EP3964290A1

EP3964290A1 - Apparatus and method for preparing liquid samples for a sample analysis

Info

Publication number: EP3964290A1
Application number: EP20194535.9A
Authority: EP
Inventors: Anjali SETH; Tom CALATAYUD; Guilhem Tourniaire; Joshua CANTLON-BRUCE; Holger Eickhoff
Original assignee: Scienion GmbH
Current assignee: Scienion GmbH
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2022-03-09

Abstract

A sample preparing apparatus 100 for preparing a plurality of liquid samples 1 for a sample analysis, for instance for mass spectrometry, comprises a carrier plate device 10 with an array 11 of reaction sites 12, wherein the carrier plate device 10 is adapted for a first arrangement condition, wherein the reaction sites 12 are exposed and the carrier plate device 10 is configured for accommodating the samples 1 and supplying reagents to the liquid samples 1, and a collection device 20 comprising a collection vessel 21 with a vessel opening and a vessel bottom 23, wherein the collection vessel 21 is adapted for collecting the liquid samples 1 from at least two of the reaction sites 12 and providing the liquid samples 1 for the sample analysis, wherein the collection device 20 is adapted for a detachable connection with the carrier plate device 10, so that the vessel opening of the collection vessel 21 faces to the reaction sites 12, and the carrier plate device 10 is further adapted for a second arrangement condition, wherein the collection vessel 21 is connected with the carrier plate device 10 and the liquid samples 1 are capable to flow from the reaction sites 12 to the collection vessel 21 by the effect of a driving force. Furthermore, a sample preparing method for preparing a plurality of liquid samples 1 for a sample analysis is described, wherein the sample preparing apparatus 100 is used.

Description

Technical Field

The present invention relates to a sample preparing apparatus and to a sample preparing method for preparing a plurality of liquid samples for a sample analysis, like e. g. for a mass spectrometry measurement. Applications of the invention are available e. g. in the field of multiplex processing of liquid samples, in particular biological samples, e. g. in proteomics investigations.

Technical Background

In the present specification, reference is made to the following prior art relating to the technical background of the invention, in particular to preparing cell samples for mass spectrometry:

[1] US 2019/0219592 A1 ;
[2] H. Specht et al. "Single-cell mass-spectrometry quantifies the emergence of macrophage heterogeneity" doi: http://dx.doi.org/10.1101/665307;
[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;
[4] Z. Y. Li et al. "Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis" in "Anal. Chem." 2018, 90, 5430-5438; and
[5] B. Budnik et al. "SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation" in "Genome Biology" (2018) 19:161, https://doi.org/10.1186/sl3059-018-1547-5.

It is generally known that there are increasing efforts in investigating the proteome, in particular analysing the entire set of proteins that is included in biological material, like biological cells or cell aggregates or cell components. Various antibodies based techniques and mass spectrometry based techniques for analysing the proteome are available, like e. g. mass cytometry (CyTOF), matrix-assisted laser desorption/ionization (MALDI), Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), antibody-based techniques or liquid chromatography mass spectrometry (LC-MS). The latter has advantages in terms of the capability of detecting a large number of proteins per cell with high specificity.
LC-MS generally includes the following procedure. Biological cells are isolated, e. g. with a fluorescence-activated cell sorter (FACS), and subjected to cell lysis for providing proteins included in the cells. The proteins are enzymatically digested into peptides fragments. In case of analysing single biological cells, these fragments typically are labelled with labelling molecules, like e. g. tandem mass tag (TMT) labels, serving as mass reporters, optionally followed by label quenching (removing unbound labelling molecules). After pooling of the labelled samples, a separation of the peptide fragments by liquid chromatography (LC) is conducted. The peptide fragments are introduced into a tandem mass spectrometer and identified by peptide fingerprinting or tandem mass spectrometry, using bioinformatics techniques. The sample preparation steps from isolating the cells to pooling may be challenging in terms of parallel handling large numbers of samples included in small liquid quantities, keeping the assignment of the peptide fragments to certain samples and avoiding cross-contamination between different samples.
Current techniques for sample preparation are described e. g. in [1] to [4], which use the same general workflow for cell isolation, lysis, digestion, TMT labelling and sample collection, but differ with regard to the sample substrates and details of the applied reagents for lysis and digestion. According to [1] and [2], lysis and digestion is applied in a micro-well plate with 384 wells, and the labelled samples are collected into a single glass HPLC insert. According to [3], home-made patterned glass slides are used for sample preparation. Using an oil layer for reducing evaporation from small liquid samples is proposed in [4], wherein a layer stack is used for creating sample receptacles and spanning self-supported oil film above each sample receptacle.
Using the conventional substrates, like the 384-well plate, the patterned glass slides or the layer stack, has the following disadvantages. Firstly, the 384-well plate is adapted for handling relatively large volumes in a range up to 100 µl. Furthermore, the substrates require complex lid handling for minimizing evaporation. As a further disadvantage, transferring the labelled fragments from the wells to the glass HPLC insert or any other common collection vessel is time consuming, includes a risk of introducing contaminations and is prone to errors. For instance, according to [1], time-consuming pipetting one by one is used for the transferring task. Furthermore, in particular the patterned glass slides are specialized devices which are not adapted to all steps of the process chain, including further preceding sample handling steps, like cell isolation. The layer stack used in [4] has a complex and costly structure. Generally, the conventional techniques are not adapted for an automation of sample preparation, resulting in limitations in terms of time consuming processes with human interaction and the risk of contaminations.
The above problems do not occur in preparing samples for mass spectrometry only, but also with other tasks of preparing liquid samples for a sample analysis by applying reagents to multiple samples and handling the samples.

