Device and method for transferring material by means of laser radiation from a starting substrate onto a target substrate

Abstract

The invention relates to a device and to a method for transferring biological material (1) from a starting substrate (3) into a target substrate (4) by means of laser radiation (2), which substrates are provided with suitable indentations (5) for this purpose. The core concept behind the cross-sectional form according to the invention of the indentation (5) is that of both generating capillary forces for fixing the cells in the indentations (5), even when inverted, and also optimising the ejection from the indentation (5) of the starting substrate (3), for example by a jet effect within the indentation (5), in order to improve the material discharge with reduced thermal energy input.

Description

[0001] Apparatus and method for transferring material from a source substrate to a target substrate by means of laser radiation

[0002] The invention relates to a device and a method for transferring material, in particular biomaterials, particles and cells, from a starting substrate to a target substrate by means of laser radiation.

[0003] In laser-induced forward transfer (LIFT), the material is transferred from a source substrate to a target substrate using laser radiation. This is a laser-assisted printing process. The method enables the high-precision transfer of a wide range of materials, such as metals, plastics, and biomaterials, even living cells.

[0004] The LI FT process also allows for the processing of extremely small amounts of liquid, as there are no dead volumes due to supply lines. Furthermore, the process is very gentle on the materials being transferred, making it particularly suitable for the positioning of biomaterials.

[0005] The laser does not act directly through radiation forces as with optical tweezers, but is used only as a means of controlled energy input and thermally triggers the material transfer, whereby the materials can be embedded in different environments (matrix) as a carrier substrate.

[0006] Many different parameters must be considered in this process. Various factors influence the process. For example, different biological materials can be transferred. The beam source used also influences the process with its wavelength, pulse duration, beam profile, and focus. Process variants are also known in which the laser beam does not enter the back of the source substrate, but rather passes through the target substrate onto the front of the source substrate, thus leading the material flow in the opposite direction to that of the laser beam.

[0007] Various effects are exploited to trigger the transfer. The material to be transferred can be melted and released from the layer through thermal expansion or through evaporated components. The material either absorbs the laser radiation itself or relies on an absorbing auxiliary medium.

[0008] The absorber layer can, for example, be made of a material that releases gas upon irradiation. These can be organic molecules that release nitrogen, such as photopolymers and triazene polymers, especially aryltriazene photopolymers. Such a layer can be consumed by the laser radiation. The absorber layer can be made of a material that evaporates upon irradiation, thus converting the energy of the laser beam into kinetic energy.

[0009] For a liquid droplet to form, sufficient energy must be introduced into the system. If too little power is applied, no transfer occurs. If too much power is applied, many small droplets form, which spread as a spray onto the receiver. Ideally, a directed micro-jet area is created.

[0010] Depending on the material to be transferred, the receiving substrate must also be prepared. Specific adhesive layers for materials specifically designed for microarray production can be used. To limit the shear forces generated during the transfer of living cells, hydrogel layers are used as a cushioning layer to absorb the impact, reduce mechanical damage, and enable cell survival.

[0011] US 7,875,324 B2 describes the laser transfer of biological materials. A pulsed laser sends laser pulses into the target substrate and through the carrier. The pulses are absorbed by the intermediate layer, transferring a portion of the transfer material to a receiving substrate. The target substrate, the receiving substrate, and the photon energy source can be movable relative to each other. Laser absorption and energy conversion by the intermediate layer results in the removal of a three-dimensional pixel of biomaterial from the target and to the receiving substrate. The technique enables the transfer of very small transferred volumes ( <pL-Maßstab), Punktgrößen von nur 20 - 50 pm und hat die Fähigkeit, Muster einzelner Zellen abzuscheiden.

[0012] US Pat. No. 6,936,311 B2 relates to a method for depositing a biomaterial, such as a living cell culture, onto a receiving substrate. The laser energy has sufficient energy to cause the desorption of the transfer material and deposit it onto the receiving substrate. The laser-absorbing layer can comprise gold, chromium, and titanium.

