Device and method for applying matrix material for matrix-assisted ionization

Abstract

The invention relates to a device and a method for applying matrix material for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization. The invention also relates to the use of a device according to the invention for applying matrix material for matrix-assisted ionization. The invention also relates to the use of a sample carrier comprising matrix material for matrix-assisted ionization, which has been applied by means of a method according to the invention, for ion spectrometric analyses.

Description

[0001] Apparatus and method for applying matrix material for matrix-assisted ionization

[0002] The present invention relates to a device and a method for applying matrix material for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization. The present invention also relates to the use of a device according to the invention for applying matrix material for matrix-assisted ionization. The present invention also relates to the use of a sample carrier comprising matrix material for matrix-assisted ionization, which was applied using a method according to the invention, for ion spectrometric analyses.

[0003] The invention is defined in the appended claims. Preferred aspects of the present invention will become apparent from the following description, including the examples.

[0004] To the extent that certain embodiments are designated as preferred for one aspect of the invention, the corresponding statements also apply to the other aspects of the present invention, mutatis mutandis. Preferred individual features of aspects of the invention (as defined in the claims and / or disclosed in the description) can be combined with one another and are preferably combined with one another, unless otherwise apparent to the person skilled in the art from the present text.

[0005] Ionization by matrix-assisted laser desorption / ionization (MALDI) was developed by Hillenkamp and Karas and is primarily used for the ionization of large molecules; see, for example, F. Hillenkamp, ​​J. Peter-Katalinic, Eds., MALDI MS — A Practical Guide to Instrumentation, Methods and Applications (Wiley, Weinheim, Germany, ed. 2, 2013). Preparation for MALDI involves encapsulating the analyte molecules in crystals of a suitable (MALDI) matrix material, usually on a sample support plate, or applying a matrix material to the sample to be analyzed. After preparing the sample to be analyzed with matrix material, a plasma cloud is generated by bombarding the prepared sample with a laser (usually a pulsed UV laser). In this plasma cloud, ionized matrix molecules ionize the evaporated analyte molecules.

[0006] The preparation of a sample to be analyzed with a matrix material can be performed in various ways. Conventional methods include the dried droplet method (= mixing the sample to be analyzed with a solution containing the matrix material and subsequent evaporation of the solvent used), thin-layer preparation (= applying the matrix material in a volatile solvent to a sample support, evaporating the solvent to form crystals of the matrix material on the sample support, and then applying a solution containing the sample to be analyzed to the crystallized matrix material), or the application of matrix material using a spraying method (by dissolving the matrix material in a solvent, applying the solution containing the matrix material as a spray or mist to the sample to be analyzed, and then evaporating the solvent).

[0007] Another option for preparing a sample to be analyzed with a matrix material is to transfer the matrix material into the gas phase and then re-solidify the matrix material on the sample surface to be prepared. The transfer into the gas phase is usually achieved by sublimation, and the re-solidification on the sample to be prepared is achieved by resublimation of the matrix material.

[0008] The ionization of analyte molecules using MALDI is of great importance, for example, in mass spectrometry imaging (MSI) of tissue thin sections and is predominantly used there as the preferred ionization method (see, for example, Dreisewerd, K., Bien, T., Soltwisch, J. (2022) "MALDI-2 and t-MALDI-2 Mass Spectrometry Imaging" in: Lee, YJ. (eds) "Mass Spectrometry Imaging of Small Molecules. Methods in Molecular Biology", vol 2437, Humana, New York, NY. https: / / doi.org / 10.1007 / 978-l-0716-2030-4_2).

[0009] For the analysis of tissue sections using MALDI-MSI, both the application of matrix material using a spray process and the deposition of matrix material from the gas phase (usually by sublimation of the matrix material and subsequent resublimation of the matrix material onto the sample) are generally considered. In particular, it is advisable to apply the matrix material to the sample to be analyzed by sublimation and subsequent resublimation (see "Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging" by Hankin et al. in J. Am. Soc. Mass. Spectrom. 2007, 18, 1646-1652). An advantage of this method of applying matrix material is that the crystal size of matrix material applied by resublimation is particularly small and very homogeneous, which enables high spatial resolution in the imaging process (see"Comparison Between Vacuum Sublimed Matrices and Conventional Dried Droplet Preparation in MALDI-TOF Mass Spectrometry" by Jaskolla et al. in J. Am. Soc. Mass Spectrom. 2009, 20, 1104-1114). For the application of matrix material by sublimation and resublimation, a container with matrix material is usually placed in a vacuum chamber (typically at 10 1 - IO -3mbar) so that the matrix material sublimates. Typical sublimation temperatures for common matrix materials are (depending on the prevailing pressure) in the range of 50–150 °C. The sample to be prepared with the matrix material is also positioned in the vacuum chamber in such a way that it comes into contact with the sublimated matrix material, thereby causing deposition or resublimation of the matrix material onto the sample. To promote the resublimation process, the sample to be prepared with the matrix material is often cooled (typically to temperatures up to 50 °C below room temperature).

[0010] Devices are already known from the prior art that are designed for the application of matrix material by evaporation or sublimation of matrix material and subsequent deposition or resublimation of the gaseous matrix material on the sample to be prepared. Reference is made to the following documents:

[0011] Konferenzbeitrag "Matrix Sublimation and Rehydration Chamber for Solvent-based Contrast MALDI Imaging Mass Spectrometry" von Egbejiogu et al. gezeigt auf der ASMS Conference 2021, MP131, Imaging Session;

[0012] "Novel Matrix Sublimation Device for MALDI Mass Spectrometry Imaging" von Fernandez at al. in Anal. Chem. 2019, 91, 1, 803-807;

[0013] "Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry" von Caughlin et al. in Journal of Visualized Experiments, 2017, 121, e55254, 1-8;

[0014] - EP2975393B1;

[0015] - US7880139B2;

[0016] - US9334569B2;

[0017] - US2016035553A1;

[0018] - US2020181758A1;

[0019] - W02020078830A1.

[0020] A disadvantage of prior art devices is that they typically require a relatively large amount of matrix material to be initially introduced, and only a small portion of this is deposited on the sample to be prepared. Typical ratios are 10:1. The reason for this imbalance between the matrix material introduced and the matrix material ultimately deposited on the sample to be prepared is that, after transitioning into the gas phase, a significant portion of the matrix material is often distributed throughout the entire apparatus and then deposited on the surfaces of the devices. This circumstance not only results in a significant loss of unused matrix material, but also requires regular, thorough cleaning of all internal components of the devices (especially before changing the matrix material to be applied to avoid unwanted contamination).The need for regular, laborious cleaning of conventional devices and the associated downtime of the deposition devices also reduces the available operating time of the devices and ultimately results in a strong time limitation of the possible throughput of samples that can be prepared with matrix material.

[0021] The distribution of gaseous matrix material throughout the entire apparatus setup also poses the risk of potential contamination of a vacuum pump connected to the device and the vacuum apparatus's exhaust air. This first point complicates maintenance and simultaneously shortens the required maintenance intervals (due to the increased contamination of the vacuum apparatus). Since some of the commonly used matrix substances are toxic or carcinogenic (such as 1,5-diaminonapthalein and 9-aminoacridine), the ingress of gaseous matrix material also places greater demands on the exhaust air removal and the decontamination of the pumps, for example, when they need to be sent in for maintenance.

[0022] Furthermore, the thickness obtained from matrix material applied to a sample often cannot be reproduced satisfactorily using devices known from the state of the art and is often non-uniform.

[0023] Against the background of the above-mentioned disadvantages of the prior art, it was a primary object of the present invention to provide devices and methods for preparing a sample with matrix material for matrix-assisted ionization - in particular for preparing a sample with matrix material for matrix-assisted laser desorption / ionization - which eliminate or at least mitigate one or more of the aforementioned disadvantages and thus contribute to an improvement with regard to (i) the ratio between the matrix material introduced and the matrix material deposited on the sample to be prepared, (ii) the uniformity of the applied matrix material, (iii) the reproducibility of the application result, (iv) the ease of cleaning the device, and / or (v) the throughput of preparable samples per unit time. Further objects arise from the following description and the patent claims.