Objective of the Invention

The objectives of the invention are to provide an improved sample preparing apparatus and sample preparing method, being capable of avoiding disadvantages or limitations of conventional techniques. In particular, sample preparation is to be provided with a capability of facilitating multiplexing the sample preparation and analysis, decreasing operation time, handling reduced sample volumes down to the nL range, minimizing or avoiding the evaporation problem, and/or facilitating the sample pooling. According to further aspects, the sample preparing apparatus is to be configured as user-friendly as possible and/or adapted for the whole process chain of a sample preparation, e. g. for mass spectrometry, in particular allowing an automation of the sample preparation.

Summary of the invention

The above objectives are respectively solved by a sample preparing apparatus and sample preparing method, comprising the features of the independent claims. Features of preferred embodiments and applications of the inventions are defined in the dependent claims.
According to a first general aspect of the invention, the above objective is solved by a sample preparing apparatus, being adapted for preparing a plurality of liquid samples for a sample analysis, comprising a carrier plate device with an array of reaction sites, wherein the carrier plate device is adapted for a first arrangement condition, wherein the reaction sites are exposed and the carrier plate device is configured for accommodating the samples and supplying reagents to the samples, and a collection device comprising a collection vessel with a vessel opening and a vessel bottom, wherein the collection vessel is adapted for collecting the samples from at least two of the reaction sites and providing the samples for the sample analysis.
According to the invention, the collection device is adapted for a detachable connection, like e. g. a connection with resilient mechanical clips or other connecting elements, with the carrier plate device, so that the vessel opening of the collection vessel faces to the reaction sites, and the carrier plate device is further adapted for a second arrangement condition, wherein the collection vessel is connected with the carrier plate device and the samples are capable to flow from the reaction sites to the collection vessel by the effect of a driving force.
According to a second general aspect of the invention, the above objective is solved by a sample preparing method for preparing a plurality of liquid samples for a sample analysis, wherein the sample preparing apparatus according to the first general aspect of the invention or an embodiment thereof is used. The sample preparing method comprises a processing phase, wherein the carrier plate device is arranged with the first arrangement condition, including the steps of supplying the liquid samples, in particular including at least one of at least one biological cell and parts thereof, to the reaction sites, supplying reagents to the liquid samples at the reactions sites, in particular including reagents inducing cell lysis and protein digestion into peptides, and supplying site specific labelling molecules to the liquid samples at each of the reaction sites, and a collection phase, including the steps of connecting the collections vessel to the carrier plate device, changing the carrier plate device so that it is arranged with the second arrangement condition and flowing the liquid samples from the reaction sites of the array of reaction sites to the collection vessel. The labelling molecules (or so called bar code molecules) comprise any specific molecule connecting with the sample and being specific for the reaction site, like e. g. TMT labelling molecules.
The inventive sample preparing apparatus comprises the carrier plate with one or more arrays of reactions sites. The term "reaction site" refers to a delimited section of the carrier plate device being capable of accommodating a liquid sample (in particular sample droplet). The carrier plate device comprises a substrate plate which preferably has a plane shape. The reaction site is a sample receptacle provided by a surface of the substrate plate. Samples at the reactions sites are spatially separated from each other, e. g. by a rim delimiting a reaction site from neighbouring reactions sites, in particular surrounding a reaction site, or by the distance between the reactions sites. Multiple reaction sites provide the array of reactions sites, which can have e. g. a matrix arrangement. The number of reaction sites per array can be selected in dependency on the degree of parallelism of sample handling to be obtained, in particular in dependency on the number of different labelling molecules to be applied.
The sample preparing apparatus further comprises the collection device with the collection vessel. The collection vessel is configured for simultaneously receiving samples from at least two, preferably all reactions sites. The collection vessel commonly accommodates all samples from the at least two reactions sites. To this end, the collection vessel of the collection device preferably is adapted for the detachable connection with the carrier plate device, in particular for a direct connection of the vessel opening with the at least two reactions sites. The vessel opening matches the outer shape of the at least two reactions sites, preferably the whole array of reactions sites, so that a liquid communication can be provided between the reactions sites and the collection vessel.
The carrier plate device has at least two arrangement conditions. In other words, the carrier plate device is capable to be switched between two different configurations or arrangements, in particular between different orientations in space. With the first arrangement condition, the reactions sites, optionally provided with a covering liquid, are exposed for the supply of the samples and reagents. Accordingly, with the first arrangement condition, the carrier plate device is preferably arranged with a horizontally oriented substrate plate with an arrangement of the reaction sites on an upper site thereof. Accordingly, the liquid samples and reagents can be located at the reactions sites by the effect of gravity. With the second arrangement condition, the collection vessel is connected with the carrier plate device and the carrier plate device generally is arranged with another spatial orientation. The spatial orientation of the second arrangement condition depends on type of the driving force, like a centrifugal force, an aspiration force and/or gravity, to be applied for moving the samples from the reactions sites to the collection vessel. As an example, the spatial orientation is declined relative to the horizontal alignment or even inversed so that the reactions sites face downwards (direction of gravity).
