection cartridge. Therefore, one or more samples prepared in one container can only be transferred to one collection cartridge.

[0026] The term "connection" generally refers to a topographical structural feature (or element) that provides an exposed mating shape, particularly with inherent liquid tightness, when connected to another connection with a complementary shape. Each connection of the reaction substrate device, particularly the open end of a container that provides the container opening, mates with the open end of a connection, particularly a collection cartridge that provides the cartridge opening.

[0027] Advantageously, the connection part additionally provides a mechanical connection between the container and the collection cartridge even during sample transfer, for example by centrifugation. Optionally, the mechanical connection between the container and the collection cartridge can also be supported by the design of the centrifugation device and / or by a fixing element that holds the container and the collection cartridge together and provides an additional clamping and / or threaded connection between them in the connected state of the container and the collection cartridge.

[0028] According to a second general aspect of the present invention, the above object is solved by a liquid sample analysis device adapted to prepare a sample for LC-MS sample analysis (e.g., proteomic mass spectrometry) and perform LC-MS sample analysis, and comprising a sample substrate and an LC-MS device according to the first general aspect of the present invention or an embodiment thereof.

[0029] According to a third general aspect of the present invention, the above object is solved by a method of performing LC-MS sample analysis (e.g., proteomic mass spectrometry), wherein a sample substrate and an LC-MS device according to the first general aspect of the present invention or an embodiment thereof are used. The method includes collecting a sample to be analyzed in a container of the sample substrate, performing a decomposition reaction on the (collected) sample in the container, and connecting each collection cartridge to a container (preferably after sample processing). The method further includes transferring (e.g., pooling) the decomposed sample from each container to the connected collection cartridge, separating the collection cartridge from the container, then connecting the collection cartridge to the LC-MS device, and performing LC-MS sample analysis using the LC-MS device.

[0030] When performing the method, the sample substrate, in particular the reaction substrate device, can be arranged at two different positions (orientation relative to gravity). In particular, when collecting the sample, performing the decomposition reaction on the sample, and connecting the collection cartridge to the container, the reaction substrate device is arranged at a first position where the container is arranged on the top side of the reaction substrate device and / or the container opening faces upward, i.e., in a direction opposite to the direction of gravity. After transferring the decomposed sample and optionally during the transfer, the sample substrate is arranged at a second position where the container is arranged on the bottom side of the reaction substrate device and / or the container opening faces downward, i.e., in the direction of gravity. Preferably, the sample substrate is moved from the first position to the second position by inverting the sample substrate. At the second position, the decomposed sample can flow from the container to the (connected) collection cartridge by the action of gravity. Preferably, this sample transfer is further supported by centrifugation.

[0031] Preferably, the samples prepared and analyzed using the sample substrate include biological materials, particularly biological cells, cell populations, and / or cell components. For example, the biological material can be a single cell, multiple cells, or at least one piece of biological material derived from a tissue section (such as a fresh frozen tissue or a paraffin-embedded FTPE (fluorinated thermoplastic elastomer) tissue section). Additionally, the decomposition reaction preferably includes cell lysis and / or protein digestion. For example, reagents are added such that the sample cells are lysed and the proteins are digested into peptide fragments.

[0032] The present invention has the following advantages. Before subsequent analysis by an LC-MS method (mass spectrometry), all steps of the sample preparation protocol can be performed using the sample substrate. In particular, the sample substrate enables the sample prepared in a container to be directly and preferably automatically transferred (e.g., pooled) to a collection cartridge such as Evotips ® etc., thereby excluding the use of additional auxiliary pipettes and / or pipetting steps from the workflow for transferring the prepared sample before introducing it into an analytical device, particularly an LC-MS device. Eliminating the pipetting step will reduce the peptide loss that typically occurs on the walls of the pipette. Particularly for single-cell or small-cell (e.g., protein) samples prepared for single-cell proteomics analysis, where the sample size is usually already very low (usually <ng), using the sample substrate according to the present invention provides an increase in the number of proteins detected from each sample.

[0033] As other advantages, compared to conventional manual techniques, directly transferring the sample prepared in a container to the collection cartridge results in a more rapid transfer. Additionally, the sample volume (“reaction volume”) used for sample preparation (sample reaction) in the container can be reduced according to the application conditions, down to a sample volume below 1 µl, particularly below 100 nl, for example down to 1 nl or even lower. After preparation, the sample volume can be increased to, for example, 1 µl to 10 µl (“transfer volume”) by a dilution step before transfer via centrifugation. The size of the container can be reduced, thereby facilitating the handling of the sample substrate and facilitating measures to prevent evaporation. By assigning the containers to the collection cartridges, multiplexing of sample preparation can be supported. As other advantages, the sample substrate can be easily manufactured, for example, from low-cost plastic materials, and / or can be provided and used as a disposable or reusable consumable.