[0024] The primary object of the present invention is achieved by a device for applying matrix material (more precisely for applying matrix material to a sample carrier and / or to a sample located on the sample carrier) for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization, comprising a vacuum chamber, a sample (carrier) receiving element for receiving one or more sample carriers (usually comprising a sample), a matrix receiving element for receiving matrix material, a cooling element for cooling the sample receiving element and / or for cooling sample carriers that can be attached in or to the sample receiving element, and a heating element for heating the matrix receiving element, in particular for heating matrix material that can be placed in the matrix receiving element above its sublimation or boiling temperature,wherein the sample receiving element and the matrix receiving element each have a base and a wall (preferably completely) extending around the base (or a wall preferably completely surrounding the base) and are each open on the side opposite the base, the sample receiving element and the matrix receiving element are each arranged in the vacuum chamber (for example on the inner sides of the vacuum chamber), and when the vacuum chamber is closed, the matrix receiving element projects with its open side first (through the open side of the sample receiving element) into the sample receiving element, so that the walls of the matrix receiving element and the sample receiving element at least partially overlap and at the same time a gap is formed at least at one point between the overlapping walls,through which the inner regions of the sample receiving element and matrix receiving element communicate with the (remaining) atmosphere of the vacuum chamber. In other words, the wall of the matrix receiving element, when the vacuum chamber is closed, projects lengthwise at least partially into the inner region of the sample receiving element, so that the part of the wall of the matrix receiving element that projects into the sample receiving element is (preferably completely) surrounded by the wall of the sample receiving element. The fact that the matrix receiving element, when the vacuum chamber is closed, projects with its open side first into the sample receiving element in such a way that a gap is formed or a gap remains, through which the inner regions of the sample receiving element and matrix receiving element communicate with the remaining atmosphere of the vacuum chamber, implies,that the wall of the matrix element projecting into the sample receiving element at least does not completely touch the bottom of the sample receiving element (since otherwise no gap would form or remain). Preferably, the wall of the matrix receiving element does not touch the bottom of the sample receiving element at any point when the vacuum chamber is closed, in particular to reduce or prevent heat transfer between the sample receiving element and the matrix receiving element.

[0025] The advantage of the above-mentioned arrangement of matrix receiving element and sample receiving element according to the invention is, on the one hand, that due to the at least partial overlap of the walls of matrix receiving element and sample receiving element when the vacuum chamber is closed (in the form of a partial protrusion of the matrix receiving element with its open side first into the sample receiving element), diffusion of gaseous matrix material during operation of the device into an area other than the two interior spaces of matrix receiving element and sample receiving element is made significantly more difficult or even completely prevented,so that during operation of the device, the matrix material passing into the gas phase reaches essentially all of the sample carriers mounted in the sample receiving element, and the probability and possibility of spreading and possible deposition of gaseous matrix material in areas of the device other than in or on the matrix receiving element and sample receiving element is at least significantly reduced, if not completely avoided. On the other hand, the gap that forms or exists at least at one point between the overlapping walls of the matrix receiving element and the sample receiving element when the vacuum chamber is closed ensures thatthat the interior of the matrix receiving element and the sample receiving element can nevertheless be subjected to a negative pressure after the vacuum chamber has been closed. The partial overlap of the walls of the matrix receiving element and the sample receiving element when the vacuum chamber is closed, while simultaneously leaving a gap at least at one point between the overlapping walls, is intended, on the one hand, to largely prevent the escape of gaseous matrix material from the interiors of the matrix receiving element and the sample receiving element during operation of the device and, at the same time, to maintain the possibility of creating a negative pressure for the application process. The gap that forms between the overlapping walls of the matrix receiving element and the sample receiving element when the vacuum chamber is closed acts in a certain way as a gap seal.into which only a very limited amount of gaseous matrix material can enter during the usual time period of an application process (during the usual time period for the preparation of a sample).

[0026] To further increase the sealing effect of the gap, it can also be designed in the form of a labyrinth seal (for example in the form of a tripping collar labyrinth), which can be achieved by appropriately shaping the wall of the matrix receiving element and / or sample receiving element in the overlapping area of ​​the walls.

[0027] For the purposes of the present invention, a vacuum chamber is understood to be a chamber or container which, when closed (usually by connecting a pump), can be provided with a negative pressure. Within the scope of the invention, a vacuum chamber is also designed such that it accommodates a matrix receiving element and a sample receiving element, as well as the arrangement of the matrix receiving element and sample receiving element, as described above and in the claims. The arrangement of the matrix receiving element and sample receiving element in a vacuum chamber enables the application of matrix material at pressures below atmospheric pressure, thereby reducing the sublimation or boiling temperature of the matrix material to be applied and thus facilitating, for example, sublimation of the matrix material.In addition, the application of a negative pressure allows the removal of any interfering substances in the device before the actual application process begins.

[0028] A vacuum chamber within the meaning of the present invention is further configured such that it can be opened at least for the introduction of matrix material and for the insertion and removal of sample carriers and closed again (for carrying out an application process under negative pressure). A vacuum chamber within the meaning of the present invention can be configured in several parts and, for example, have a lower part (preferably for receiving or attaching the matrix receiving element) and an upper part, preferably configured as a lid (preferably for receiving or attaching the sample receiving element).

[0029] The terms sublimation and boiling temperature are defined in the context of the present invention (according to their common understanding in the present technical field) as those temperatures at which a solid or liquid substance changes into the gas phase at a prevailing pressure.

[0030] For the purposes of the present invention, a matrix receiving element can be understood as any type of receiving element, such as a container, which meets the requirements defined above and in the claims and is suitable for receiving a matrix material.

[0031] Matrix material can be introduced into the matrix receiving element in both solid and liquid form. Another option for loading the matrix receiving element with matrix material is to dissolve the desired matrix material(s) in a solvent, such as acetone, and then add it in dissolved form to the matrix receiving element. One advantage of adding matrix material in dissolved form can be improved and more precise dosing of the matrix material. If matrix material is added to the matrix receiving element in dissolved form, it is usually necessary to wait until the solvent used has completely or almost completely evaporated before beginning the application process.

[0032] Examples of suitable matrix materials that can be used in a device according to the invention are 2-aminobenzoic acid (o-aminobenzoic acid, anthranilic acid), 3-aminobenzoic acid (m-aminobenzoic acid), 4-aminobenzoic acid (p-aminobenzoic acid), 2,3-dihydroxybenzoic acid (2,3-DHB), 2,4-dihydroxybenzoic acid (2,4-DHB, ß-resorcylic acid), 2,5-dihydroxybenzoic acid (2,5-DHB, gentisic acid, hydroquinonecarboxylic acid), 2,6-dihydroxybenzoic acid (2,6-DHB, γ-resorcylic acid), 3,4-dihydroxybenzoic acid (3,4-DHB, protocatechuic acid), 3,5-dihydroxybenzoic acid (3,5-DHB, α-resorcylic acid), super-DHB (a 90:10 mixture of 2,5-DHB and 2-Hydroxy-5-methoxy-benzoic acid), 3,5-Dimethoxy-4-hydroxy-cinnamic acid (Sinapinic acid), 4-Hydroxy-3-methoxycinnamic acid (Ferulic acid), 3,4-Dihydroxycinnamic acid (Caffeic acid), CHCA (a-Cyano-4-hydroxycinnamic acid), Pyridine-2-carboxylic acid (Picolinic acid, PA), Pyridine-3-carboxylic acid (Nicotinic acid), Pyridine-4-carboxylic acid (Isonicotinic acid), 3-Hydroxypicolinic acid (3-HPA),Butanedioic acid (succinic acid), glycerin (glycerol, propane-1,2,3-triol), 1,5-diaminonapthalein (1,5-DAN), 9-aminoacridine (9AA), 2,4,6-trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone, 2-nitrobenzonitrile (2-NBN, o-nitrobenzonitrile), 3-nitrobenzonitrile (3-NBN, m-nitrobenzonitrile), 4-nitrobenzonitrile (4-NBN, p-nitrobenzonitrile), 5-methyl-2-nitrobenzonitrile, 2-bromo-2-nitro-l,3-propanediol (Bronopol), 4-nitrobenzaldehyde (p-nitrobenzaldehyde), 2,3,7,8-Tetrahydroxychromeno[5,4,3-cde]chromene-5,10-dione (ellagic acid), 3-phenylacrylic acid (cinnamic acid) and its derivatives, 6-aza-2-thiothymidine, urea, 1,6-hexanedioic acid (adipic acid), 1,3-propanedioic acid (malonic acid), 2,5-dihydroxyacetophenone (2,5-DHAP) and 2-(4-hydroxyphenylazo)benzoic acid (HABA).