In the processing phase of the inventive method, the carrier plate device is in the first arrangement condition, so that the liquid samples, reagents and labelling molecules can be supplied to the reaction sites, e. g. with a droplet deposition technique, preferably using at least one piezoelectric droplet dispenser. Advantageously, the sample preparing apparatus is capable to be used in an automated manner. In particular, the processing phase can be conducted in a droplet handling and processing machine. In the collection phase of the inventive method, the carrier plate device is in the second arrangement condition, wherein the collection vessel is connected with the carrier plate device and the liquid samples, previously subjected to preparing reactions for a subsequent sample analysis, flow to the collection vessel. Advantageously, the collection phase can be conducted in a droplet handling and processing machine as well, preferably in the same machine like the processing phase. Thus, the sample preparing apparatus is adapted for use in the droplet handling and processing machine during all phases of sample preparation, further facilitating the automation of the preparation process.
The invention has the following further advantages. Due to the provision of reaction sites and the capability of the collection device to be directly connected with the carrier plate device, the liquid transfer from the reaction sites to the collection vessel is facilitated and accelerated. The sample volumes can be reduced depending on the application conditions, down to samples with a volume below 1 µL, in particular below 100 nL, down to e. g. 1 nL, or even below. The size of the array of reaction sites can be reduced, thus facilitating the handling of the sample preparing apparatus and facilitating measures against evaporation. By the assignment of the array of reaction sites to the collection vessel, the multiplexing of the sample preparation is supported. This advantage is even more pronounced, if multiple arrays of reactions sites and multiple collection vessels are provided as outline below. As a further advantage, the sample preparing apparatus can be easily manufactured, e. g. from a low-cost plastics material. The sample preparing apparatus can be provided and used as a single-use or reusable consumable.
As a further advantage, the application of the invention is possible for preparing the plurality of liquid samples for various types of sample analyses. Preferably, the samples include biological cells or components thereof. For example, a single cell, multiple cells or cell components can be arranged at each of the reactions sites. The samples can be prepared e. g. for a subsequent mass spectrometry, optionally combined with a liquid chromatographic separation of sample components, or for another analysis, e. g. a genomic analysis, including a sequencing measurement.
If one single biological cell is supplied to at least one of the reaction sites (i. e. one single cell per reaction site), advantages in terms of cell specific analyses can be obtained. If multiple biological cells are supplied to at least one of the reaction sites (i. e. multiple cells per reaction site), advantages in terms of reducing background noise in the subsequent analysis are obtained. With a further variant, one of the reactions sites may include multiple cells and each of the remaining reaction sites may include single cells. Advantageously, the reaction site with more than one cell can be used as a reference or as a carrier channel for the analysis of the remaining samples.
According to another preferred embodiment of the invention, at least one of the reactions sites and the collection vessel have hydrophobic inner surfaces. Advantageously, hydrophobic inner surfaces facilitate the transfer of liquid samples from the reactions sites to the collection vessel.
Particularly preferred, the inner surfaces of at least one of the reactions wells and the collection vessel are made of polytetrafluoroethylene (PTFE) or polypropylene (PP). These materials have particular advantages in terms of providing sufficiently inert and stable surfaces which do not influence the samples.
According to a further preferred embodiment of the invention, the collection vessel has an inner shape narrowing from the vessel opening towards the vessel bottom. Accordingly, a cross-sectional dimension, like a diameter, at the vessel bottom of the collection vessel is smaller than the cross-sectional dimension at the vessel opening. Advantageously, the narrowed vessel bottom facilitates the formation of a compact droplet from the liquid samples collected from the reaction sites. The free surface of the collected liquid is reduced compared with a collection on an extended flat vessel bottom, so that evaporation from the liquid is reduced and taking the collected liquid from the collection vessel is facilitated.
Particularly preferred, the collection vessel has an inner shape of an inverse pyramid or cone. The vessel bottom may be provided by a point-shaped end of the inverse pyramid or cone or, in case of a truncated inverse pyramid or cone, by a flat truncated section thereof. The pyramid or cone shape has particular advantages in terms of providing a smooth surface from the vessel opening down to the vessel bottom, thus facilitating the liquid collection from the reaction sites.
