Glass elements and systems for inspecting biological materials and methods of making same

Abstract

The invention relates to a glass element (1) for receiving and / or transporting a biological material (60), in particular cells and / or cellular components of a human or animal or plant, in particular proteins and / or DNA and / or RNA, comprising a plurality of microchannels (2), the microchannels connect one surface (U) of the glass element (1) to the opposite surface (O) of the glass element (1) or terminate in a blind hole. The microchannel (2) narrows from the lower side (U) towards the upper side (O). In particular, the microchannel (2) is funnel-shaped at least in some regions. The invention also relates to a system (10) having such a glass element (1), which system is suitable for inspecting a biological material (60) in a spatially resolved manner and / or for detecting single molecules by means of digital PCR.

Description

Technical Field

[0001] The invention relates to a glass element with microchannels, which can be used in particular for the detection and examination of biological materials in a spatially resolved manner. Such biological materials are, for example, human, animal or plant proteins, antibodies and / or DNA of cells as components of cell clusters, such as tissue samples or cell cultures. The invention also relates to a system for the examination of biological materials and a method for producing a glass element.

[0002] The aforementioned examinations are usually referred to as cell and / or DNA diagnostics. The glass element according to the invention allows the examination of biological materials in a spatially resolved manner, also referred to as "spatial diagnostics" or "spatial biology". The aforementioned invention allows the examination of expression patterns of DNA and proteins of cells in a spatially resolved manner, wherein information about cell clusters, such as tissue samples or cell cultures, is retained. The aforementioned invention also allows the examination of single molecules, such as biomarkers in biological or medical samples, such as blood, serum or urine samples, cell supernatants, etc. Background Art

[0003] US 2015 / 0122656 A1 discloses a glass element for separating DNA from a liquid medium. In this method, a channel is drilled into a glass plate by means of a laser beam. The method is based on the introduction of a filament into the glass by means of successive pulses of an ultrashort pulse laser. By shifting the focal point of the laser, a channel with a diameter of 1 μm is formed from the filament through the substrate. This is said to allow the produced channel to have particularly smooth inner walls, and the deviations of the channel from the ideal cylindrical shape are essentially negligible.

[0004] For examination in a spatially resolved manner, it is desirable to arrange as many channels as possible as close to one another in the glass element. It is also desirable to adjust the diameter of the channels so as to achieve a sufficient collection efficiency of the biological material. However, each channel represents a weakening of the glass element and there must be sufficient glass volume between the channels in order to achieve a sufficient minimum stability to ensure that the glass element can be used effectively. There is therefore a conflict between collection efficiency and spatial resolution. Summary of the invention

[0005] The invention solves this problem by means of a glass element according to the independent claim. A corresponding glass element is provided by the method according to the invention. Preferred embodiments are defined in the dependent claims.

[0006] The invention comprises a glass element for accommodating and / or conveying biological material (60), in particular human or animal or plant cells and / or cell components, in particular proteins and / or DNA and / or RNA, wherein the glass element has a plurality of microchannels, which preferably connect one surface of the glass element to the opposite surface of the glass element or terminate in blind holes, wherein the microchannels narrow from the bottom to the top. Preferably, the microchannels are at least partially funnel-shaped.

[0007] In application, the lower side of the glass element is the side facing the biological material to be examined. The glass element is usually a plate-like element, a so-called slide. The microchannel can connect a surface of the glass element to the opposite surface of the glass element, especially connect the lower side to the upper side. The microchannel is therefore continuous. However, alternatively, the microchannel can also be formed as a blind hole, wherein the microchannel does not run through a side, which is defined as the upper side. The bottom of the blind hole is therefore located in the area of ​​the surface and / or near the surface. The microchannel widens from one side of a surface of the glass element toward the opposite surface, and preferably the microchannel is at least partially funnel-shaped.

[0008] Advantageous glass elements therefore provide that the diameter of the channel inlet of the microchannel on the bottom side is greater than the diameter of the channel outlet or channel end in the region of the top side.