[0034] Further advantageously, the application of the present invention can prepare multiple liquid samples for various types of sample analysis. For example, multiple single cells, multiple cells, and / or cell components can be arranged in respective reaction containers. Samples can be prepared, for example, for subsequent mass spectrometry analysis, optionally in combination with liquid chromatography separation of the sample components, and / or for other analyses, such as genomic analysis including sequencing measurements.

[0035] In addition, the sample substrate according to the present invention can be incorporated into various sample collection devices to reduce the number of manual operations required and improve the quality / sensitivity of, for example, proteomics mass spectrometry results. For example, the sample substrate can be used in combination with the sample collection device (also known as proteoCHIP) disclosed in [7].

[0036] According to an alternative variant of the present invention, the containers can be arranged in a matrix and / or an array, where the containers are spaced apart from each other. The number of containers and the type of arrangement can be selected according to the degree of parallelism of the sample processing to be obtained, for example, according to the number of different labeling molecules to be applied. Additionally, the shape of the reaction containers can be selected according to, for example, the application conditions of the sample preparation device and the sample liquid handling techniques applied.

[0037] According to a preferred embodiment of the present invention, each cartridge connection portion matches the shape of the sample input portion of the LC-MS device. Advantageously, after the sample has been transferred from the container to the collection cartridge, the collection cartridge can be removed from the reaction substrate device and directly placed in the LC-MS device without further transferring the sample to other containers to perform LC-MS analysis. For example, in the case of using Evotips ® as the collection cartridge, Evotips ® is capable of being directly connected to an Evosep ® liquid chromatography system for mass spectrometry analysis.

[0038] According to another preferred embodiment of the present invention, one of the container connection portion and the cartridge connection portion is respectively formed by a protruding edge at the container opening of the container or the cartridge opening of the collection cartridge, and the other of the container connection portion and the cartridge connection portion is respectively provided by an inner wall portion at the container opening or the cartridge opening. When connecting, the protruding edge and the inner wall portion are in direct contact, for example, where the inner wall portion surrounds the protruding edge. The connection combination of the edge and the inner wall has particular advantages in terms of being integrated into the materials of the reaction substrate device, particularly the container and the collection cartridge, and particularly in terms of a reliable liquid-tight connection between plastic materials. Preferably, the edge has a truncated conical shape.

[0039] Particularly preferably, the protruding edge has a conical shape. Advantageously, the conical shape further facilitates the simplified connection of the container connection portion and the cartridge connection portion.

[0040] According to a preferred variant of the present invention (hereinafter: the first embodiment or single-board embodiment of the present invention), the reaction substrate device includes a single board having a plurality of containers. The containers can be formed as holes within the single board. Thus, the reaction substrate device can be a microplate, where each hole is provided with a container connection portion. Such a single-board embodiment is particularly preferably used for preparing samples containing label-free reactions.

[0041] According to another preferred variant of the invention (hereinafter: the second embodiment or the double-plate embodiment of the invention), the reaction substrate device comprises a stack of two plates, the two plates comprising a carrier plate and a funnel plate. The carrier plate comprises a plurality of arrays of at least two receiving spaces of the container, wherein each array has a carrier plate connection portion with an opening of the container portion. The funnel plate comprises a plurality of funnel-shaped opening channels, wherein each funnel-shaped opening channel comprises a first end and a second end providing a container connection portion, the first end having a funnel connection portion with a funnel connection opening. The carrier plate connection portion and the funnel connection portion are matched to each other by form fit such that they are adapted (in particular, liquid-tight) to connect the carrier plate and the funnel plate, thereby forming a plurality of containers, each container being composed of an array of one of the at least two receiving spaces and a funnel-shaped opening channel that are connected to each other and are in direct liquid communication via the container portion opening and the funnel connection opening. With the second embodiment, each container is provided by combining an array of receiving spaces with a funnel-shaped opening channel.

[0042] The double-plate embodiment comprising a stack of a carrier plate and a funnel plate is particularly preferably used for preparing a sample containing a multiplexed reaction. For example, the carrier plate can correspond to the carrier plate device disclosed in [6] (which is incorporated herein by reference), particularly in terms of the shape, size and usage method of the carrier plate device.