[0033] In the sense of the present invention, the bottom and wall of the matrix receiving element are substantially, preferably completely, impermeable to gaseous matrix material and are connected to one another in such a way that matrix material passing into the gas phase can leave the interior of the matrix receiving element substantially, preferably exclusively, only via the open side opposite the bottom.

[0034] For the purposes of the present invention, a sample (carrier) receiving element can be understood as any type of receiving element that meets the requirements defined above and in the claims and is suitable for receiving one or more sample carriers. The type of receiving of sample carriers, which usually contain a sample to be analyzed, can be designed in various ways. In a preferred embodiment, the base of the sample receiving element has one or more recesses into which the sample carriers can be inserted. In a further preferred embodiment, one or more holders into which the sample carriers can be inserted are attached to the base of the sample receiving element.Therefore, a device according to the invention is preferred, wherein the base of the sample receiving element has one or more holding elements and / or recesses for the reversible attachment and / or insertion of sample carriers. However, other types of sample (carrier) holding are also conceivable, for example, gluing the sample carriers to the base of the sample receiving element.

[0035] The sample carriers that can be accommodated by the sample receiving element can have different shapes within the scope of the invention. Typically, the sample receiving element is designed and configured to accommodate sample carriers typical for matrix-assisted ionization, in particular for matrix-assisted laser desorption / ionization (MALDI). In this case, the sample carriers can be, for example, a stainless steel plate or a glass slide, preferably having an indium tin oxide (ITO) coating. Holders or recesses on or in the base of the sample receiving element are designed such that they can accommodate sample carriers of conventional dimensions, for example sample carriers with dimensions of 75 mm x 25 mm.

[0036] Analogous to the matrix receiving element, within the meaning of the present invention, the bottom and wall of the sample receiving element are preferably also substantially, particularly preferably completely, impermeable to gaseous matrix material. The sample receiving element is designed and arranged in the device in such a way that, during operation of the device, gaseous matrix material entering the interior of the sample receiving element cannot usually leave it, or can only leave it to an insignificant extent, via the bottom or wall of the sample receiving element.In the case of preferred embodiments in which the base of the sample receiving element has one or more recesses for receiving sample carriers, these recesses are covered during operation of the device by corresponding sample carriers or otherwise (for example by a cooling element arranged on the outside of the base of the sample receiving element during operation of the device), so that even in such embodiments no or only insignificant amounts of gaseous matrix material can pass through the base of the sample receiving element into the remaining area of ​​the device during operation of the device.

[0037] An essential aspect of the present invention (as already explained above) is that, when the vacuum chamber is closed, the matrix receiving element projects into the sample receiving element with its open side facing forward, so that the walls of the matrix receiving element and the sample receiving element at least partially overlap, and at the same time, at least at one point between the overlapping walls, a gap is formed, through which the inner regions of the sample receiving element and the matrix receiving element communicate with the atmosphere of the vacuum chamber. This means that the vacuum chamber of a device according to the invention is designed such that the matrix receiving element and the sample receiving element belonging to the device can be arranged accordingly.At the same time, this expresses that the matrix receiving element and sample receiving element of a device according to the invention are coordinated with one another in such a way that a corresponding arrangement is possible when the vacuum chamber is closed (i.e., for example, the dimensions of the matrix receiving element are coordinated with the sample receiving element of a device in such a way that the matrix receiving element can actually protrude into the sample receiving element with its open side first, so that at the same time a gap remains or is formed at least at one point between the overlapping walls). Devices according to the invention are preferably designed such that, when the vacuum chamber is closed, the matrix receiving element is located in the lower region of the vacuum chamber and the sample receiving element is located in the upper region of the vacuum chamber.Since, when the vacuum chamber is closed, the matrix receiving element projects into the sample receiving element with its open side first, the device must therefore, at least in this preferred embodiment, be designed to hold the sample receiving element above the matrix receiving element. This is usually achieved by attaching the sample receiving element to the upper region of the vacuum chamber, preferably to the lid of the vacuum chamber, in such a way that, when the vacuum chamber is closed, the sample receiving element is held by the vacuum chamber above the matrix receiving element in such a way that the sample receiving element projects into the sample receiving element with its open side first, as explained in more detail above and in the claims. However, it is also conceivable within the scope of the invention for the sample receiving element to be held above the matrix receiving element in a different way.For example, additionally or alternatively, the dimensions of the matrix receiving element and the sample receiving element can be coordinated in such a way that the walls of the matrix receiving element and the sample receiving element (when the vacuum chamber is closed) contact each other at one or more points, so that the sample receiving element (when the vacuum chamber is closed) is held above the matrix receiving element due to a frictional resistance or a clamping effect between the walls of the matrix receiving element and the sample receiving element (also when the vacuum chamber is closed).

[0038] The advantage of a predominant arrangement (with a closed vacuum chamber) of the matrix receiving element in the lower region and the sample receiving element in the upper region of the vacuum chamber is that the matrix material, which is often provided in the matrix receiving element as a powdered or coarse-grained solid, can be easily placed on the bottom of the matrix receiving element without having to be secured to the bottom of the matrix receiving element in any way. Conversely, at least in those preferred embodiments in which the sample receiving element is arranged above the matrix receiving element (and thus "overhead"), the bottom of the sample receiving element must be oriented to hold the sample carriers to be fastened in or to the bottom and prevent them from falling.This can be achieved, for example, by clamping holders on the base of the sample receiving element or – in cases where the sample carriers are accommodated through recesses in the base of the sample receiving element – ​​by supporting structures or holders on the edge of the recesses, onto which the sample carriers can be placed and held. Magnetic sample carriers can also be held in position by magnets.

[0039] The cooling element of the device according to the invention can be designed in various ways within the scope of the invention and, depending on the embodiment, can serve to cool the sample receiving element and / or the sample carriers that can be attached in or on the sample receiving element. Possible cooling of sample carriers that can be attached in or on the sample receiving element results in better and more targeted deposition of gaseous matrix material on the sample carrier. Possible cooling of the sample receiving element, in particular of the wall of the sample receiving element, in turn has the advantageous effect that the temperature of the sample receiving element or the temperature of the wall of the sample receiving element during an application process of matrix material remains below the sublimation temperature of the matrix material.of the matrix materials used can be held, so that any gaseous matrix material that enters the gap between the sample receiving element and the matrix receiving element is deposited on the wall of the sample receiving element upon contact. In this case, the wall of the sample receiving element can thus act as a cold trap, further hindering or reducing any discharge of matrix material into other areas of the device.

[0040] Preferably, the cooling element is designed and arranged such that essentially the bottom of the sample receiving element or the sample carriers (and samples located thereon) that can be attached to or in the sample receiving element can be cooled, while simultaneously cooling the wall of the sample receiving element. Preferably, the cooling element (when the vacuum chamber is closed) is arranged directly on the outside of the bottom of the sample receiving element and is designed, for example, in the form of a Peltier element or fan.