[0009] This can be seen as the result of the widening of the microchannel. The channel has an opening on one side of the glass element, the diameter of which is greater than the diameter of the opening on the opposite side or the end of the blind hole. Typically, these sides are the main sides of the plate-shaped glass element. Its lower side is defined as the side facing the biological material to be examined in the application, and the upper side is the opposite side facing the inspection unit. The widening part of the channel is usually located on the lower side and therefore faces the biological material to be examined. Particularly advantageously, the microchannel has a circular or elliptical diameter. In the sense of this specification, a microchannel refers to a diameter that is in the range of a few micrometers to several hundred micrometers.

[0010] By widening the microchannels on the underside of the glass element and thus its essentially funnel shape, the collection efficiency of the biological material is increased, while between the microchannels on the upper side of the glass element there is more glass material, which ensures the mechanical stability of the glass element. Thus, the openings of the channels on the underside of the glass element can be arranged very close to each other, thereby achieving a good spatial resolution, but always providing good mechanical stability.

[0011] Likewise possible and belonging to the invention is a double funnel in which widening of the microchannels is present both on the upper side and on the lower side of the glass element. In the middle region of the microchannels, the microchannels are designed, so to speak, to be converging and / or cylindrical, and there is sufficient glass material between these regions and therefore sufficient glass material in the volume of the glass element.

[0012] An advantageous embodiment relates to a glass element, wherein in the region of the surface of the glass element the microchannels have a diameter of 5 to 200 μm, preferably 7 to 130 μm, particularly preferably 10 to 100 μm, at least in the region of the channel inlet or the channel outlet.

[0013] By selecting these diameters, the biomaterial can enter the microchannel on one side of the glass element, as described, usually the lower side, and be transported toward the opposite surface. The selection of the diameter helps or determines that only the biomaterial to be examined, which is usually less than 1 μm in length, but not undesired larger cells and / or cell components, can enter the microchannel.

[0014] Advantageously, the glass element provides that the diameter of the channel inlet is 5 μm to 200 μm, preferably 10 μm to 100 μm.

[0015] As described, this can be a conical region that tapers toward the opposite surface, or the following widening region can be a cylindrical region with a largely constant diameter or a region that widens toward the opposite surface. It is also possible that the cylindrical region is followed by a widening region.

[0016] The opening angle of the funnel-shaped region of all described microchannels is advantageously 0.1° to 30°, particularly advantageously 2° to 18°. Advantageously, the thickness of the glass element is 0.1 mm to 3 mm.

[0017] The selection of the given opening angles enables efficient production, in particular by means of the method described below. These values ​​also contribute to ensuring that the biological material to be examined can be easily transported from the channel inlet to the other surface without causing blockages.

[0018] The choice of the opening angle allows the material to be examined to be separated between two or more channels with increasing concentration within the channel or biological material at the upper side of the glass element. The ratio of the concentration increase to the square of the channel diameter (R 下 ) 2 / (R 上 ) 2 The sensitivity of the analysis is increased by fluorescence spectroscopy and targeted interaction with functional molecules on the channel wall is possible. Particularly advantageously, the concentration increases when the radius ratio is greater than 5.

[0019] The method according to the invention described below enables production within a wide range, but advantageously in particular at very thin thicknesses.

[0020] According to the above description, an advantageous glass element provides that the funnel-shaped region in the region of the channel inlet is followed by a cylindrical or funnel-shaped region, in particular in the region of the channel outlet and / or in the region of the end of the blind hole.

[0021] The aforementioned features enable glass elements to have a very high density of microchannels. The density of microchannels is also referred to as microchannel density and denotes the number of microchannels per unit area on the surface of the glass element, for example the number of microchannels per square millimeter. Since the channel inlet is always mapped to the end of the channel outlet or blind hole, the microchannel density on the upper side of the glass element is equal to the microchannel density on the lower side of the glass element. This can therefore be described as a universal microchannel density.

[0022] Particularly advantageously, the glass element according to the invention has a microchannel density of at least 20 / mm 2 or at least 50 / mm 2 , especially at least 400 / mm 2 , especially at least 10000 / mm 2 , these values ​​serve as lower limits. The upper limits can advantageously be up to 16000 / mm 2 .