[0013] It is also already known from the state of the art to use microtiter plates for high-throughput screening, which can contain up to several thousand wells. Filling such wells can be automated for large sample volumes using pipetting robots based, for example, on extrusion or inkjet printing.

[0014] The invention is based on the objective of significantly improving the transfer of cells, particles, or biomaterials. In particular, the transfer should be more targeted, gentle on the material, and also with different orientations of the source substrate relative to the target substrate. Furthermore, a large volume should be transferable without increasing the energy input.

[0015] This object is achieved according to the invention with a device according to the features of claim 1. The further embodiment of the invention can be found in the subclaims.

[0016] According to the invention, the starting substrate and / or target substrate has a plurality of contoured recesses defined by a bottom surface and a wall surface, which are designed to accommodate material volumes in the picoliter and nanoliter range. This enables a surprisingly simple, gentle yet reliable transfer of the material, particularly together with a carrier liquid, while simultaneously reducing the energy input of the laser radiation and increasing the transferred volume. By equipping the starting substrate with recesses that promote the material flow, particularly by concentrating or focusing it, not only is an optimal microbeam generated, which avoids losses due to spray, but the energy required for transfer is also reduced.Furthermore, the target substrate can also be equipped with corresponding recesses designed in such a way, in particular with such a small opening cross-section, that the incoming material can be held solely by the action of capillary forces. A particular advantage is the comparatively gentle impact of the material in the recess of the target substrate, which is caused by a deceleration due to capillary forces. Since the diameter of the recesses only needs to be slightly larger than the droplet diameter, the air displacement when the droplet enters the recess can be greater, which can lead to deceleration and thus a gentler transfer.

[0017] According to the invention, material can be transferred from the source substrate to the target substrate in a surprisingly simple manner because orienting one of the substrates in an overhead position, i.e., with the recess opening facing downward, is easily possible and requires no additional measures. Capillary forces hold the material in the overhead position of the source substrate until it is ejected by the thermal energy input of the laser. In the overhead position of the target substrate, the incoming material is also held by capillary forces.

[0018] This allows the relative orientation of the source substrate to the target substrate to be changed even during the transfer process, because the effects of gravity have only a minor effect.

[0019] For the same volume, wells with a higher aspect ratio can be filled further without increasing the risk of liquids mixing between the wells. On flat substrates without wells, mixing cannot be avoided because liquid media would run into each other. The wells therefore allow better utilization of the array area. In addition, wells with a high aspect ratio improve the liquid surface to volume ratio, which reduces evaporation, for example. For the same volume, liquids in wells with a high aspect ratio have a longer optical path, which improves the sensitivity of optical examination, for example. Wells with a small footprint can be arranged closer together, allowing multiple samples to be analyzed simultaneously using the same optical evaluation unit.

[0020] For samples on flat substrates, the surface area for interaction with, for example, antibodies bound to the substrate surface is smaller. This reduces the absolute number of interactions, and also increases the time required for a certain amount of binding to occur. The three-dimensional surface of substrates with recesses has greater contact with the volume of the liquid, resulting in more binding. This is advantageous because, for example, a detection threshold can be reached more quickly or when cells are stimulated with antigens, receptor interactions, etc.

[0021] The recesses are not limited to an extremely small cross-sectional area, previously unknown in the prior art. Particularly advantageously, the wall surfaces of the recesses are contoured in such a way that opposing wall sections between the base surface and the opening are not parallel, at least in some sections, but rather inclined to one another. In particular, the recesses are not cylindrical, at least in some sections, but rather conically shaped, for example, whereby several conical sections with different opening angles or even opposing opening angles can adjoin one another in the direction of the main axis.Of course, at least one cross-sectional plane, in particular in the region of the bottom surface or the opening of the recess, can have any other shape and geometry deviating from the circular shape, for example oval or polygonal, wherein pairs of opposite wall sections can run parallel and inclined to one another along the same section of the longitudinal axis.