[0041] The cooling element and / or the sample receiving element are also preferably designed and arranged such that (at least when the vacuum chamber is closed) the best possible and extensive thermal contact is maintained between the sample carriers attachable to or in the sample receiving element and the cooling element or sample receiving element. This promotes uniform temperature control or cooling of the sample carriers and thus a more uniform deposition of matrix material on the sample carrier to be prepared.

[0042] The subject matter of the invention also includes embodiments which have more than one cooling element (for example for the separate cooling of several sample carriers located in or on the sample receiving element or for the separate cooling of sample carriers or the bottom of the sample receiving element and the wall of the sample receiving element).

[0043] The heating element of the device according to the invention serves to heat the matrix receiving element or to heat one or more matrix materials located in the matrix receiving element in order to transfer the matrix material into the gas phase. After the matrix material has transitioned into the gas phase, it can diffuse towards the sample receiving element and the sample carriers located there and then ideally deposit primarily on the sample carriers (particularly if the sample carriers are cooled significantly below the sublimation or boiling temperature of the matrix material used). Preferably, the heating element serves to heat both the base and the wall of the matrix receiving element so that matrix material, once transferred into the gas phase, does not deposit on the wall of the matrix receiving element.

[0044] The heating element preferably enables simultaneous heating of the base and wall(s). During operation of the device, both the base and the wall(s) of the matrix receiving element are typically heated to a temperature above the sublimation temperature of the respective matrix material used, so that the matrix material in the matrix receiving element, on the one hand, transitions into the gas phase and, on the other hand, upon contact with the wall of the matrix receiving element, does not deposit on it, but instead moves further toward the (preferably cooled) sample carrier and is deposited thereon. The heating element can be designed in various ways within the scope of the invention, for example in the form of a fan heater, an induction heater, a resistance heater, and / or in the form of heating cartridges.Preferably, the heating element comprises a glass ceramic plate (Ceran® field) which can be heated by an IR radiation source.

[0045] In addition to the components mentioned above, a device according to the invention can also comprise further components. Preferably, a device according to the invention additionally comprises one or more of the following components: one or more control units, for example, for controlling the pressure in the vacuum chamber, for controlling the temperature or power of the cooling element, and / or for controlling the temperature or power of the heating element; a control element, preferably a control element for monitoring and / or regulating the quantity and / or spatial distribution of the matrix material applied to the sample carrier(s) to be prepared.

[0046] The presence of a control unit allows for more precise and adaptable adjustment of operating parameters, such as pressure, temperature of the sample carriers to be prepared, and the temperature of the matrix receiving element or the base of the matrix receiving element, depending on the type of application process. Elements for monitoring and / or controlling the amount of matrix material applied to the sample carriers allow, for example, a better assessment of when an application process is complete or whether or to what extent a change in operating conditions appears necessary to achieve the most complete and uniform application of matrix material to a sample carrier.

[0047] A device according to the invention is preferred, wherein the sample receiving element, preferably the base of the sample receiving element, is designed to be able to receive more than one sample carrier simultaneously, wherein the sample receiving element, preferably the base of the sample receiving element, can preferably receive two or three sample carriers simultaneously.

[0048] The possibility of preparing more than one sample carrier (more than one sample) simultaneously advantageously increases the maximum possible throughput of preparable sample carriers (preparable samples).

[0049] A device according to the invention is preferred, wherein the matrix receiving element, preferably the base of the matrix receiving element, is designed to receive two or more than two different matrix materials separately from one another, preferably two or three different matrix materials, and / or wherein the matrix receiving element comprises one or more inner walls through which several identical or different matrix materials can be placed spatially separated from one another, wherein the one or more inner walls are preferably designed and matched to the sample receiving element such that the matrix receiving element has a separate chamber (open towards the sample carrier) for each of several sample carriers that can be attached in and / or on the sample receiving element,in which matrix material for application to a specific one of the several sample carriers can be placed spatially separated from matrix material for application to an adjacent sample carrier.

[0050] Particularly in the case of preferred embodiments in which the base of the sample receiving element is configured to accommodate more than one sample carrier simultaneously, the separate accommodation of matrix material in the matrix receiving element for each sample carrier attachable to the sample receiving element enables better control of the amount and type of matrix material to be applied to a sample carrier while nevertheless simultaneously preparing several sample carriers or samples. The presence of inner walls orSeparate chambers in the matrix element for each sample carrier that can be attached to the sample receiving element contribute to the fact that the matrix material intended and presented for a respective sample carrier to be prepared hardly mixes, or in the best case not at all, with other matrix materials in other chambers even when changing into the gaseous state and is guided directly to and deposited on the sample carrier for which the presented matrix material is intended.

[0051] The presence of separate chambers in the matrix receiving element for each sample carrier that can be attached to the sample receiving element also makes it possible, for example in cases where a device designed for the simultaneous preparation of several sample carriers is used for the preparation of only one sample carrier, for the matrix material provided to be spread out during transition to the gas phase primarily or exclusively in the direction of the sample carrier to be prepared, thus hardly being able to deposit or not depositing at any locations other than on the sample carrier to be prepared, thus enabling the desired high ratio of matrix material deposited on a sample carrier to matrix material provided.

[0052] Further preferred within the scope of the present invention are devices wherein a plurality of sample carriers that can be simultaneously received by the sample receiving element can be cooled separately from one another and / or a plurality of matrix materials that can be separately received by the matrix receiving element can be heated separately from one another. Such a preferred embodiment of the device according to the invention makes it possible, for example, to carry out temperature control at only one of possibly several locations in the device for the preparation of a sample carrier or, in the case of the simultaneous preparation of several sample carriers, to specifically select the temperature conditions for the respective application process and, for example, to adapt them to the respective type and quantity of the matrix material provided for a sample carrier to be prepared.

[0053] A device according to the invention is preferred, wherein the bottoms (or the bottom surfaces) of the sample receiving element and the matrix receiving element are opposite one another when the vacuum chamber is closed, wherein the bottoms (or the bottom surfaces) of the sample receiving element and the matrix receiving element are preferably aligned (largely) parallel to one another when the vacuum chamber is closed.

[0054] The advantage of such a preferred alignment of the bottoms (bottom surfaces) of the sample receiving element and matrix receiving element is that it facilitates the access or diffusion of matrix material that is changing into the gas phase in the matrix receiving element towards the bottom and the one or more sample carriers to be prepared located there, which ultimately shortens the time for the deposition of matrix material on a sample carrier to be prepared and at the same time contributes to as much gaseous matrix material as possible reaching the sample carrier and depositing on it (and not elsewhere).

[0055] In the case of a sample receiving element with several sample carriers that can be accommodated therein simultaneously and a matrix receiving element with several chambers for the spatially separate accommodation of matrix material for each sample carrier that can be mounted in the sample receiving element, the floors of the respective chambers are preferably arranged directly opposite the respective positions for receiving the sample carriers when the vacuum chamber is closed, and / or the floor shapes of the chambers are preferably adapted to the shape of the sample carriers that can be accommodated in the sample receiving element opposite when the vacuum chamber is closed. Such arrangements and adaptations of the sample receiving element and matrix receiving element further contribute to ensuring that the path of each matrix material that enters the gas phase and is intended for deposition on a specific sample carrier is also primarily directed towards said sample carrier.

[0056] A device according to the invention is preferred, wherein, when the vacuum chamber is closed, the distance between the floors (or the floor surfaces) of the sample receiving element and the matrix receiving element corresponds at least to the length of the longest side of the floor of the sample receiving element and / or the floor of the matrix receiving element and / or the distance between the floor of the matrix receiving element and the sample surface of the one or more sample carriers that can be attached in and / or on the sample receiving element corresponds at least to the length of the longest side of the sample carriers that can be attached in and / or on the sample receiving element.

[0057] This serves to ensure that the matrix material provided in the matrix receiving element, after the transition into the gas phase, has sufficient space and time to distribute itself as homogeneously as possible in the interior area formed by the matrix receiving element and the sample receiving element, in order to thereby support a subsequent uniform or homogeneous deposition of the matrix material that has passed into the gas phase over the entire surface of the sample carrier to be prepared.