[0023] Particularly advantageous is a glass element in which the channel wall contains a structure with a plurality of rounded, dome-shaped recesses, which in particular have a depth of less than 5 μm and in particular have an extent of 5 to 20 μm, and advantageously a roughness of 50 nm to 1 μm. This value is the average roughness value Ra.

[0024] The inventors have found that it is advantageous, surprisingly, for the walls of the microchannel to be constructed as non-smooth as possible, but rather to have a structure. The aforementioned depth and roughness represent advantageous embodiments. Depth here refers to the deepening in a direction perpendicular to the axis of the channel, while extension refers to the length of the recess parallel to the axis of the channel.

[0025] These structures can be efficiently fabricated by the ultrashort pulse laser fabrication method described below, followed by a subsequent etching step.

[0026] The dome-shaped recess provides an advantage that when the gel is filled into the microchannel, the gel can not only be easily filled but also can be well maintained attached to the channel.

[0027] Another particular advantage of the structure of the channel wall, in particular the dome-shaped recess, is the adjustability of the electroosmotic flow (EOF). Electroosmotic flow is the movement of a liquid along a polar surface, such as a buffer solution in a glass capillary, in an electric field due to electrical charges.

[0028] The choice of roughness allows tuning the contribution of the EOF to the movement of the biomaterial in the channel. A greater roughness of the polar surface causes a reduction in the velocity of the EOF and a possible reversal of the direction of movement of the polar biomaterial in the glass element compared to a surface with a lower roughness.

[0029] The choice of the channel wall roughness and the simultaneously adjustable opening angle allows the reverse The channels are selectively closed by the EOF and the applied electric field.

[0030] An advantageous glass element provides that the channel outlets form an outlet matrix in which the position of a channel inlet can be assigned to each channel outlet.

[0031] In particular, this makes it possible to carry out a spatially resolved examination of biological material. This is in particular because the positions of the channel outlets or alternatively the blind holes form an outlet matrix in which the position of the channel inlet can be assigned to each channel outlet.

[0032] In other words, this means that the positions of the inlet matrix can be assigned to the positions of the outlet matrix. In the simplest case, the center point of the channel inlet on the lower side of the glass element at least substantially coincides with the position of the channel outlet or the end of the blind hole on the upper side of the glass element. This corresponds to an embodiment of a microchannel in which the axis of the channel extends vertically through the main surface of the glass element. However, it is also possible for the channel axis to extend obliquely through the main surface of the glass element. However, even in this case, the position of the channel inlet can be assigned to the position of the channel outlet or the position of the end of the blind hole, so that the analysis of the biological material to be examined at the channel outlet or at the end of the blind hole can clearly lead to its position with respect to the inlet matrix and / or the channel inlet origin.

[0033] Advantageously, the glass element comprises, at least in the region of the microchannel, a glass which is characterized in that the glass has a low intrinsic fluorescence in the wavelength range of 300 nm to 700 nm.

[0034] The intrinsic fluorescence should be less than 10% of the incident light intensity. -6 Due to the low intrinsic fluorescence, the fluorescence does not interfere with the optical examination of biological materials. A suitable range for intrinsic fluorescence is, for example, 10% of the incident light intensity. -8 Up to 10 -6 .

[0035] Particularly suitable glass compositions are mentioned in the examples below. Generally, glasses which are characterized by high chemical resistance and / or high water resistance are particularly suitable. Alkali-free glasses are particularly advantageous.

[0036] Suitable glass materials may be, in particular, alkali-free glass and borosilicate glass. In particular, commercially available glasses with the product names AF32, AF35, AS87, D263, D263T, B270, MEMPAX, Willow, G-Leaf, EN-A1, and BDA-E may be considered.