[0043] A particular advantage of the second embodiment is provided by the dual function of the reaction substrate in receiving and pooling the sample. Using the funnel plate, a plurality of samples separately prepared in different receiving spaces of the array can be pooled into a common collection cylinder.

[0044] According to another preferred embodiment of the invention, the sample substrate may comprise at least one of the following features. The volume of the container is in the range of 100 nl to 10 µl. The volume of the collection cylinder is in the range of 10 µl to 1 ml. The inner diameter of one of the container connection portion and the cylinder connection portion is equal to the outer diameter of the other of the container connection portion and the cylinder connection portion. The inner diameter of one of the container connection portion and the cylinder connection portion is in the range of 1 mm to 10 mm, and the outer diameter of the other of the container connection portion and the cylinder connection portion is in the range of 1 mm to 10 mm. The sample substrate and / or the entire reaction substrate device is made of glass, plastic, silicon, polytetrafluoroethylene, polypropylene and / or polycarbonate. All components of the reaction substrate device may be made of the same material, or different components may be made of different materials.

[0045] Preferably, in the case of an outstanding container connection portion, the longitudinal length (height) of the container connection portion is selected in the range of 1 mm to 10 mm, particularly 2 mm to 10 mm. Advantageously, the stable connection of the connection portion during centrifugation is particularly improved by using these parameters.

[0046] The preferred embodiments, variations, and features of the present invention described above can be combined with each other as needed. The features disclosed in the context of the sample substrate and its embodiments also represent the preferred features of the method of the present invention and its embodiments, and vice versa. Therefore, the above aspects, as well as the inventions and preferred features, particularly those regarding the implementation of the method, also apply to the sample substrate. Description of the Drawings

[0047] Hereinafter, further advantages and details of the present invention will be described with reference to the drawings, which are schematically shown:

[0048] Figures 1 to 3 : A single board of a reaction substrate device according to a first embodiment of the present invention shown from different perspectives;

[0049] Figure 4 : A sample substrate according to the first embodiment;

[0050] Figure 5 : A carrier board of a reaction substrate device according to a second embodiment of the present invention;

[0051] Figures 6 to 9 : A funnel board of a reaction substrate device according to the second embodiment shown from different perspectives;

[0052] Figure 10 : A sample substrate according to the second embodiment; and

[0053] Figures 11 to 12 : An alternative single board according to the first embodiment. Detailed Description of the Invention

[0054] Hereinafter, the features of the preferred embodiments of the present invention will be described by way of example with reference to sample preparation for LC-MS / MS analysis. It should be emphasized that the application of the present invention is not limited to these examples, but rather, for example, it can be correspondingly used for other analyses by using other samples and reagents, particularly in the field of proteomics sample preparation. In addition, although the sample substrate is schematically shown, the details can be modified according to specific application conditions, such as the number, shape, and size of the containers and / or the accommodation spaces and / or the number, shape, and size of the collection cylinders. Details of the sample processing for proteomics research are not described because these details are known per se from conventional techniques, such as [1] to [4].

[0055] By way of example, a container connection portion that protrudes into a cartridge connection portion is referred to, that is, an embodiment in which the container connection portion is introduced into the cartridge connection portion. The present invention can be implemented in the same manner with alternative shapes, particularly in a manner where the cartridge connection portion that protrudes into the container connection portion, that is, where the cartridge connection portion is introduced into the container connection portion.

[0056] Figure 1 , Figure 2 and Figure 3 show a first embodiment (single-plate embodiment) of the single plate 10 of the reaction substrate device of the sample substrate 100. Specifically, from different perspectives (i.e., a perspective view ( Figure 1 ), a side view ( Figure 2 ), and a top view ( Figure 3 )), the single plate 10 is shown.

[0057] The reaction substrate device includes the single plate 10, and the single plate 10 includes a plurality of containers 12 such as holes, preferably in a conical shape. Each container 12 has a separate accommodation space configured to accommodate a liquid sample and a container connection portion 15 having a container opening 13. Alternatively, each container 12 can be subdivided into a plurality of accommodation spaces. The containers 12 are spaced apart from each other. Preferably, each container opening 13 is the only opening of the corresponding container 12. The container connection portion 15 is formed by a protruding edge of the container 12.