With a further advantageous embodiment of the invention, at least one of the carrier plate device and the collection device has a coupling section being adapted for a liquid tight connection of the reaction sites and the collection vessel. Accordingly, there is at least one coupling section fixed to or detachably connected with the carrier plate and/or the collection vessel. The term "coupling section" refers to any topographic structural feature or element providing a mutual matching of a portion of the carrier plate surrounding the reaction sites of the array with a portion of the collection vessel surrounding or providing the vessel opening. Advantageously, the at least one coupling section provides an inherent liquid tightness in the second arrangement condition, when the collection vessel is connected with the carrier plate device, and additional measures preventing sample loss and cross contaminations are not required. Optionally, the at least one coupling section can be provided as a connecting element for the detachable connection of the carrier plate and the collection device.
According to a particularly preferred variant, both of the carrier plate device and the collection device have coupling sections, which comprise a rim surrounding one of the array of reaction sites and the vessel opening and a notch surrounding the other one of the array of reaction sites and the vessel opening, wherein the rim and the notch are adapted for a form-fit connection. The rim-notch combination has particular advantages in terms of integration into the materials of the carrier plate and the collection vessel and a reliable liquid tight connection in particular between plastics materials.
Another advantage of the invention results from the variety of design options for providing the reactions sites. The shape of the reactions sites can be selected in dependency on application conditions of the sample preparing apparatus and the applied sample liquid handling technique. Preferably the reaction sites comprise at least one of sample wells and sample surface sections. Accordingly, each reaction site is a sample receptacle provided by at one of a plane surface section of the substrate plate or a vessel (e. g. a recess included in the substrate plate) of the carrier plate device, like a well.
According to a particularly preferred embodiment of the invention, the carrier plate device comprises at least one further array of reaction sites, and the collection device comprises at least one further collection vessel, wherein each of the collection vessels is adapted for a detachable connection with one of the arrays of reaction sites and, when the carrier plate device is in the second arrangement condition, the samples are capable to flow from each of the reaction sites to one of the collection vessels by the effect of the driving force. Preferably, the sample preparing apparatus has multiple arrays of reactions sites, to which collection vessels simultaneously can be connected. Particularly preferred, the samples are capable to flow simultaneously from each of the reaction sites to one of the collection vessels. As a main advantage of providing multiple arrays of reaction sites, the number of sample prepared in parallel can be substantially increased. For routine applications, the sample preparing apparatus may include e. g. up to 12 arrays, but even more arrays are possible as well. As an example, if each array has 4 ^∗ 4 reactions sites and the sample preparing apparatus has 12 arrays, 16 ^∗ 12 = 192 samples can be simultaneously prepared for the subsequent sample analysis.
According to another advantageous modification of the invention, the sample preparing apparatus comprises at least one holder device being adapted for accommodating at least one of the carrier plate device and the collection device. Thus, handling of the sample preparing apparatus can be facilitated in an advantageous manner. Preferably, the holder device is a single component configured for fulfilling two functions. Firstly, the holder device is adapted for holding the carrier plate during the sample preparation in the processing phase. Secondly, when the carrier plate is connected to the collection plate, the holder device is adapted for holding the collection plate in the collection phase, e. g. in a centrifuge. Alternatively, the holder device can comprise at least two holders, one for the processing phase, e. g. dispensing the liquid, and one for the collection phase, e. g. transferring the liquid from reaction sites to the collection device.
If, according to another preferred embodiment of the invention, the sample preparing apparatus further comprises a temperature setting device which is adapted for heating or cooling at least one of the carrier plate device and the collection device, advantages for setting reaction and/or storing conditions, in particular a reaction and/or storing temperature, can be obtained. The temperature setting device preferably comprises a resistance heater and/or a Peltier element. The temperature setting device can be fixedly connected with at least one of the carrier plate device and the collection device. Thus, temperature setting can be provided with increased precision and shortened adjustment times. Alternatively, with a particularly preferred variant, the temperature setting device can be coupled with the at least one holder device, thus facilitating the provision of the carrier plate and/or the collection device as consumables.
Advantageously, the reaction sites can be covered with a covering liquid before providing the liquid samples in the reaction sites, wherein the covering liquid has a mass density lower than water and the covering liquid is immiscible with water. Providing the covering liquid, in particular providing a prefilled configuration of the reaction plate, reduces the evaporation from the reactions sites. The inventors have found that e. g. biological cells or cell components are kept in a locally aqueous phase when supplied to the prefilled reactions sites, so that the sample does not contact the covering liquid, and the aqueous phase is surrounded, in particular covered, by the covering liquid. Preferably, the covering liquid comprises an oil, like e. g. an alkane (like hexadecane and/or dodecane) and/or a fluorocarbon oil.
With an advantageous variant, the reaction sites with at least one of the reagents and the labelling molecules are covered with the covering liquid at a temperature below a solidification temperature of the covering liquid, and the liquid samples are supplied to the reaction sites at a temperature above the solidification temperature of the covering liquid. Thus, the carrier plate with prefilled and safely closed reactions sites can be prepared and stored as a complete sample processing kit.