[0058] A device according to the invention is also preferred, wherein, when the vacuum chamber is closed, the width (or the extent) of the gap forming at least at one point between the overlapping walls is smaller than the mean free path of the gaseous particles in the inner regions of the sample receiving element and matrix receiving element prevailing immediately before the start of the application of matrix material and / or the distance between the open side of the matrix receiving element and the bottom of the sample receiving element (or the distance of the wall of the matrix receiving element to the bottom of the sample receiving element) is smaller than the mean free path of the gaseous particles in the inner regions of the sample receiving element and matrix receiving element prevailing immediately before the start of the application of matrix material and / orthe extent) of the gap forming at least at one point between the overlapping walls is less than 1 cm, preferably less than 0.85 cm, particularly preferably in the range from 0.1 cm to 1 cm, very particularly preferably in the range from 0.1 cm to 0.85 cm, and / or the distance between the open side of the matrix receiving element and the bottom of the sample receiving element (or the distance of the wall of the matrix receiving element to the bottom of the sample receiving element) is in the range from 0.1 cm to 1 cm, preferably in the range from 0.1 cm to 0.5 cm, particularly preferably in the range from 0.1 cm to 0.2 cm.

[0059] For the purposes of the present invention, the mean free path is conventionally defined as the average distance traveled by a particle without colliding with other particles. Aligning the width or extent of the gap formed at least at one point between the overlapping walls with the mean free path of the gaseous particles in the inner regions of the sample receiving element and matrix receiving element (orin the interior region formed by the sample receiving element and matrix receiving element), as described above and in the claims as preferred, has the advantage that, if gaseous matrix material particles enter the gap, there is a high probability that these gaseous matrix material particles will come into contact with the wall of the sample receiving element and (particularly if the wall of the sample receiving element is cooled to a temperature below the sublimation or boiling temperature of the matrix material) that these gaseous matrix material particles will be deposited on the wall of the sample receiving element. This advantageously further reduces the probability of gaseous matrix material being carried out and deposited in other regions of the device.

[0060] The gap prevailing between the overlapping walls is designed and dimensioned in such a way that it limits and / or largely prevents the escape of gaseous matrix material from the matrix receiving element and sample receiving element, but at the same time this gap nevertheless maintains a connection to the atmosphere outside the interior of the sample receiving element and matrix receiving element, which, for example, enables the setting and maintenance of sublimation-capable printing conditions in the interior of the receiving elements.

[0061] The prevailing distance (when the vacuum chamber is closed) between the open side of the matrix receiving element and the bottom of the sample receiving element (or the distance between the wall of the matrix receiving element and the bottom of the sample receiving element) is generally dimensioned to be as small as possible, since this (i) keeps any discharge of gaseous matrix material from the interior of the sample receiving element and matrix receiving element as small as possible and at the same time (ii) keeps the area or distance in which gaseous matrix material can come into contact with the preferably cooled wall of the sample receiving element as small as possible and thus reduces the amount of matrix material depositing on the wall of the sample receiving element (i.e. the amount of matrix material depositing instead on the sample carrier to be prepared is advantageously further increased).

[0062] A preferred device according to the invention is one in which, when the vacuum chamber is closed, the sample receiving element and the matrix receiving element do not touch each other at any point. This prevents or reduces undesirable temperature exchange between the preferably cooled wall of the sample receiving element and the preferably or usually heated wall of the matrix receiving element (i.e., heating of the wall of the sample receiving element by the wall of the matrix receiving element or cooling of the wall of the matrix receiving element by the wall of the sample receiving element).

[0063] A device according to the invention is also preferred, wherein, when the vacuum chamber is closed, the gap formed between the overlapping walls extends completely around the matrix receiving element, wherein the width of the gap extending completely around the matrix receiving element is preferably largely uniform at every point. This ensures, on the one hand, that there is no contact between the walls of the matrix receiving element and the sample receiving element, and, on the other hand, that the gap preferably has an optimal dimension at every point.

[0064] Preferably, the wall of the sample receiving element (with the vacuum chamber closed) overlaps the wall of the matrix receiving element to the greatest extent possible. This creates or maintains the longest possible gap, through which any gaseous matrix material entering the gap must pass before escaping into other areas of the device. The longer the path traveled by the gaseous matrix material through the gap, the more likely it is that the gaseous matrix material will come into contact with the preferably cooled wall of the sample receiving element, which in turn increases the probability of deposition of gaseous matrix material entering the gap on the wall of the sample receiving element and thus reduces the probability of matrix material being discharged into other areas of the device.In this context, a device according to the invention is preferred, wherein, when the vacuum chamber is closed, the wall height of the sample receiving element is longer than, preferably at least twice as long as, the distance from the bottom of the sample receiving element to the open side of the matrix receiving element and / or the ratio of the wall height of the sample receiving element to the distance between the bottom of the sample receiving element and the open side of the matrix receiving element is greater than 1, preferably at least 2:1.

[0065] These dimensions have proven to be particularly effective for preventing, as completely as possible, the deposition of matrix material in areas of the device other than the interior of the sample receiving element and matrix element or the walls of the receiving elements.

[0066] A device according to the invention is preferred, wherein the matrix receiving element and / or the sample receiving element, preferably the inner surface of the matrix receiving element and / or the sample receiving element, has a non-stick coating, preferably a non-stick coating comprising or consisting of polytetrafluoroethylene (PTFE).

[0067] The presence of such a non-stick coating promotes a homogeneous application of the matrix material to the sample carrier to be prepared. Particularly when the matrix material is introduced as a solution containing matrix material, the non-stick coating ensures that the solution is evenly distributed across the base of the matrix receiving element. If the solvent evaporates before the application process, the matrix material is deposited in a fine-grained and homogeneous manner on the base of the matrix receiving element. A homogeneous deposition or distribution of the matrix material on the base of the matrix receiving element, in turn, forms an ideal starting point for achieving a homogeneous distribution of the matrix material, which is subsequently transferred into the gas phase, across the entire interior of the matrix receiving element and the sample receiving element, and thus for a homogeneous deposition of the gaseous matrix material on the sample carrier.

[0068] The presence of a non-stick coating also allows for easier cleaning of

[0069] Sample receiving element and matrix receiving element after an application process has been completed, as this makes it easier to remove any matrix material adhering to the sample receiving element and / or matrix receiving element after an application process.

[0070] Furthermore, such a non-stick coating, in particular a coating comprising or consisting of PTFE, contributes to the chemical inertness of the surfaces of the sample receiving element and / or matrix receiving element, so that even at elevated temperatures no reactions take place between, for example, the matrix receiving element and the matrix material provided, and the surfaces of the receiving elements preferably do not serve as catalysts for any reactions.

[0071] A device according to the invention is preferred, wherein the bottom of the matrix receiving element and / or the bottom of the sample receiving element is flat and / or the wall of the matrix receiving element runs perpendicular to the bottom of the matrix receiving element and / or the wall of the sample receiving element runs perpendicular to the bottom of the sample receiving element and / or the bottoms (or the bottom surfaces) of the matrix receiving element and the sample receiving element, when the vacuum chamber is closed, are each designed and aligned with one another in such a way that the distance between the bottom (or the bottom surface) of the matrix receiving element and the bottom (or the bottom surface) of the sample receiving element is (largely) the same at every point.

[0072] The aforementioned preferred configurations of the bases and walls of the matrix receiving element and / or the sample receiving element advantageously ensure better direct access of the matrix material provided in the matrix receiving element to the sample carriers to be prepared (and thus the most complete possible deposition of the matrix material on the sample carriers) or ensure an even more homogeneous distribution of the gaseous matrix material over the entire surface of the sample carriers to be prepared (and thus a more homogeneous deposition of the matrix material on the sample carriers). A device according to the invention is preferred, wherein the sample receiving element and / or the matrix receiving element can be inserted into and removed from the vacuum chamber at least partially reversibly and non-destructively.In other words, a device according to the invention is preferred wherein the sample receiving element and / or the matrix receiving element—or at least parts of the sample receiving element and / or the matrix receiving element—is / are designed to be replaceable, or the connection between the sample receiving element and / or the matrix receiving element (or parts thereof) and the vacuum chamber is designed to be detachable. This enables easy loading of the receiving elements and allows for easy cleaning of the corresponding elements.