[0058] In the illustrated example, an array of 14 containers 12 is provided, and the array is arranged in two rows, with 7 containers 12 in each row. As an example, the inner diameter of each container 12 at the container opening 13 is 4.3 mm and the outer diameter is 4.8 mm, the depth of each container 12 is 1 mm, and the distance between the centers of the containers 12 is 9 mm. The height of the container connection portion 15 is, for example, 2 mm.

[0059] In addition, each container 12 has an internal shape that narrows from the container opening 13 towards the container bottom. As an example, the inner diameter of each container 12 at the container bottom is 2.5 mm, and / or the opening angle of the internal shape towards the container opening 13 is 52° (refer to Figure 2 ). Alternatively, a cylindrical internal shape can be provided.

[0060] The carrier plate device 10 further includes a main board portion 11, preferably having a planar shape. The main board portion 11 provides a substrate that defines the external lateral dimensions of the single plate 10, and various geometries can be used according to application conditions. The external lateral dimensions can be selected to be equal to the size of a microscope slide, for example, approximately 75 mm * 25 mm, with a thickness of approximately 1 mm. Alternatively, another larger external lateral dimension can be used, for example, matching the specific substrate size processed in a sample collection (and processing) machine, such as Figure 11 and Figure 12 shown, 8.6 cm * 12.7 cm, with 8 * 12 containers arranged. For Figure 1 and Figure 12 embodiments, the height of the container connection portion 15 is, for example, 2 mm.

[0061] The carrier plate device 10 can include a standard microplate or nanoplate, where each hole is provided with a container connection portion as described above.

[0062] The containers 12 are arranged on the main board part 11. Preferably, all containers 12 are arranged on one side of the main board part 11, wherein when samples to be analyzed are collected in the containers 12, the opposite side of the main board part 11 can be used to place the single board 10 on a table, for example.

[0063] Figure 4 The sample substrate 100 according to the first embodiment is shown, wherein the sample substrate 100 further comprises a plurality of Evotips ® Each collecting tube has a tube connection portion 42 with a tube opening 43 (see also the second embodiment of the present invention) Figure 10 (b)) and is configured to receive a liquid sample from one container 12. Preferably, the cartridge opening 43 is the only opening of the respective collecting cartridge 40. Each collecting cartridge 40 preferably has the shape of a pipette tip, a cone and / or a column.

[0064] The specific feature of the sample substrate 100 is that each container connection part 15 matches each cartridge connection part 42 by form fit. Therefore, one collection cartridge 40 and one container 12 can be directly connected to each other by form fit via the corresponding container connection part 15 and the corresponding cartridge connection part 42. Therefore, by forming a connection such as a form fit, direct liquid communication is provided via the container opening 13 of the container connection part 15 and the cartridge opening 43 of the cartridge connection part 42. Preferably, each container connection part 15 and each cartridge connection part 42 are suitable for direct connection to each other.

[0065] The sample substrate 100 is Figure 4 1 and 10. It is shown in a simplified manner, including only two collecting cartridges 40, one connected to the container 12, and the other arranged adjacent to the single plate 10, to illustrate the detachable connection between the collecting cartridge 40 and the container 12 provided by the sample substrate 100 according to the present invention.

[0066] In the example shown, each container connection part 15 is formed by a protruding edge at the container opening 13 of the container connection part 15, in particular, by an outer wall part forming the side wall of the corresponding container 12. The protruding edge preferably has a conical shape. Thus, the side wall performs a dual function in forming the container 12 on the main plate part 11 and in connection with the collecting barrel 40.

[0067] Accordingly, each cartridge connection 42 is provided by an inner wall portion at the cartridge opening 43 of the cartridge connection 42, specifically, by an inner wall portion forming the side wall of the collection cartridge 40. Thus, the side wall of the collection cartridge 40 also performs a dual function in forming the collection cartridge 40 and connecting to the container 12.

[0068] According to the illustrated example, the collection cylinder 40 can be connected to the container 12 in a simple manner by placing the cylinder connection part 42 on the container connection part 15. This can be done manually or, in particular, by using a machine in an automated process.

[0069] Alternatively, each cylinder connection part 42 can be formed by a protruding edge at the cylinder opening 43 of the cylinder connection part 42, such as by an outer wall part forming the side wall of the collection cylinder 40. Correspondingly, each container connection part 15 can be provided by an inner wall part at the container opening 13 of the container connection part 15, such as by an inner wall part forming the side wall of the container 12.