[0022] The depressions of the starting substrate and / or target substrate have depressions that preferably have an aspect ratio (depth to width) of greater than 1 or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0023] The depressions can preferably have a circular cross-section or a rectangular cross-section. The diameter of circular depressions is preferably between 10 and 1000 pm or more, preferably 50 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, or 1000 pm. The depth of the depressions is preferably between 20 and 1100 pm or more, preferably 50 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm, or 1100 pm.

[0024] The depressions preferably have a center-to-center distance of 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, or 500% or more of the diameter or edge length. The distance from the edge of a depression to the nearest edge of an adjacent depression is preferably 2 pm, 5 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, or 1000 pm or more. The density of the depressions is preferably between 0.25 per mm 2 and 6,944 per mm 2 , preferably 0.5 per mm 2 , 1 per mm 2 , 5 per mm 2 , 10 per mm 2 , 20 per mm 2 , 30 per mm 2 , 40 per mm 2 , 50 per mm 2 , 60 per mm 2 , 70 per mm 2 , 80 per mm 2 , 90 per mm 2 , 100 per mm 2 , 200 per mm 2 , 300 per mm2 , 400 per mm 2 , 500 per mm 2 , 600 per mm 2 , 700 per mm 2 , 800 per mm 2 , 900 per mm 2 , 1,000 per mm 2 , 2,000 per mm 2 , 3,000 per mm 2 , 4,000 per mm 2 , 5,000 per mm 2 , 6,000 per mm 2 .

[0025] For example, by enclosing various wall sections of rotationally symmetrical depressions with a central longitudinal axis of the depression or the axis of symmetry at an angle of inclination, jet effects are created that significantly increase the flow velocity of the material being transferred while maintaining unchanged energy input. For this purpose, the depression has a constriction with a cross-sectional area that is smaller than the base area of ​​the depression.

[0026] The wall surface of the recess is not limited to flat surfaces; rather, according to a further preferred embodiment of the invention, at least individual wall sections can have a concave or convex, non-flat profile, thereby achieving both flow optimization and an improvement in capillary effects. The pressure profile can thus be determined by the design of the wall surface.

[0027] According to a further preferred embodiment of the invention, an absorber layer is assigned to the wall or bottom surface of the recess of at least the starting substrate, onto which the laser radiation is directed. This layer can also be implemented, for example, as a coating on the bottom surface or an opposite rear surface.

[0028] In addition, the absorber can also be liquid or flowable and, due to the capillary effects within the recess, can also be used in an overhead position with an opening at the bottom, whereby the unintentional leakage of the absorber liquid is excluded.

[0029] Of course, the absorber liquid can also be used in a conventional position of the well with the bottom surface facing downwards. In another variant, the density of the absorber liquid is greater than the density of the material or carrier liquid, so that it migrates downwards under the influence of gravity to form the base layer within the well and, if necessary, displace and lift the material from the bottom surface. As the absorber material sinks to the bottom of the well and lifts existing cells, the laser radiation can be focused there, reducing the risk of cell damage from the laser radiation. Since the absorber layer may hinder microscopic analysis of the well contents, the absorber material can only be introduced shortly before the LI FT process is carried out.This is not easily possible with flat substrates according to the state of the art due to the running of liquids.

[0030] Preferably, at least some of the depressions have a bottom surface that is at least partially transparent, so that the optical detection of the sample takes place through the bottom surface, wherein at least a substantial proportion of the depressions of the sample carrier have an aspect ratio of the height of the depression to the diameter or the edge length of the opening greater than 1.

[0031] In a further particularly preferred embodiment of the invention, the starting substrate and / or the target substrate is designed as a glass substrate, wherein the depressions in the starting substrate and / or target substrate are produced by means of laser radiation. The focus of the laser radiation undergoes spatial beam shaping along a beam axis of the laser radiation, thereby creating modifications in the glass substrate, so that the depressions in the glass substrate are subsequently created by the action of an etching medium and by successive etching as a result of the anisotropic material removal in the respective region of the modifications in the glass substrate. The substrate can be made of silicon, wherein the depressions are produced by the Bosch process (reactive deep ion etching). The substrate can also be made of metal, plastic, and other materials.The substrate can consist of several materials that are bonded together, for example a silicon plate with recesses in the form of through holes and a bonded glass substrate as the bottom of the recesses.