[0037] According to an exemplary embodiment, the composition of the glass includes the following components expressed in weight percentage:

[0038] Element (weight%) <![CDATA[SiO2]]> 63-85 <![CDATA[Al2O3]]> 0-10 <![CDATA[B2O3]]> 5-20 <![CDATA[Li2O+Na2O+K2O]]> 2-14 MgO+CaO+SrO+BaO+ZnO 0-12 <![CDATA[TiO2+ZrO2]]> 0-5 <![CDATA[P2O5]]> 0-2

[0039] According to another exemplary embodiment, the composition of the glass includes the following components:

[0040] Element (weight%) <![CDATA[SiO2]]> 60-84 <![CDATA[Al2O3]]> 0-10 <![CDATA[B2O3]]> 3-18 <![CDATA[Li2O+Na2O+K2O]]> 5-20 MgO+CaO+SrO+BaO+ZnO 0-15 <![CDATA[TiO2+ZrO2]]> 0-4 <![CDATA[P2O5]]> 0-2

[0041] In another embodiment, the composition of the glass includes the following components:

[0042] Element (weight%) <![CDATA[SiO2]]> 58-65 <![CDATA[Al2O3]]> 14-25 <![CDATA[B2O3]]> 6-10.5 MgO+CaO+SrO+BaO+ZnO 8-18 ZnO 0-2

[0043] Another suitable composition for the glass is given as follows:

[0044] Element (weight%) <![CDATA[SiO2]]> 50-81 <![CDATA[Al2O3]]> 0-5 <![CDATA[B2O3]]> 0-5 <![CDATA[Li2O+Na2O+K2O]]> 5-28 MgO+CaO+SrO+BaO+ZnO 5-25 <![CDATA[TiO2+ZrO2]]> 0-6 <![CDATA[P2O5]]> 0-2

[0045] According to another embodiment, the composition of the glass includes the following components:

[0046]

[0047]

[0048] To all of the above-mentioned glass components, coloring oxides such as Nd2O3, Fe2O3, CoO, NiO, V2O5, MnO2, CuO, Cr2O3 can be added if necessary.

[0049] As2O3, Sb2O3, SnO2, SO3, Cl, F and / or CeO2 can be added as refining agents in an amount of 0-2 wt. %, and the total amount of the total components is respectively 100 wt. %.

[0050] It can also be advantageous and included in the invention that the surface energy of the walls of the microchannel is set so that the biological material to be examined is transported further through the walls or adheres to the walls. The surface energy can be set, for example, by a coating.

[0051] Thus, advantageous glass elements provide that at least the walls of the microchannel are coated, in particular with a coating that repels the biological material to be examined or with a coating for the adhesion of the biological material to be examined. Preferably, in particular for at least local optimization of the surface energy, the coating comprises polymers, silanes, oligonucleotides, antibodies, proteins, antigens or nitrocellulose or inorganic coatings, in particular metal particles, in particular gold and / or iron-based or polymer-based nanoparticles or microparticles or combinations thereof.

[0052] As mentioned above, the coating may be present only in sections of the microchannel, for example at the channel inlet, or different coatings may be present in the channel, in particular along the channel axis.

[0053] The coating can generally have a thickness of a monomolecular layer or can have a plurality of coatings with a thickness in the nm range up to completely filling the channel. In the simplest case, the coating is applied via a dip coating process. Coating by means of vacuum processes, such as chemical vapor deposition (CVD) or atomic layer deposition (ALD), is particularly suitable for small channel diameters. It may be advantageous to selectively apply the coating in the channel, for example by lifting off the material outside the channel or by selective sputtering on the surface of the glass element.

[0054] The aforementioned advantageous structure of the wall of the microchannel interacts particularly advantageously with the coating and / or the filler, in particular because the structure increases the surface area of ​​the channel wall. In the case of an attached coating, a larger surface area is available for the biomaterial to be examined; in the case of a coating that continues to pass through, the coating inhibits or prevents the biomaterial from clogging the structure and / or the coating at least helps to inhibit or prevent the biomaterial from clogging the structure. In the case of a filler, the adhesion of the filler in the channel can be ensured or at least improved.

[0055] In an advantageous glass element, the microchannels are filled with gel. The glass element advantageously has pores. Depending on the choice and / or design of the gel, the pores can have different diameters, so that the pores can accommodate and / or transport biomaterials of different sizes.

[0056] Particularly useful for the present invention are polyacrylamide gels and / or agarose gels. Agarose gels usually have larger pores than polyacrylamide gels and can be particularly used for analyzing DNA and / or larger proteins. Polyacrylamide gels usually with smaller pores are generally used for analyzing smaller proteins.