[0070] The sample substrate 100 can be used to prepare samples for LC-MS analysis and / or perform LC-MS sample analysis (e.g., proteomic mass spectrometry), where the sample substrate 100 is particularly capable of preparing single-cell or small amounts of cell (e.g., protein) samples for single-cell proteomic analysis, where the sample amount is typically very low (usually <ng). All steps of the sample preparation protocol before subsequent sample analysis can be performed in the sample substrate 100.

[0071] In the first step, the sample to be analyzed is collected in the container 12. In particular, the reaction substrate device 100, i.e., the single plate 10, is set at the first position in the absence of multiple collection cylinders 40. Preferably, the single plate 10 can be placed, for example, on a table, where the container 12 is arranged on the top side of the single plate 10 and / or the container opening 13 faces upward. In this way, the sample can be supplied to the container 12 via the container opening 13.

[0072] The sample can include biological materials such as biological cells, cell populations, and / or cell components. Preferably, the biological material includes a single cell, multiple cells, or at least one piece of biological material derived from a tissue section (such as a fresh frozen tissue or a paraffin-embedded tissue section).

[0073] In the second step, a decomposition reaction is performed on the sample in the container 12, where the decomposition reaction preferably includes cell lysis and / or protein digestion. For example, a reagent is supplied to the container 12, which will cause the sample cells to lyse and the proteins to be digested into peptide fragments. As an example, a droplet processing and processing machine such as an automatic pipetting robot can be used to supply liquid samples and reagents through, for example, a piezoelectric droplet dispenser. Alternatively, liquid samples and reagents can be supplied by manual pipetting.

[0074] The first two steps can be carried out at different temperatures, for example, 5 to 100 °C.

[0075] In the third step, each collection cylinder 40 is connected to a container 12 while the single plate 10 remains in the first position. Thus, when the corresponding cylinder connection portion 42 is connected to the container connection portion 15 by form fit, the cylinder opening 43 of the collection cylinder 40 faces downward. Accordingly, the collection cylinder 40 is arranged on the top side of the single plate 10.

[0076] In the fourth step, the decomposed samples are transferred (e.g., pooled) from the respective containers 12 to the connected collection cylinders 40. In particular, after the collection cylinder 40 and the container 12 are connected to each other, the entire sample substrate 100 is inverted. In other words, the sample substrate 100 is moved to the second position, where the container 12 and the collection cylinder 40 are arranged on the bottom side of the single plate 10. In this way, the decomposed samples can flow from the respective containers 12 to the connected collection cylinders 40 by gravity.

[0077] In practice, the sample transfer (e.g., pooling) is further facilitated by centrifuging the sample substrate 100, for example, in a laboratory centrifuge, such that the decomposed samples from all the containers 12 flow to the bottom of their respective connected collection cylinders 40.

[0078] In the fifth step, the collection cylinder 40 containing the decomposed sample is separated from the reaction substrate device, i.e., the single plate 10, and connected to the LC-MS device. The LC-MS device has an input portion for sample input. According to a preferred embodiment of the present invention, due to the shape matching between the cylinder connection portion and the sample input portion of the LC-MS device, the collection cylinder 40 can be directly connected to the LC-MS device (without additional pipetting steps). Preferably, the LC-MS / MS device is a tandem mass spectrometer including a liquid chromatography unit and a mass spectrometer. In the case of Evotips ® being used as the collection cylinder 40, the LC-MS device can be an Evosep ® liquid chromatography system for mass spectrometry.

[0079] Finally, in the sixth step, LC-MS sample analysis is performed using the LC-MS device. For example, the peptide fragments in the decomposed sample are ionized, and the first spectrometer platform (survey scan) separates the ions by the mass-to-charge ratio (m / z) of the ions. Ions with a specific m / z ratio are selected. These ions are split into smaller fragment ions, and these fragment ions are introduced into the second spectrometer platform (identification scan) for separation and detection by their m / z ratio. Alternatively, the mass spectrometer can rely on time-of-flight (TOF) technology. During the LC-MS sample analysis, chromatographic separation may occur in the collection cylinder 40.

[0080] According to the second double-plate embodiment of the present invention, the reaction substrate device of the sample substrate 200 includes a stack of two plates, i.e., a stack of a carrier plate 20 and a funnel plate 30.

[0081] Figure 5 Such a carrier plate 20 is shown, which comprises a plurality of preferably rectangular arrays 22, each array having at least two receiving spaces 23 for receiving a liquid sample. In the example shown, each of the plurality of arrays 22 is an array of nine receiving spaces 23, each receiving space being configured to receive a liquid sample.