Brief description of the drawings

Further advantages and details of the invention are described in the following with reference to the attached drawings, which schematically show in:

Figure 1:: a carrier plate of a sample preparing apparatus according to a first embodiment of the invention,
Figure 2:: a carrier plate of a sample preparing apparatus according to a second embodiment of the invention;
Figure 3:: a collection device of the sample preparing apparatus according to the first embodiment of the invention;
Figure 4:: further details of the carrier plate and the collection device of the sample preparing apparatus according to a first embodiment of the invention;
Figure 5:: the sample preparing apparatus being arranged in (A) the first arrangement condition just before changing to the second arrangement condition and (B) the second arrangement condition;
Figure 6:: a perspective view of a frame of the collection device;
Figure 7:: a perspective view of a holder device of the collection device;
Figure 8:: a perspective view of a lid of the collection device; and
Figure 9:: an illustration of features of preferred embodiments of a sample preparing method according to the invention.

Description of preferred embodiments

Features of preferred embodiments of the invention are described in the following with exemplary reference to sample preparation for the LC-MS/MS analysis. It is emphasized, that the application of the invention is not restricted to this embodiment, but correspondingly possible with other analyses, e. g. by using other samples and reagents. Furthermore, while the sample preparing apparatus is schematically shown, details, like e. g. the number, shape and size of the arrays and reactions sites and/or the number, shape and size of the collection device(s) can be modified in dependency on the particular applications conditions. Details of the sample processing for proteomics investigations are not described as far as they are known per se from conventional techniques, like e. g. [1] to [4].
Figures 1 and 2 show perspective views of a first and a second embodiment of a carrier plate device 10 in a first arrangement condition thereof, in particular with multiple exposed arrays 11 of reaction sites 12. In the illustrated example, twelve arrays 12 are provided, each having sixteen reaction sites 11. As practical examples, conical reaction sites 12 have a diameter of 1,5 mm, a depth of 1 mm and a distance between centres of reaction sites 11 of 1,75 mm, and cylindrical reaction sites 12 have a diameter of 1 mm, a depth of 1,75 mm and a centre-centre-distance of 1,5 mm.
Each array 11 has a coupling section 14 formed by a rim surrounding the upper side of the array 11. The coupling sections 14 of neighbouring arrays 11 are spaced apart from each other. The coupling sections 14 fulfil a double function in terms of delimiting neighbouring arrays 11 against each other and coupling with the collection device (see e. g. Figure 4B).
The carrier plate device 10 has a main plate section 15. The main plate section 15 provides a substrate defining the outer lateral size of the carrier plate device 10. The outer lateral size preferably is selected to be equal to a size of a microscope slide, i. e. about 75 mm ^∗ 26 mm, with a thickness of about 1 mm. Alternatively, another, e. g. larger outer lateral size can be used, being matched to a specific substrate size processed in a liquid sample processing machine. The coupling sections 14 have a thickness of e. g. 0,75 mm, a height of e. g. 1 mm and a mutual distance between neighbouring array 11 of e. g. 10 mm.
According to the first embodiment (Figure 1), a platform plate section 16 is arranged on the main plate section 15 (see also Figure 4A). The platform plate section 16 is designed for providing the arrays 12 of reactions sites 11, wherein various geometries can be used in dependency on the application conditions. The reactions sites 11 comprise recesses in the platform plate section 16, and they have e. g. a cylindrical or conical shape. With an example, each array has a size of 9 mm ^∗ 9 mm with a mutual distance between neighbouring arrays of 1 mm, and the platform plate section 16 (including the coupling sections 14) has a thickness of 2,5 mm.