[0057] Gels, in particular the mentioned gels, can be used as carrier gels in electrophoresis processes, often also referred to as gel electrophoresis, in the sense of the present invention. Using gel electrophoresis, in particular, different small molecules, in particular DNA, RNA and proteins, can be separated from one another and analyzed in a subsequent step. In this case, the term gel electrophoresis usually includes the transport and / or movement of the substances to be examined through the gel, without separation being necessary.

[0058] The molecules to be separated and / or transported are moved here through a carrier gel, which is advantageously located in a microchannel. The movement can be supported by applying an electric field as further described. Depending on the size and charge and / or intensity of the applied electric field, the molecules travel different distances and / or travel at different speeds.

[0059] In typical electrophoresis, the electrophoresis forms a characteristic band pattern in a microchannel. The molecules can be made visible by dyeing, and a characteristic color of the molecule can be assigned to the molecule and thus the molecule can be identified.

[0060] However, in the case of glass elements according to the invention, in particular thin glass elements, it is also conceivable to use gels and / or alternative substances for electrophoretically transporting the material to be examined from the bottom side of the glass element to the top side of the glass element. The analysis is then carried out on the top side, for example by means of fluorescence spectroscopy or by means of nanopores through DNA sequencing. These are standard procedures and will not be described in detail here.

[0061] Glass elements are particularly advantageous in which at least one DNA- or RNA-sensitive dye, in particular a plurality of dyes, is present in the region of the channel outlet or at the bottom of the blind hole.

[0062] If DNA and / or RNA in the biological material to be examined is transported through the microchannel, then when the microchannel is formed in this way, the DNA and / or RNA reaches the channel outlet and / or the bottom of the blind hole. Here, the DNA or RNA can react with the DNA or RNA-sensitive dye applied there. The characteristic fluorescence, in particular its wavelength, now indicates the presence of the corresponding DNA or RNA chain.

[0063] Possibly advantageous staining substances may be, for example, ethidium bromide, propidium iodide, crystal violet, 4',6-diamidino-2-phenylindole and / or 7-aminoactinomycin D.

[0064] An advantageous embodiment of the glass element provides that in the region of the channel outlet or at the bottom of the blind hole there are molecules for the polymerase chain reaction, in particular DNA, primers, nucleotides and / or the enzyme DNA polymerase. This embodiment is particularly suitable for single-molecule examinations, in which the material to be examined is first amplified in the microchannel and / or the blind hole, as described herein. It can be seen that the aforementioned molecules can also be introduced into the microchannels and / or blind holes, in particular when the material to be examined is introduced into the microchannels and / or blind holes.

[0065] The specific advantage of this embodiment is that the amplification of cells and / or DNA and / or RNA in the material to be inspected in the microchannel (including the blind hole) is carried out in a very small volume. As a result, the time required for amplification can be reduced. Similarly, the material to be inspected can be inspected, including detection, in this small volume, which leads to good evaluation, especially good signal quality. In particular, rare populations can be distinguished or subtle differences between cells can be identified with improved accuracy. New biomarkers or characteristic molecules (Signaturmoleküle) can be distinguished in this way. In addition, by examining individual molecules or cells in the sample, the heterogeneity of molecules or cells in the sample can be explained. In particular, individual differences between patients can be made visible in order to develop personalized treatment plans based on the specific characteristics of cells or molecules. Overall, this provides a reasonable and reliable inspection, which has the potential for application in personalized medicine.

[0066] The glass element described is, so to speak, a preliminary product of the system according to the invention, with which spatially resolved diagnostics and / or single-molecule diagnostics or single-cell diagnostics can be performed. As described, the biological material can be analyzed and / or amplified in a subsequent process on the glass element or, for example, by means of DNA sequencing. In this way, the expression pattern of genes and / or proteins and the distribution pattern of vDNA markers of individual cells can be determined simultaneously, wherein the position information in the cell cluster or tissue sample is retained.