[0082] In addition, each array 22 has a carrier plate connection 25 with a container opening 24. The arrays 22 are spaced apart from one another or adjacent to one another. Preferably, the container opening 24, which is in particular square or rectangular, is the only opening of the respective array 22. In the example shown, an arrangement of 12 arrays 22 is provided, which comprises two rows of 6 arrays 22 each.

[0083] The carrier plate 20 further comprises a main carrier plate portion 21 preferably having a planar shape. The main carrier plate portion 21 provides a substrate defining the outer lateral dimensions of the reaction substrate device. Characteristics such as measures of the main carrier plate portion 21 may be the same or at least similar to those of the main plate portion 11 according to the first embodiment of the present invention.

[0084] The arrays 22 are arranged on the main carrier plate 21. Preferably, all arrays 22 are arranged on one side of the main carrier plate 21, wherein the opposite side of the main carrier plate 21 can be used to place the reaction substrate device, for example on a table, when collecting samples to be analyzed in the arrays 22.

[0085] Figure 6 , Figure 7 , Figure 8 and Figure 9 The funnel plate 30 according to the second embodiment is shown. Figure 6 )、Side view( Figure 7 )、Top view( Figure 8 ) and bottom view ( Figure 9 )) shows the funnel plate 30.

[0086] The funnel plate 30 comprises a plurality of funnel-shaped open channels 32, wherein each funnel-shaped open channel 32 comprises a first end and a second end providing a container connection portion 35, the first end having a funnel connection portion 36 with a funnel connection opening 37. Each container connection portion 35 is provided with a container opening 33. Preferably, the container connections 15, 35 of the first and second embodiments are identical, and the container openings 13, 33 of the first and second embodiments are necessarily identical. In the embodiment shown, an array of funnel-shaped open channels 32 is provided, the array being arranged in rows of 6 funnel-shaped open channels 32, corresponding to Figure 5 The arrangement of the carrier plate containers 22 of the carrier plate 20 is shown.

[0087] As an example, the square cross-section of each container opening 33 is 8.2 mm * 8.2 mm, and the distance between the centers of the funnel-shaped opening channels 32 is 9 mm. Additionally, the total depth of each funnel-shaped opening channel 32 is approximately 7 to 8 mm, and its internal shape tapers, with an opening angle of 82° towards the container opening 33. The cross-section of each funnel-shaped opening channel 32 can be constant up to a depth of approximately 3 mm at the container opening 33, forming a funnel connection portion 36. The overall arrangement of the funnel-shaped opening channels 32 has an external lateral dimension of approximately 55 mm * 15 mm and a uniform thickness of approximately 7 to 8 mm (excluding the container connection portion 35).

[0088] Furthermore, in the example shown, the container connection portion 35 has the same or at least similar characteristics, particularly dimensions, as the container 12 (and thus, the container connection portion 15) of the first embodiment. The container connection portion 35 can have a constant internal shape with a cross-section of 2.5 mm.

[0089] Thus, each funnel-shaped opening channel 32 is adapted to fluidly connect the funnel connection opening 37 of the funnel connection portion 36 with the container opening 33 of the corresponding container connection portion 35.

[0090] The funnel plate 30 further includes a main funnel plate portion 31 which preferably has a planar shape. The funnel-shaped opening channels 32 are arranged within and / or preferably perpendicularly through the main funnel plate portion 31. Thus, the first end of the funnel-shaped opening channel 32 is arranged on one side of the main funnel plate portion 31 and the second end of the funnel-shaped opening channel 32, i.e., the container connection portion 35, is arranged on the opposite side of the main funnel plate portion 31. Preferably, the characteristics such as dimensions of the main funnel plate portion 31 are the same or at least similar to those of the main carrier plate portion 21. In the example shown, the main funnel plate portion 31 has the same external lateral dimension as the carrier plate portion 21, approximately 75 mm * 25 mm, and a thickness of approximately 1 mm.

[0091] Figure 10 Different views of a sample substrate 200 according to a second embodiment are shown which further includes a plurality of collection cylinders 40. The collection cylinders 40 are preferably the same for both the first and second embodiments.

[0092] Similar to Figure 4 the sample substrate 200 is shown in a simplified manner in Figure 10 and only includes three collection cylinders 40, two of which are separately connected to a container respectively, and the third is arranged adjacent to the reaction substrate device 200 to show the detachable connection of the collection cylinders 40 and the containers provided by the sample substrate 200 according to the present invention.