According to the second embodiment (Figure 2), the platform plate section is omitted, and the reactions sites 11 are provided directly on the main plate section 15, e. g. by a surface thereof. The reactions sites 11 are surface sections (see dotted circle) being delimited from the neighbouring reaction site by the mutual distance only. The coupling sections 14 comprise rims surrounding the arrays 11 on the main plate section 15.
Preferably, the carrier plate device 10 of Figures 1 and 2 is made by CNC machining or hot embossing or injection molding of a plastics material, particularly preferred as an integral component. The carrier plate device 10 is made e. g. from PTFE or from another plastics material coated with PTFE.
Figure 3 shows a perspective view of an embodiment of a collection device 20 with a plurality of collection vessels 21, each with a vessel opening 22 and a vessel bottom 23 (not shown in Figure 3, see Figure 4B). The number, arrangement and size of the collection vessels 21 is matched to the number and size of the arrays 12 of reaction sites 11 of the carrier plate device 10. Each vessel opening 22 is surrounded by a coupling section 24A, 24B provided by a notch between neighbouring collection vessels 21 and an inner side of a rim surrounding all collection vessels 21. The notch and the inner side of the rim are shaped for a liquid-tight form-fit connection with the rim-shaped coupling section 14 of the carrier plate device 10.
The carrier plate device 10 has a first arrangement condition, which is shown in Figure 4A with an enlarged view of a reaction site 12. In the first arrangement condition, all reaction sites 12 are exposed for supplying a liquid sample 1, e. g. including a single biological cell 2. Optionally, the liquid sample 1 is covered by the covering liquid 3, so that evaporation from the liquid sample 1 is suppressed. The inner surface 13 of the reaction site 12 is hydrophobic. To this end, it is preferably made of or at least coated with PTFE or another coating material having hydrophobic properties matched to the hydrophobic properties of PTFE. The inner volume of the reaction site 12 is e. g. 500 nL for the conical reactions sites or up to 1500 nL for the cylindrical reactions sites.
During the processing phase (see Figure 4A and Figure 9), wherein the liquid samples 1 and reagents are supplied to the reactions sites 12, the carrier plate device 10 is kept in the first arrangement condition.
After the processing phase, the collection device 20 is connected with the carrier plate device 10 for preparing the sample preparing apparatus 100 for the subsequent collection phase, where the samples are transferred to the collection vessels 21. This situation of the collection device 20 connected with the carrier plate device 10, while the carrier plate device 10 still is in the first arrangement condition, is shown in Figure 5A. By employing the coupling sections 14, 24A, 24B, the collection device 20 has a liquid tight connection with the carrier plate device 10.
Subsequently, when the collection vessel 21 is connected with the carrier plate device 10, the whole sample preparing apparatus 100 is turned or otherwise moved, so that the carrier plate device 10 is changed to the second arrangement condition, wherein the liquid samples 1 are capable to flow from the reaction sites 12 to the collection vessel 21 by the effect of e. g. a centrifugal force and/or gravity. This situation of the collection device 20 connected with the carrier plate device 10, wherein the carrier plate device 10 is in the second arrangement condition, is schematically shown in Figures 4B and 5B.
In practice, the sample preparing apparatus 100 preferably is subjected to a centrifugation, e. g. in a laboratory centrifuge, so that the liquid samples 1 from all reactions sites 11 belonging to a common array 11 flow to the vessel bottom 23 of the collection vessel 21 related to this array 11 (see Figure 4B).
Figures 6, 7 and 8 illustrate further preferred components of the inventive sample preparing apparatus 100, comprising a holder device 30 and a temperature setting device 40. The holder device 30 includes a frame 31 (Figure 6) for accommodating multiple collection devices, a holder box 32 (Figure 7) for holding the carrier plate devices and a lid 33 (Figure 8) for closing the holder box 32. The frame 31 has three receptacles each being adapted for accommodating of one of the collection devices (not shown in Figure 6). The holder box 32 receives the frame 31, which functions as an adaptor for aligning the collection devices relative to the carrier plate devices. Furthermore, the holder box 32 is provided with the temperature setting device 40, being integrated into the box body.