[0067] A system for examining biological material, in particular human or animal or plant cells and / or cell components, in particular proteins and / or DNA and / or RNA, comprises a glass element with a plurality of microchannels, in particular the aforementioned glass element, wherein the glass element in an operating state serves to accommodate biological material in the microchannels and / or to convey biological material through the microchannels from the bottom side of the glass element to the region of the top side of the glass element, the system having an evaluation device by means of which the biological material can be examined in an operating state. In the invention, the evaluation device can be assigned to the glass element or arranged downstream of the glass element.

[0068] The evaluation unit usually detects the presence of biomaterial and / or its components transported through the microchannel and / or present in the microchannel. In particular, the evaluation unit can include an electronic image sensor, which can in particular record the intrinsic fluorescence of the aforementioned dyeing substances. Thus, the design of the microchannel as a blind hole is also included.

[0069] An advantageous system therefore provides that in the region of the channel outlet (22) and / or in the blind hole (25), in particular at the bottom of the blind hole, at least one DNA- or RNA-sensitive dye is present, the color information of which can be detected by the evaluation device, in particular in a spatially resolved manner.

[0070] In the case of DNA examination, a common staining substance that binds to DNA is, for example, ethidium bromide.

[0071] Advantageously, the system comprises a sample carrier to which the biological material to be examined is attached in an operating state. The sample carrier may typically be a sample plate or a slide.

[0072] Particularly advantageous is a system in which a conveying device is assigned to the glass element, by means of which the biomaterial is introduced into the microchannels of the glass element in the operating state.

[0073] The transport device is generally a device that exerts a force on the biological material to be examined, at least supports the movement of the biological material and / or transports the biological material into the microchannel of the glass element and toward the opposite surface thereof.

[0074] Advantageously, a system is provided in which the transport device comprises: a negative pressure unit, by means of which negative pressure is applied to the outlet side of the glass element in an operating state; and / or a device for generating an electric potential gradient across the thickness of the glass element, whereby the biological material to be examined is introduced into the microchannel by an ion flow or by an electric field and / or is transported through the microchannel.

[0075] Since DNA is negatively charged in principle due to its phosphate residues, it migrates to the anode during gel electrophoresis. It is therefore particularly advantageous to attach the anode to the upper side of the advantageous glass element or to assign an anode to this upper side of the glass element. If a cathode is provided on the lower side or in the vicinity of the lower side, an electric field is generated by applying a voltage and anions migrate to the anode and cations to the cathode.

[0076] When examining DNA, it is of course possible to amplify the DNA fragments beforehand and / or in the channel of the glass element, in particular by means of a polymerase chain reaction.

[0077] Depending on the biological material to be examined, it is of course also possible and included in the invention to arrange an anode on the lower side of the glass element and a cathode on the upper side and / or to assign an anode and a cathode to the lower side and the upper side respectively. Other arrangements are likewise possible and included in the invention. For example, it may also be sufficient to move the medium in which the biological material to be examined is located by means of a conveying device.

[0078] It is also advantageous if the conveying device comprises a scraper unit, by means of which the biological material to be examined can be introduced into the microchannel, in particular into the blind hole, by coating.

[0079] An advantageous system comprises a microfluidic system as a conveying device, which introduces the biological material to be examined into the blind holes and / or microchannels by means of a fluid flow.

[0080] A particularly advantageous embodiment of the system is that the evaluation device comprises a closed or nanoporous membrane on the outlet side of the microchannel, which membrane consists in particular of a non-conductor or semiconductor or a combination of non-conductive, conductive and / or semiconductive components, in particular of glass, silicon, graphene and / or biomolecules, in particular lipid molecules, DNA origami structures or transmembrane proteins, with the aid of which biological material can be examined, in particular sequence or binding properties or size or optical properties, in particular fluorescence.

[0081] In an alternative embodiment, the system comprises an evaluation device in the form of a closed or nanoporous membrane on the outlet side of the microchannel, the membrane consisting in particular of a nonconductor or semiconductor or a combination of nonconductive, conductive and / or semiconductive components, in particular of glass, silicon, graphene and / or biomolecules, in particular lipid molecules or transmembrane proteins. Using this evaluation device, the biological material to be examined can be examined, in particular sequence or binding properties or size or optical properties, in particular fluorescence.