[0093] As Figure 10As shown in views (a), (b), and (c), the carrier plate 20 and the funnel plate 30 can be detachably connected to each other. In particular, each carrier plate connection portion 25 and each funnel connection portion 36 match each other by form fit, such that they are adapted to connect the carrier plate 20 and the funnel plate 30.

[0094] Due to this form fit connection, a container is formed, each container consisting of an array 22 of accommodation spaces connected to each other and a funnel-shaped opening channel 32, wherein direct liquid communication is provided between the corresponding array 22 and the funnel-shaped opening channel 32 via the container part opening 24 and the funnel connection opening 37.

[0095] Therefore, direct communication is provided between the corresponding carrier plate container 22 (such as the area of the accommodation space 23, etc.) and the collection cylinder 40 via the funnel-shaped opening channel 32.

[0096] Similar to the sample substrate 100 described above, the sample substrate 200 can be used to prepare samples for LC-MS analysis and / or perform LC-MS sample analysis. In particular, the sample substrate 200 is also capable of preparing single-cell or small-cell (e.g., protein) samples for single-cell proteomics analysis, where the sample amount is usually very low (usually <ng). All steps of the sample preparation protocol before subsequent sample analysis can be performed in the sample substrate 200.

[0097] In the first and second steps, the sample to be analyzed is collected in a container, specifically, in the accommodation space 23 of the array 22, and a decomposition reaction is performed on the sample in the accommodation space 23. In particular, similar to the single plate 10 according to the first embodiment, the carrier plate 20 is arranged in the first position without a plurality of collection cylinders 40. Preferably, the carrier plate 20 can be placed, for example, on a table, where the array 22 is arranged on the top side of the carrier plate 20 and / or the container part opening 24 faces upward. In this way, the sample and, for example, reagents can be supplied to the array 22 via the container part opening 24.

[0098] The first two steps can be performed with the funnel plate 30 connected or not connected to the carrier plate 20. In other words, the funnel plate 30 can be connected to the carrier plate 20 after the first two steps have been performed.

[0099] In the third step, each collection cylinder 40 is connected to a container while the carrier plate 20 remains in the first position. There are two possibilities for performing the third step. According to one possibility, the funnel plate 30 is connected to the carrier plate 20 which remains in the first position. Thus, the funnel plate 30 is arranged on the top side of the reaction substrate device, whereby the container connection portion 35 is also arranged on the top side of the reaction substrate device. Accordingly, when the corresponding cylinder connection portion 42 is connected to the container connection portion 35 by form fit, the cylinder opening 43 of the collection cylinder 40 faces downward. Or, according to another possibility, the funnel plate 30 and the carrier plate 20 are still not connected, wherein first the collection cylinder 40 is connected to the container connection portion 35 by form fit, and then the entire arrangement of the funnel plate 30 and the collection cylinder 40 is placed on top of the carrier plate 20 such that the funnel plate 30 can be connected to the carrier plate 20.

[0100] In the fourth step, the decomposed samples are transferred from the respective containers to the connected collection cylinders 40. In particular, after the collection cylinder 40 and the container are connected to each other, the entire sample substrate 200 is inverted so that the sample substrate 200 moves to the second position. The decomposed samples can flow from the respective containers to the connected collection cylinders 40 by gravity and preferably additionally by centrifugation.

[0101] The possible fifth and sixth steps can correspond to the steps as described previously with respect to the first embodiment.

[0102] For the implementation of the present invention in its various embodiments, the features of the present invention disclosed in the above description, drawings, and claims, whether individually, or in combination or sub - combination, are of importance. The present invention is not limited to the above - preferred embodiments. On the contrary, it is also possible to use various variants and derivatives of the concept of the present invention, and thus they fall within the scope of protection of the present invention. Additionally, the present invention also claims the subject matter and features of the dependent claims, which are independent of the features and claims to which they refer.

Claims

1. A sample substrate (100; suitable for preparing a sample for LC-MS analysis 200), comprising: A reaction substrate device (10; 20, 30) having a plurality of containers (12; 22, 32), each of the containers having a container connection portion (15; 35) with a container opening (13; 33) and at least one accommodation space configured to accommodate a liquid sample; and A plurality of collection cylinders (40), each of the collection cylinders having a cylinder connection portion (42) with a cylinder opening (43) and configured to accommodate the liquid sample from one of the containers (12; 22, 32), Characterized in that Each of the container connection portions (15; 35) is matched with each of the cylinder connection portions (42) by form fit such that they are adapted to connect the reaction substrate device (10; 20, 30) and the plurality of collection cylinders (40), thereby providing direct liquid communication between the containers (12) and the collection cylinders (40) via the openings (13, 43; 33, 43) of the containers (12) and the collection cylinders (40).