The frame 31, holder box 32 and lid 33 can be detachably connected to each other, using magnetic forces. To this end, at least one of the components is provided with at least one permanent magnet and the remaining components are provided with at least one permanent magnet and/or ferromagnetic materials. Alternatively, the components can be hold together using mechanical connectors, like resilient clips.
In practical use, the carrier plate devices can be provided in the holder box 32 in the first arrangement condition, so that the liquid samples and reagents can be supplied to the reactions sites. After the processing phase, the collection devices, coupled with the frame 31, are set on the carrier plate devices (as shown in Figure 5A). Subsequently, the lid 33 is closed and the holder device 30 including three sample preparing apparatuses is set into a centrifuge for further processing.
Figures 9A to 9D schematically illustrate the main steps of the inventive sample preparing method according to preferred embodiments of the invention.
According to Figure 9A (processing phase), when the carrier plate device 10 is in the first arrangement condition, liquid samples and reagents are supplied using a schematically shown droplet handling and processing machine 200 with a piezoelectric droplet dispenser 210, like e. g. the cellenONE cell isolation and dispensing platform (manufacturer Scienion AG, Germany). The carrier plate device 10 can be prefilled with a covering liquid, comprising e. g. an oil droplet at each reaction site, or the covering liquid droplets are supplied to the reaction sites in a preparation step. Subsequently, the liquid samples 1 with single cells 2 (see Figure 4A) are supplied to the reactions sites with the piezoelectric droplet dispenser 210.
Further in the processing phase, reagent dispensing is conducted using the droplet handling and processing machine 200. Reagents inducing cell lysis, e. g. RapiGest (trademark) or DTT (see [3]), are supplied to each reaction site. Optionally, alkylation and/or reduction agents can be supplied. Finally, reagents inducing protein digestion into peptide fragments, e. g. trypsin, are supplied to each reaction site.
Subsequently, TMT labelling molecules are dispensed using the droplet handling and processing machine 200 to label the peptide fragments. The labelling molecules are different at every reaction site in order to be able to identify which peptide fragment was coming from which reaction site (e.g. which cell) after the mass spectrometry analysis.
Finally, still in the processing phase, quenching of the TMT labelling molecules can be provided, using e. g. hydroylamine. As a result, peptide fragments are ready for analysis with LC-MS/MS.
For the analysis, the content of each of the reaction sites 11 of one array 12 (see e. g. Figure 1) needs to be combined. The collection phase, as shown in Figure 9B comprises pooling of the 16plex samples from each array of reaction sites into a common related collection vessel 21 by centrifugation. The collection device 20 is connected with the carrier plate device 10, and the whole sample preparing apparatus 100 is turned to the second arrangement condition and placed in a centrifuge. After centrifugation, peptide fragments coming from 12^∗ 16 single cells are ready for injection in the LC-MS/MS apparatus 300 (Figures 9B, 9C)
The LC-MS/MS apparatus 300 is a tandem mass spectrometer, including a liquid chromatography unit, like e. g. the mass analyzer "LTQ Orbitrap Elite" (manufacturer Thermo Fisher, USA) equipped with a "NanoAcquity" HPLC pump (manufacturer Waters, USA) (see e. g. [5]). Peptide fragments are subjected to an ionisation, and a first spectrometer stage (survey scan) separates the ions by their mass-to -charge ratio (m/z). Ions of particular m/z ratios are selected. These ions are split into smaller fragment ions, and these fragment ions are introduced into the second spectrometer stage (identification scan) for separation by their m/z-ratio and detection.
The features of the invention disclosed in the above description, the drawings and the claims can be of significance both individually as well as in combination or sub-combination for the realization of the invention in its various embodiments.