[0073] The reversible and non-destructive insertion and removal of the sample holding element and / or matrix holding element (or parts thereof) into the vacuum chamber enables not only easy cleaning, but also the cleaning of used holding elements during simultaneous operation of the device using another set of holding elements. It also allows for the easy exchange of holding elements of different designs for different purposes. For example, this allows for the flexible insertion and exchange of holding elements, each designed for the preparation (holding) of sample carriers of a specific size or for the simultaneous preparation of a different number of sample carriers.

[0074] A device according to the invention is preferred, wherein the size and shape of the base (or the base surface) of the matrix receiving element is adapted to the size and shape of sample carriers that can be attached to the base of the sample receiving element and / or inserted into the base of the sample receiving element, such that when the vacuum chamber is closed, the base surface of the matrix receiving element is largely congruent, preferably congruent, with the opposite surface to be coated of attachable and / or insertable sample carriers, wherein preferably no area of ​​the base surface of the matrix receiving element protrudes beyond the opposite surfaces to be coated of attached and / or inserted sample carriers.

[0075] This advantageously further improves the ratio of matrix material provided to matrix material deposited on the sample carrier to be prepared. Part of the invention is also a method for applying matrix material for matrix-assisted ionization (more precisely, for applying matrix material to a sample carrier and / or to a sample located on the sample carrier), in particular for applying matrix material for matrix-assisted laser desorption / ionization, comprising the following steps: a) providing a device according to the invention or preferably according to the invention (as defined above and in the claims), comprising a sample receiving element, a matrix receiving element, and a vacuum chamber, b) providing one or more matrix materials, c) providing one or more sample carriers, wherein the one or more sample carriers preferably comprise one or more samples,d) Attaching the one or more sample carriers in and / or on the sample receiving element, preferably attaching the one or more sample carriers in and / or on the base of the sample receiving element, wherein the attachment of sample carriers comprising a sample is preferably carried out in such a way that the surface(s) of the one or more sample carriers containing the sample point into the inner region of the sample receiving element, e) Placing the one or more matrix materials in the matrix receiving element, preferably evenly distributing the one or more matrix materials on the one or more bases of the matrix receiving element, f) Closing the vacuum chamber containing the sample receiving element and the matrix receiving element, g) If necessary, setting a negative pressure in the vacuum chamber, h) If necessary, cooling the sample receiving element, i) Heating the one or more matrix materials,so that the one or more matrix materials enter the gas phase and are subsequently deposited at least partially, preferably largely completely, particularly preferably completely, on the one or more sample carriers. When attaching the one or more sample carriers in and / or on the sample receiving element, care should be taken, particularly in the case of cooling of the sample carriers, to ensure good, flat thermal contact between the sample carrier and the sample receiving element.

[0076] In the case of providing a plurality of different matrix materials and providing a matrix receiving element which has a plurality of chambers for the spatially separated placement of matrix materials, preferably only one specific one of the plurality of different matrix materials is placed in each chamber of the matrix receiving element or is evenly distributed on the base piece of the respective chamber.

[0077] Part of the invention is also the use of a device according to the invention or preferably according to the invention (as defined above and in the claims) for applying matrix material for matrix-assisted ionization (more precisely for applying matrix material to a sample carrier and / or to a sample located on the sample carrier), in particular for applying matrix material for matrix-assisted laser desorption / ionization.

[0078] The invention also includes the use of a sample carrier comprising matrix material for matrix-assisted ionization (in particular matrix material for matrix-assisted laser desorption / ionization), which has been applied using a method according to the invention (as defined above and in the claims), for ion spectrometric analyses. In particular, for ion mobility analyses, mass analyses, or combinations of ion mobility and mass analyses, for example, of thin tissue sections.

[0079] The invention is explained in more detail below using examples. The following examples are intended to describe and explain the invention in more detail without limiting its scope.

[0080] For a better understanding of the invention, reference is also made to the accompanying drawings. The elements in the drawings are not necessarily drawn to scale, but are primarily intended to illustrate the principles of the invention (largely schematically). In the drawings, corresponding elements are designated by like reference numerals throughout the different views. In the following:

[0081] Figure 1: Schematic, exemplary, cross-sectional drawing of a sample receiving element and a matrix receiving element of a device according to the invention with a closed vacuum chamber (vacuum chamber not shown in the figure).

[0082] Figure 2A: Illustration of a device according to the invention in the closed state.

[0083] Figure 2B: Representation of the device according to the invention from Figure 2A in the open state (side view).

[0084] Figure 2C: Illustration of the device according to the invention from Figure 2A in the open state (top view) and with the connection between the sample receiving element and the cooling element open.

[0085] Figure 3: Example of a pixel intensity histogram measured from a sample carrier prepared with 2,5-DHAP using the device according to the invention.

[0086] Figure 4A: Image of a sample carrier prepared with 2,5-DHAP using the device according to the invention.

[0087] Figure 4B: Image of a sample carrier prepared with 2,5-DHAP using a state-of-the-art reference device.

[0088] Figure 1 shows, by way of example, a cross-sectional drawing of a sample receiving element 12 and a matrix receiving element 13 of a device according to the invention for applying matrix material for matrix-assisted ionization in a closed vacuum chamber. The vacuum chamber of the device according to the invention is not shown in Figure 1. As can be seen from Figure 1, when the vacuum chamber is closed, the matrix receiving element 13 projects into the sample receiving element 12 with its open side first in such a way that the walls of the matrix receiving element 13 and the sample receiving element 12 at least partially overlap and, at the same time, at least at one point between the overlapping walls, a gap is formed via which the inner regions of the sample receiving element 12 and the matrix receiving element 13 are in communication with the atmosphere of the vacuum chamber.The sample receiving element 12 shown in Figure 1 has a recess onto which a sample carrier 15 can be placed, so that the sample carrier 15 is held by the sample receiving element 12, the sample carrier 15 covers the recess of the sample receiving element 12 and one side of the sample carrier (which usually has a sample to be analyzed) is in contact with the interior of the sample receiving element 12 and the matrix receiving element 13 and any matrix material located in this interior.

[0089] Also visible in Figure 1 is a cooling element 11 in the form of a Peltier element, which is arranged directly behind the sample receiving element 12 and is in contact with the sample receiving element 12 and the sample carrier 15 located on the sample receiving element 12. Also visible in Figure 1 is a heating element 14 in the form of a heating plate or heating dish, which is arranged directly below the matrix receiving element 13.

[0090] Figure 2A shows an example of a device 10 according to the invention for applying matrix material for matrix-assisted ionization. The device 10 is shown in the closed state in Figure 2A. This illustration shows the vacuum chamber 16 of the device 10 as well as a handle 18 attached to the vacuum chamber 16. In the embodiment shown, the vacuum chamber 16 has a lower part and an upper part designed as a lid, which is connected to the lower part of the vacuum chamber 16 via hinges. Also located on the vacuum chamber 16 is a closure 17, which ensures that the vacuum chamber 16 remains closed throughout the entire application process.

[0091] Figure 2B shows the device 10 according to the invention from Figure 2A in the open state. In this illustration, in addition to the vacuum chamber 16, the sample receiving element 12 arranged in the vacuum chamber 16 and the matrix receiving element 13 likewise arranged in the vacuum chamber 16 can be seen. The matrix receiving element 13 is located in the lower part of the vacuum chamber in this illustration and, according to the embodiment shown, has three chambers, in each of which matrix material can be introduced separately. The sample receiving element 12 is attached to the inside of the upper part (the lid) of the vacuum chamber 16 and has three recesses for attaching a sample carrier each. Thus, with the device 10 according to the invention shown, three sample carriers can be prepared simultaneously.When the vacuum chamber 16 is closed, the three recesses in the sample receiving element 12 and the chamber bottoms of the matrix receiving element 13 are positioned relative to each other such that each chamber bottom faces a sample carrier that can be mounted in or on the recess (TI). Furthermore, the area of ​​each chamber bottom and the area of ​​the sample carriers that can be mounted in or on the recesses are coordinated and largely identical.