2. The sample substrate (100; 200) according to claim 1, wherein Each of the cylinder connection portions (42) is shaped to match the sample input portion of an LC-MS device.

3. The sample substrate (100; 200) according to any one of the above claims, wherein Each of the collection cylinders (40) has the shape of a pipette tip or a cone.

4. The sample substrate (100; 200) according to any one of the above claims, wherein One of the container connection portion (15; 35) and the cylinder connection portion (42) is formed by a protruding edge at the container opening (13; 33) or the cylinder opening (43), and the other of the container connection portion (15) and the cylinder connection portion is provided by an inner wall portion at the container opening (13; 33) or the cylinder opening (43).

5. The sample substrate (100; 200) according to claim 4, wherein The protruding edge has a conical shape.

6. The sample substrate (100; 200) according to any one of the above claims, wherein Each of the container connection portions (15; 35) and each of the cylinder connection portions (42) are adapted to be directly connected to each other.

7. The sample substrate (100) according to any one of the above claims, wherein The reaction substrate device (10) includes a single board (10) having the plurality of containers (12).

8. The sample substrate (200) according to any one of claims 1 to 6, wherein The reaction substrate device (20, 30) includes a stack of two boards, the two boards including a carrier board (20) and a funnel board (30), wherein The carrier board (20) includes a plurality of arrays (22) of at least two of the accommodation spaces (23) of the containers (32), each of the arrays (22) having a carrier board connection portion (25) with a container portion opening (24), and The funnel plate (30) includes a plurality of funnel-shaped open channels (32), wherein each of the funnel-shaped open channels (32) includes a first end and a second end providing one of the container connections (35), and the first end has a funnel connection portion (36) with a funnel connection opening (37), wherein the carrier plate connection portion (25) and the funnel connection portion (36) match each other by form fit such that they are adapted to connect the carrier plate (20) and the funnel plate (30), thereby forming the plurality of containers (22, 32), and each of the containers is composed of an array (22) of the at least two accommodation spaces (23) and one of the funnel-shaped open channels (32) that are connected to each other and in direct liquid communication via the container portion opening (24) of the array (22) and the funnel connection opening (37).

9. The sample substrate (100; 200) according to any one of the preceding claims, wherein the volume of the container (12; 22, 32) is in the range of 100 nl to 10 µl.

10. The sample substrate (100; 200) according to any one of the preceding claims, wherein the volume of the collection cylinder (40) is in the range of 10 µl to 1 ml.

11. The sample substrate (100; 200) according to any one of the preceding claims, wherein the inner diameter of one of the container connection portion (15; 35) and the cylinder connection portion (42) is equal to the outer diameter of the other of the container connection portion (15; 35) and the cylinder connection portion (42).

12. The sample substrate (100; 200) according to any one of the preceding claims, wherein the inner diameter of one of the container connection portion (15; 35) and the cylinder connection portion (42) is in the range of 1 mm to 10 mm, and the outer diameter of the other of the container connection portion (15; 35) and the cylinder connection portion (42) is in the range of 1 mm to 10 mm.

13. The sample substrate (100; 200) according to any one of the preceding claims, wherein the sample substrate (100; 200) is made of at least one of glass, plastic, silicon, polytetrafluoroethylene, polypropylene, and polycarbonate.

14. A liquid sample analysis device adapted to prepare a sample for LC-MS sample analysis and perform the LC-MS sample analysis, comprising: the sample substrate (100; 200) according to any one of the preceding claims, and an LC-MS device.

15. A method for performing LC-MS sample analysis, wherein a sample substrate (100; 200) and an LC-MS device according to any one of claims 1 to 13 are used, and the method includes: collecting a sample to be analyzed in the container (12; 22, 32) of the sample substrate (100; 200), performing a decomposition reaction on the sample in the container (12; 22, 32), connecting each of the collection cylinders (40) to one of the containers (12; 22, 32), Transfer the decomposed sample from each of the containers (12; 22, 32) to the connected collection cylinder (40), Connect the collection cylinder (40) to the LC-MS device, and Perform the LC-MS sample analysis using the LC-MS device.

16. The method according to claim 15, wherein the sample comprises biological material, in particular biological cells, cell populations and / or cell components, and the decomposition reaction comprises cell lysis and protein digestion.

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