[0032] The recesses can be created using laser drilling and other abrasive processes.

[0033] Furthermore, the object of the invention is also achieved with a method in which the laser beam is directed onto a wall surface of the recess. The invention takes advantage of the fact that the material collects with a certain probability in the area of ​​the bottom surface. A particularly material-friendly energy input is accordingly achieved by directing the laser beam onto the wall surface at a distance from the bottom surface, which, due to its contouring, in particular the cross-sectional reductions or expansions, allows the ejection of the material even when the focal point of the energy input is located between the material and the opening of the recess. The invention permits various embodiments. To further clarify its basic principle, one of them is shown in the drawing and is described below. This shows a schematic representation in

[0034] Fig. 1 shows different relative orientations of a starting substrate with respect to a target substrate, wherein at least one of the substrates has a plurality of recesses for the material;

[0035] Fig. 2 a starting or target substrate in a cross-sectional view with differently contoured depressions;

[0036] Fig. 3 shows a starting or target substrate in a plan view with several recesses with different cross-sectional shapes;

[0037] Fig. 4 different positions of possible focal points of the laser radiation within the recesses in a cross-sectional view;

[0038] Fig. 5 the targeted passivation of a selected cell within a recess;

[0039] Fig. 6 the transfer of single cells between the source and target substrate;

[0040] Fig. 7 the transfer of individual cells between the starting and the target substrate using an auxiliary material;

[0041] Fig. 8 the introduction of absorber layers before and after the introduction of a cell into the recess.

[0042] The invention is explained in more detail below with reference to the figures. Figure 1 shows the basic idea of ​​the transfer of biological material 1 in particular, such as the individual cells shown, from a starting substrate 3 into a target substrate 4 by means of laser radiation 2, which are equipped with suitable recesses 5 for this purpose. The essential idea of ​​the cross-sectional shape of the recess 5 according to the invention is, on the one hand, the generation of capillary forces for fixing the cells in the recesses 5, even in the overhead position, and, on the other hand, the optimization of the ejection from the recess 5 of the starting substrate 3, for example through a nozzle effect within the recess 5 due to a constriction E, in order to thus improve the material discharge with reduced thermal energy input.This material removal is achieved by means of the laser-induced forward transfer, also known as the LIFT process, whereby the cells are transferred directly or bound to so-called beads as an auxiliary substance or transfer aid 6, as shown in Figure 7, from the recess 5 or from the surface of the starting substrate 3 into the recess 5 or onto the surface of the target substrate 4. As shown, one of the substrates 3, 4 can also be flat. It is also possible to transfer only the beads that have bound, for example, cell biomolecules, such as DNA.

[0043] The object of the invention is the immobilization of the cells, particularly in the direction of the long extension of the substrate 3, 4. With flat substrates 3, 4, the application of the LIFT method can lead to movement of cells in the immediate vicinity of the transfer. Therefore, before each individual transfer, it is necessary to detect where cells are located and where the laser radiation 2 should be focused to avoid directly irradiating cells or transferring multiple cells. The use of recesses 5 makes it possible to determine the position 12 of the cells once and thus create a cell map. This map can then be used to focus the laser radiation 2 to suitable positions 12 during transfer. In longer experiments with cells, cell migration also always occurs. The recesses 5 limit this to a significantly smaller area per cell.

[0044] The laser radiation 2 can act either from above or from below. To transfer the material 1, the laser energy is converted into heat, which suddenly vaporizes the liquid and propagates the material 1. A medium, such as water, or reagents or added absorbers 7 can serve as the absorber 7 for the laser energy. The pressure propagation is channeled and concentrated by a wall surface 8 of the recess 5, and the material 1 is focused toward the opening 9 of the recess 5 and deflected in a targeted manner.