[0092] According to the embodiment shown, the sample receiving element 12 and the matrix receiving element 13 are each mounted interchangeably in or on the vacuum chamber 16 and can each be inserted into and removed from the vacuum chamber 16 without causing any damage (for example, for the purpose of cleaning the receiving elements).

[0093] As shown in Figure 2B, a cooling element 11 is also arranged between the sample receiving element 12 and the lid of the vacuum chamber 16 for cooling the sample receiving element 12 and the sample carriers that can be mounted therein or thereon. Sample carriers to be prepared can be placed into the recesses of the sample receiving element 12 or attached thereto by removing or opening the sample receiving element 12 - as shown in Figure 2C. After inserting sample carriers into the recesses provided for this purpose in the sample receiving element 12, the sample receiving element 12 is moved towards the cooling element 11 such that the sample carriers located in the recesses of the sample receiving element 12 are in contact with the cooling element 11 to ensure ideal cooling of the sample carriers.At the same time, the contact between the cooling element 11 and the sample receiving element 12 together with the sample carriers also ensures a more secure hold of the sample carriers in the recesses of the sample receiving element 12.

[0094] In cases where sample carriers are placed in only one or two of the recesses of the sample receiving element 12 for an application process, the contact between the sample receiving element 12 and the cooling element 11 during operation of the device 10 also ensures that, despite this, no or hardly any gaseous matrix material can spread and deposit uncontrollably through one of the recesses without a sample carrier into the remaining areas of the device 10, since all or at least almost all of the gaseous matrix material that reaches recesses without a sample carrier is instead deposited on the cooling element 11. The side of the cooling element 11 in contact with the sample receiving element 12 is also designed such that any areas that come into contact with matrix material can be easily wiped off and thus easily cleaned of any matrix material deposited thereon.Figure 2C also allows a view into the lower part of the vacuum chamber 16 and shows the heating element 14 located below the matrix receiving element 13 as well as the pump connection 19 arranged on the rear wall of the lower part of the vacuum chamber 16.

[0095] Application of matrix material using a device according to the invention and a device known from the prior art:

[0096] Tests were conducted to apply matrix material to sample carriers using a device according to the invention (as shown in Figures 2A to 2C) and using the commercially available HTX Sublimator™ device from HTX Technologies, LLC. One sample carrier was prepared for each application process. In the tests using the device according to the invention, application tests were conducted using the middle sample carrier holder (recess) and one of the outer sample carrier holders (more precisely, using the left-hand recess of the sample receiving element when looking at the closure 17 of the device).

[0097] Application experiments were carried out using both 2,5-dihydroxyacetophenone (2,5-DHAP) and 2,5-dihydroxybenzoic acid (2,5-DHB) as matrix material.

[0098] Glass slides (MALDI IntelliSlides™) were used as sample carriers.

[0099] For each device (or for each used mounting location in the device according to the invention), three application tests were carried out for each matrix material.

[0100] For the deposition experiments, 2,5-DHAP or 2,5-DHB was first dissolved in acetone at a concentration of approximately 20 mg / mL. For the preparation of sample carriers, 250 pL of each of the two prepared solutions were added to the preheated matrix receiving element of the HTX Sublimator™ or, in the case of the device according to the invention, to the respective chamber of the preheated matrix receiving element assigned to the sample carrier to be prepared. Once the solvent had evaporated, the apparatus was closed, the desired vacuum was set, the sample carrier and the sample receiving element were cooled to a temperature significantly below the sublimation temperature, and the deposition process was started by increasing the temperature in the matrix receiving element to a temperature above the sublimation temperature of the respective matrix material.

[0101] The exact parameters used for the respective application tests can be found in Table 1 below and were selected to be as identical as possible for the tests with the device according to the invention and the tests with the HTX Sublimator™. Only the pressure applied for the application tests is slightly higher for the tests with the HTX Sublimator™ compared to the tests with the device according to the invention. This can be explained by device-specific characteristics of both devices and, in particular, by the fact that the HTX Sublimator™ setting is limited to the applied pressure.For the tests with both devices, the application conditions were nevertheless selected such that, for the respective matrix material to be applied, the temperature in the matrix receiving element was significantly above the sublimation temperature of the matrix material, and the temperature of the sample carriers to be prepared was significantly below the sublimation temperature of the matrix material. After the end of the application tests, no matrix material remained in the matrix receiving element.

[0102] Table 1

[0103] The sample carriers prepared with matrix material were each examined with regard to the quantity and homogeneity of the matrix material deposited on them.

[0104] The amount of matrix material deposited on the sample slides was determined gravimetrically by weighing the glass slides before and after the application process and calculating the difference. To determine the reference value for the maximum amount of matrix material that could be deposited on a sample slide, 250 pL of the acetone and matrix material solution used were pipetted onto a previously weighed heated reference slide. The reference slide was weighed again after pipetting and complete evaporation of the acetone, and the difference between the two weighings was determined. The relative amount of matrix material transferred from the matrix receiving element to the glass slide was then obtained by calculating the quotient of the amount of matrix material deposited during the application process and the amount of matrix material deposited on the reference slides.

[0105] To determine the homogeneity of the matrix material deposit on a sample slide, the glass slides prepared with matrix material were scanned with a flatbed scanner (Canon, LiDE 120). Pixel intensity histograms were extracted from the resulting JPG files using a user-defined Python script for each examined slide. From these pixel intensity histograms, the median as well as the 2.5th and 97.5th percentiles were calculated. An example of such a pixel intensity histogram, measured from a sample slide prepared with 2,5-DHAP using the device according to the invention, and the values ​​determined from it are shown in Figure 3. The interval containing 95% of the pixel intensities was then calculated from the difference between the 97.5th percentile and the

[0106] The 2.5th percentile (95R value) is calculated and can be interpreted as a measure of homogeneity. Images from homogeneously coated slides have pixels with approximately equal intensities and therefore exhibit a small interval (a small 95R value), while inhomogeneously coated slides exhibit a wider interval (a larger 95R value). The median of the intensity histograms is an additional measure of the amount of matrix deposited on the slides and is higher the more matrix material was deposited on the sample slide.

[0107] As already mentioned above, three application tests were conducted per device (or per holder location used in the device according to the invention) and for each matrix material used. For all prepared sample carriers obtained from these tests, the ratio of the amount of matrix material deposited on a sample carrier to the amount of matrix material applied was determined, as were the median and 95R values ​​explained above. Values ​​from sample carriers prepared under the same conditions were averaged. The resulting mean values, along with their respective standard deviations, are listed in Table 2 below.

[0108] Table 2 As can be seen from Table 2, using the device according to the invention, the introduced amount of matrix material can be deposited almost completely onto the sample carrier to be prepared. In contrast, using the device known from the prior art, only a fraction of the introduced matrix material reaches the sample carrier. The fact that significantly more of the introduced matrix material could be deposited onto the sample carriers using the device according to the invention is also confirmed by the determined median values, which are at least twice as high for sample carriers prepared using the device according to the invention compared to the sample carriers prepared with the comparison device.

[0109] The R95 values ​​given in Table 2 – which are significantly lower for measurements on sample carriers prepared using the device according to the invention than for measurements on sample carriers prepared using the comparative device known from the prior art – also indicate that a significantly more uniform application of the matrix material can be achieved using the device according to the invention. This impression is also confirmed by the appearance of the sample carriers prepared with matrix material. As an example, Figure 4A shows the image of a sample carrier prepared with 2,5-DHAP using the device according to the invention, and Figure 4B shows the image of a sample carrier prepared with 2,5-DHAP using the comparative device known from the prior art.The image of the sample carrier prepared using the device according to the invention shows a uniform (gray) surface without significant contrasts, with the exception of the four black squares at the corners, which originate from the support elements for the sample carrier, and the white circle located in the center of the left side of the sample carrier and originates from the sample carrier itself, more precisely from an optical reference point (teach mark) located on the sample carrier. In contrast, the image of the sample carrier prepared using the comparison device known from the prior art (in addition to the four black squares at the corners and the white circle on the left side) shows several inhomogeneities in the form of contrasts (perceivable as spots), particularly on the right side of the sample carrier. Some of these inhomogeneities are indicated by arrows in Figure 4B.