Claims

Sample preparing apparatus (100), being adapted for preparing a plurality of liquid samples (1) for a sample analysis, comprising
- a carrier plate device (10) with an array (11) of reaction sites (12), wherein the carrier plate device (10) is adapted for a first arrangement condition, wherein the reaction sites (12) are exposed and the carrier plate device (10) is configured for accommodating the samples (1) and supplying reagents to the liquid samples (1), and

- a collection device (20) comprising a collection vessel (21) with a vessel opening (22) and a vessel bottom (23), wherein the collection vessel (21) is adapted for collecting the liquid samples (1) from at least two of the reaction sites (12) and providing the liquid samples (1) for the sample analysis,
characterized in that
- the collection device (20) is adapted for a detachable connection with the carrier plate device (10), so that the vessel opening (22) of the collection vessel (21) faces to the reaction sites (12), and

- the carrier plate device (10) is further adapted for a second arrangement condition, wherein the collection vessel (21) is connected with the carrier plate device (10) and the liquid samples (1) are capable to flow from the reaction sites (12) to the collection vessel (21) by the effect of a driving force.
Sample preparing apparatus according to claim 1, wherein
- at least one of the reactions sites (12) and the collection vessel (21) have hydrophobic inner surfaces (13, 23).
Sample preparing apparatus according to claim 2, wherein
- the inner surfaces (13, 23) are made of or at least coated with PTFE or PP or another coating material having hydrophobic properties matched to the hydrophobic properties of PTFE.
Sample preparing apparatus according to one of the foregoing claims, wherein
- the collection vessel (21) has an inner shape narrowing from the vessel opening (22) towards the vessel bottom (23).
Sample preparing apparatus according to claim 4, wherein
- the collection vessel (21) has an inner shape of an inverse pyramid or cone.
Sample preparing apparatus according to one of the foregoing claims, wherein
- at least one of the carrier plate device (10) and the collection device (20) has a coupling section (14, 24A, 24B) being adapted for a liquid tight connection of the reaction sites (12) and the collection vessel (21).
Sample preparing apparatus according to claim 6, wherein
- both of the carrier plate device (10) and the collection device (20) have coupling sections, which comprise a rim (14) surrounding one of the array (11) of reaction sites (12) and the vessel opening (22) and a notch (24A) and an inner side of a rim (24B) surrounding the other one of the array (11) of reaction sites (12) and the vessel opening (22), and

- the rim and the notch (14, 24A, 24B) are adapted for a form-fit connection.
Sample preparing apparatus according to one of the foregoing claims, wherein
- the reaction sites (12) comprise at least one of sample sites and sample surface sections.
Sample preparing apparatus according to one of the foregoing claims, wherein
- the carrier plate device (10) comprises at least one further array (11A) of reaction sites (12), and

- the collection device (20) comprises at least one further collection vessel (21A), wherein

- each of the collection vessels (21, 21A) is adapted for a detachable connection with one of the arrays (11, 11A) of reaction sites (12), and

- when the carrier plate device (10) is in the second arrangement condition, the liquid samples (1) are capable to flow from each of the arrays of reaction sites (12) to one the collection vessels (21) by the effect of the driving force.
Sample preparing apparatus according to one of the foregoing claims, further comprising
- at least one holder device (30) being adapted for accommodating at least one of the carrier plate device (10) and the collection device (20).
Sample preparing apparatus according to one of the foregoing claims, further comprising
- a temperature setting device (40) being adapted for heating or cooling at least one of the carrier plate device (10) and the collection device (20).
Sample preparing method for preparing a plurality of liquid samples (1) for a sample analysis, wherein the sample preparing apparatus (100) according to one of the foregoing claims is used, comprising:
- a processing phase, wherein the carrier plate device (10) is arranged with the first arrangement condition, including the steps of supplying the liquid samples (1), in particular including at least one of biological cells (2) and parts thereof, to the reaction sites (12), supplying reagents to the liquid samples (1) at the reactions sites, in particular including reagents inducing cell lysis and protein digestion into peptides, and supplying site specific labelling molecules to the liquid samples (1) at each of the reaction sites (12), and

- a collection phase, including the steps of connecting the collections vessel to the carrier plate device (10), changing the carrier plate device (10) so that it is arranged with the second arrangement condition and flowing the liquid samples (1) from the reaction sites (12) of the array (11) of reaction sites (12) to the collection vessel (21).
Sample preparing method according to claim 12, wherein
- one single biological cell (2) is supplied to at least one of the reaction sites (12).
Sample preparing method according to one of the claims 12 to 13, wherein
- multiple biological cells (2) are supplied to at least one of the reaction sites (12).
Sample preparing method according to one of the claims 12 to 14, wherein
- the reaction sites (12) are covered with a covering liquid (3) before providing the liquid samples (1) in the reaction sites (12), wherein the covering liquid (3) has a mass density lower than water and the covering liquid (3) is immiscible with water.
Sample preparing method according to claim 15, wherein
- the reaction sites (12) with at least one of the reagents and the labelling molecules are covered with the covering liquid (3) at a temperature below a solidification temperature of the covering oil, and

- the liquid samples (1) are supplied to the reaction sites (12) at a temperature above the solidification temperature of the covering liquid (3).