[0045] The wall surface 8 reduces the tendency to splash, allowing higher energy to be applied and thus a larger volume to be transported from the depression 5, thus increasing the efficiency of material transfer compared to a flat substrate surface. In addition, it is almost impossible for unwanted, unselected particles or cells to be transported because they are physically separated by the depressions 5. In contrast, with flat substrates according to the prior art, "transfer volumes" can arise or the material can be mixed by lateral flows. According to the invention, the cross-sectional constrictions shown in variant 2 in Figure 2 with the constriction E within the depressions 5 bring the diverging flows back together so that they emerge in a concentrated or focused manner.In the cross-sectional shape of the recess 5 shown in variant 3 in Figure 2, a high pressure is generated in the area of ​​a bottom surface 10, which is required to set the material 1 in motion. The increasing cross-sectional area reduces the pressure to reduce the risk of splashing. In contrast, Figure 2 in variant 4 shows a cross-sectional shape with a strong focus of the material flow due to a steadily decreasing cross-sectional area and thus a long path for even better focus.

[0046] In addition to the parameters laser power, wavelength, absorber 7, liquid for adjusting the printing process, the shape of the recesses 5 offers further possibilities to enlarge the parameter space and to optimize the transfer of the material 1 between the starting substrate 3 and the target substrate 4, and in particular to avoid the undesired and undetected mixing of cells.

[0047] The different cross-sectional shapes of the recesses 5 shown in Figure 3 also create further possibilities for influencing the transfer process with an even larger parameter space.

[0048] As can be seen in Figure 2 below, the material properties can be adapted to the various functions, namely the observation of material 1 through transparent material properties on the one hand and the absorption of laser energy on the other. This adaptation can also be advantageously achieved by using different lasers. The choice of laser source depends on the liquids used and any absorber materials used. Therefore, any type of laser source can be used.

[0049] For example, pulsed laser sources can be used. The pulse duration can range from 100 fs to 100 ps. The wavelength is selected so that the laser radiation 2 is absorbed as close as possible to the cells and not by the cells themselves. For example, water can serve as absorber 7, and a laser wavelength in the range of 2 pm can be used. Alternatively, absorber materials adapted to the wavelength of the laser source can be used. In principle, any wavelength from ultraviolet to infrared can be used. Furthermore, the glass substrate 3, 4 can be provided with an absorbing coating, for which gold is particularly suitable due to its inert and highly absorbent properties.

[0050] As shown in Figure 4, the energy input can occur in different areas of the bottom surface 10 or the wall surface 8 of the recess 5, with a pressure bubble 11 forming within the recess 5 being clearly visible in Figure 4. At some positions 12 in the immediate vicinity of the cell, local focusing may be undesirable because, as can be seen in Figure 5, this can lead to passivation of the cell. Due to the inclination of the wall surface 8, the pressure bubble 11 is directed downward and reflected.

[0051] As can be seen in Figure 7, so-called beads can also be printed or flushed into the wells 5 as transfer aids 6, for example by inkjet or LIFT, to which the material 1 to be transported is bound. This allows for reliable transport of the material 1 even with inaccurate focusing. By using a wavelength that is not absorbed by the cells, the desired transport is achieved by means of the beads contained in the wells 5 without impairing the cells.

[0052] As shown in Figure 8, a separate absorber layer can be created by introducing a hydrogel containing absorber 7. This layer is introduced, for example, before the cells are introduced into the well 5, which is particularly suitable for fluorescent cells. The absorber layer can also be introduced after the cell. Because it has a higher density, the cell floats, and the laser energy can be focused on the volume of the absorber layer without the risk of damaging the cell. For this purpose, a highly concentrated sugar solution can be used, for example, which is only introduced into the well 5 for removal.