[0110] In summary, using the device according to the invention, sample carriers can be prepared in a reproducible manner and with significantly less loss of applied matrix material, which also have a comparatively more homogeneous distribution of the applied matrix material.

Claims

Patent claims:

1. A device for applying matrix material for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization, comprising a vacuum chamber, a sample receiving element for receiving one or more sample carriers, a matrix receiving element for receiving matrix material, a cooling element for cooling the sample receiving element and / or for cooling sample carriers that can be mounted in or on the sample receiving element, and a heating element for heating the matrix receiving element, in particular for heating matrix material that can be placed in the matrix receiving element above its sublimation or boiling temperature, wherein the sample receiving element and the matrix receiving element each have a base and a wall extending around the base and are each open on the side opposite the base, the sample receiving element and the matrix receiving element each being arranged in the vacuum chamber,and when the vacuum chamber is closed, the matrix receiving element projects into the sample receiving element with its open side first, so that the walls of the matrix receiving element and the sample receiving element at least partially overlap and, at the same time, at least at one point between the overlapping walls, a gap is formed, via which the inner regions of the sample receiving element and the matrix receiving element are connected to the atmosphere of the vacuum chamber.

2. Device according to claim 1, wherein the sample receiving element, preferably the bottom of the sample receiving element, is designed to be able to receive more than one sample carrier simultaneously, wherein the sample receiving element, preferably the base of the sample receiving element, can preferably accommodate two or three sample carriers simultaneously.

3. Device according to one of the preceding claims, wherein the matrix receiving element, preferably the base of the matrix receiving element, is designed to receive two or more than two different matrix materials separately from one another, preferably two or three different matrix materials, and / or wherein the matrix receiving element comprises one or more inner walls through which a plurality of identical or different matrix materials can be placed spatially separated from one another, wherein the one or more inner walls are preferably designed and matched to the sample receiving element such that the matrix receiving element has a separate chamber for each of a plurality of sample carriers that can be attached in and / or to the sample receiving element, in which chamber matrix material for application to a specific one of the plurality of sample carriers can be placed spatially separated from matrix material for application to an adjacent sample carrier.

4. Device according to one of the preceding claims, wherein the bottoms of the sample receiving element and the matrix receiving element are opposite one another when the vacuum chamber is closed, wherein the bottoms of the sample receiving element and the matrix receiving element are preferably aligned parallel to one another when the vacuum chamber is closed.

5. Device according to one of the preceding claims, wherein, when the vacuum chamber is closed, the distance between the bottoms of the sample receiving element and the matrix receiving element corresponds at least to the length of the longest side of the bottom of the sample receiving element and / or the bottom of the matrix receiving element and / or the distance between the bottom of the matrix receiving element and the sample surface of the one or more sample carriers that can be mounted in and / or on the sample receiving element corresponds at least to the length of the longest side of the sample carriers that can be mounted in and / or on the sample receiving element and / or the width of the gap that forms at least at one point between the overlapping walls is smaller than the mean free path of the gaseous particles in the inner regions of the sample receiving element and matrix receiving element that prevails immediately before the start of the application of matrix material and / or the distance between the open side of the matrix receiving element and the bottom of the sample receiving element is smaller than the mean free path of the gaseous particles in the inner regions of the sample receiving element and matrix receiving element that prevails immediately before the start of the application of matrix material and / or the width of theat least at one point between the overlapping walls forming a gap is less than 1 cm, preferably less than 0.85 cm, particularly preferably in the range from 0.1 cm to 1 cm, most preferably in the range from 0.1 cm to 0.85 cm, and / or the distance between the open side of the matrix receiving element and the bottom of the sample receiving element is in the range from 0.1 cm to 1 cm, preferably in the range from 0.1 cm to 0.5 cm, particularly preferably in the range from 0.1 cm to 0.2 cm, and / or the sample receiving element and the matrix receiving element do not touch at any point and / or the gap formed between the overlapping walls runs completely around the matrix receiving element, wherein the width of the gap running completely around the matrix receiving element is preferably largely uniform at every point and / or the wall height of the sample receiving element is longer than, preferably at least twice as long as, the distance from the bottom of the sample receiving element to the open side of the matrix receiving element and / or the ratio of the wall height of the sample receiving element to the distance between the bottom of the sample receiving element and the open side of the matrix receiving element is greater than 1, preferably at least 2:

1.

6. Device according to one of the preceding claims, wherein the matrix receiving element and / or the sample receiving element, preferably the inner surface of the matrix receiving element and / or the sample receiving element, has a non-stick coating, preferably a non-stick coating comprising or consisting of polytetrafluoroethylene.

7. Device according to one of the preceding claims, wherein the bottom of the matrix receiving element and / or the bottom of the sample receiving element is flat and / or the wall of the matrix receiving element runs perpendicular to the bottom of the matrix receiving element and / or the wall of the sample receiving element runs perpendicular to the bottom of the sample receiving element and / or the bottoms of the matrix receiving element and the sample receiving element are each designed and aligned with each other when the vacuum chamber is closed so that the distance between the bottom of the matrix receiving element and the bottom of the sample receiving element is the same at every point.

8. Device according to one of the preceding claims, wherein the sample receiving element and / or the matrix receiving element can be inserted into and removed from the vacuum chamber at least partially reversibly and non-destructively.

9. Device according to one of the preceding claims, wherein the base of the sample receiving element has one or more holding elements and / or recesses for the reversible attachment and / or insertion of sample carriers.

10. Device according to one of the preceding claims, wherein the size and shape of the base of the matrix receiving element is adapted to the size and shape of sample carriers that can be attached to the base of the sample receiving element and / or inserted into the base of the sample receiving element, such that when the vacuum chamber is closed, the base surface of the matrix receiving element is largely congruent with the opposite surface to be coated of attachable and / or insertable sample carriers, preferably congruent, wherein preferably no area of ​​the base surface of the matrix receiving element protrudes beyond the opposite surfaces to be coated of attached and / or inserted sample carriers.

11. A method for applying matrix material for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization, comprising the following steps: a) providing a device as defined in any one of claims 1 to 10, comprising a sample receiving element, a matrix receiving element and a vacuum chamber, b) providing one or more matrix materials, c) providing one or more sample carriers, wherein the one or more sample carriers preferably comprise one or more samples, d) Attaching the one or more sample carriers in and / or on the sample receiving element, preferably attaching the one or more sample carriers in and / or on the base of the sample receiving element, wherein the attachment of sample carriers comprising a sample is preferably carried out in such a way that the surface(s) of the one or more sample carriers containing the sample point into the inner region of the sample receiving element, e) Placing the one or more matrix materials in the matrix receiving element, preferably evenly distributing the one or more matrix materials on the one or more bases of the matrix receiving element, f) Closing the vacuum chamber containing the sample receiving element and the matrix receiving element, g) If necessary, setting a negative pressure in the vacuum chamber, h) If necessary, cooling the sample receiving element, i) Heating the one or more matrix materials,so that the one or more matrix materials pass into the gas phase and are then deposited at least partially, preferably largely completely, particularly preferably completely, on the one or more sample carriers.

12. Use of a device as defined in any one of claims 1 to 10 for applying matrix material for matrix-assisted ionization, in particular for applying matrix material for matrix-assisted laser desorption / ionization.

13. Use of a sample carrier comprising matrix material for matrix-assisted ionization, which has been applied by a method as defined in claim 11, for ion spectrometric analyses.

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