[0053] REFERENCE MARKS LIST

[0054] 1 material

[0055] Laser beam

[0056] 2 lung initial

[0057] 3 substrate

[0058] 4 Target substrate

[0059] 5 Deepening

[0060] Transfer aid

[0061] 6 medium

[0062] 7 absorbers

[0063] 8 wall area

[0064] 9 Opening

[0065] 10 floor area

[0066] 11 Pressure bladder

[0067] 12 positions

[0068] E bottleneck

Claims

PATENT CLAIMS E 1. Device for transferring material (1), in particular biomaterials, cells and other particles, from a starting substrate (3) to a target substrate (4), characterized in that the starting substrate (3) and / or target substrate (4) has a plurality of depressions (5) delimited by a bottom surface (10) and one or more wall surfaces (8).

2. Device according to claim 1, characterized in that the transfer of the material (1) takes place by means of electromagnetic radiation, in particular laser radiation (2).

3. Device according to claim 1 or 2, characterized in that the recesses (5) are designed to accommodate a material volume of less than 1 microliter.

4. Device according to at least one of the preceding claims, characterized in that at least individual depressions (5) in the starting substrate (3) and / or target substrate (4) are designed to be cylindrical and / or conical at least in sections and / or with an angle of inclination (taper angle) of the wall surface (8) relative to the normal to the cross-sectional plane or substrate plane of between 1° and 30°.

5. Device according to at least one of the preceding claims, characterized in that at least a substantial portion of the recesses (5) of the starting substrate (3) and / or target substrate (4) has an aspect ratio of the height of the recess (5) to the cross-sectional area of ​​the recess (5) preferably greater than 1.

6. Device according to at least one of the preceding claims, characterized in that the recesses (5), in particular the aspect ratio, are dimensioned such that the transferred material (1) is held in the recess (5) by capillary forces.

7. Device according to at least one of the preceding claims, characterized in that at least individual wall sections have a non-planar course with a concave and / or convex shape.

8. Device according to at least one of the preceding claims, characterized in that the inclination of the wall sections between the bottom surface (8) and the openings (9) of the recess (5) changes at least once.

9. Device according to at least one of the preceding claims, characterized in that the recess (5) has a constriction (E) with a cross-sectional area smaller than the bottom surface (10) of the recess (5) spaced between the opening (9) and the bottom surface (10).

10. Device according to at least one of the preceding claims, characterized in that the recess (5) has a non-rotationally symmetrical, in particular polygonal cross-sectional shape in the main direction of the recess (5), in particular orthogonal to the main extension plane of the starting substrate (3) and / or target substrate (4), and / or transverse to the main direction of the recess (5).

11. Device according to at least one of the preceding claims, characterized in that the bottom surface (10) has a coating and / or an absorber layer which absorbs the laser radiation (2).

12. Device according to at least one of the preceding claims, characterized in that the starting substrate (3) and / or the target substrate (4) have at least in sections a coating which absorbs the transmission energy.

13. Device according to at least one of the preceding claims, characterized in that the starting substrate (3) and / or the target substrate (4) is designed as a glass substrate.

14. Device according to at least one of the preceding claims, characterized in that the depressions (5) in the starting substrate (3) and / or target substrate (4) designed as a glass substrate are produced by means of laser radiation, the focus of which undergoes a spatial beam shaping along its beam axis, and by means of the laser radiation (2) modifications are produced in the glass substrate along the beam axis, so that subsequently the depressions (5) in the glass substrate are formed by the action of an etching medium and by successive etching as a result of the anisotropic material removal in the respective area of ​​the modifications in the glass substrate.

15. Method for transferring material (1), in particular biomaterials and cells, by means of laser radiation (2) from a starting substrate (3) to a target substrate (4) according to at least one of the preceding claims, characterized in that the laser radiation (2) is directed onto the bottom surface (10) and / or the wall surface (8) of the recess (5).

16. The method according to claim 15, characterized in that in a first step the position of the cells is determined, in particular by means of a camera, an optical sensor, etc., and then the cells are transferred, wherein the laser radiation (2) is irradiated at a distance or with an offset to the position of each cell.

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