[0082] As described, the microchannels can be filled with gels to support transport in particular. The gel itself can include a dye that binds to the biological material to be examined. In the case of a DNA test, the DNA-binding dye is, for example, ethidium bromide. The sample can also be placed in a sample buffer, which in turn contains a dye, such as crystal violet. Alternatively, the gel can be stained after electrophoresis by a DNA-binding dye, for example by methylene blue, a full dye or ethidium bromide. Fluorescent dyes such as ethidium bromide usually require irradiation of the agarose gel with UV light and a UV filter on the camera. RNA testing is certainly also possible.

[0083] Advantageous systems provide that during operation, biological material is amplified in microchannels and / or blind holes, often referred to as digital PCR. In particular, molecules for the polymerase chain reaction are present in the region of the channel outlet or at the bottom of the blind hole.

[0084] If the blind holes contain molecules for the polymerase chain reaction (PCR), the DNA or RNA molecules contained in the material to be examined are amplified. These molecules include, for example, DNA, primers, nucleotides and the enzyme DNA polymerase.

[0085] Due to the temperature-stable properties of glass elements, standard PCR at up to 100°C is possible in addition to the room temperature PCR which is usually carried out at up to 40°C. At these temperatures, thermal expansion or position shifts of the blind holes relative to each other can be expected not to have an adverse effect on the spatial information in contrast to plastics. Furthermore, glass with its good thermal conductivity has the advantage over plastics that the heat is evenly distributed in the glass element and a uniform PCR over the entire sample can be ensured. Furthermore, glass has the advantage over plastics of low intrinsic fluorescence, so that high signal intensities are achieved compared to the background.

[0086] As described, since the outlet matrix of the microchannels on the upper side of the glass element can be assigned to the inlet matrix of the microchannels on the lower side of the glass element, the described system can derive the position of the corresponding inlet of the biomaterial on the lower side from the position of the examination result on the upper side. It can be said that the positioning of the microchannels on the glass element can be assigned to the starting position of the biomaterial in the sample. The glass element according to the invention and / or the system according to the invention thus enable a spatially resolved examination of a sample of biomaterial. This is also called "spatial biology" or "spatial diagnosis".

[0087] The glass element can be produced efficiently and in large quantities by the method according to the invention described below. The method comprises the following method steps:

[0088] - Provide ultrashort pulse lasers,

[0089] - providing a glass element substrate,

[0090] - directing a pulsed laser beam of an ultrashort pulse laser onto the glass element substrate, wherein:

[0091] - the wavelength of the laser beam and the material of the glass element matrix are matched to one another so that the workpiece is substantially transparent to the laser beam, and wherein,

[0092] - focusing the laser beam into a focal region which is elongated in the beam direction and which is at least partially located in the glass element matrix, wherein

[0093] the intensity of the laser beam and the extent of the focus are so great that the laser beam leaves filament-like defects in the workpiece and a plurality of such filament-like defects are introduced therein,

[0094] - Etching filamentary damage into microchannels.

[0095] The glass element substrate can generally be a glass plate of desired size. A pulsed laser beam from an ultrashort pulse laser is directed onto the glass element substrate, wherein the wavelength of the laser beam and the material of the glass element substrate are matched to each other so that the workpiece is substantially transparent to the laser beam.

[0096] The laser beam is focused into a focal region which is elongated in the beam direction and which is at least partially located in the glass element matrix. Different optical structures can be used for this purpose, for example Bessel optics with an axicon, optics with chromatic aberration or optics with spherical aberration.

[0097] The intensity of the laser beam and the extension of the focus are set so high that the laser beam leaves filamentary damage in the workpiece, wherein a plurality of such filamentary damages are introduced. The filamentary damages can be said to represent linear damage in the glass, which is etched into microchannels in a subsequent method step.

[0098] The filament-shaped damage is produced by nonlinear optical effects with self-focusing of the laser beam. For this purpose, ultrashort laser pulses in the picosecond range are required.

[0099] An advantageous method is to use a sequence of laser pulses to produce the filament-shaped lesions.

[0100] In this so-called burst operating mode, the laser energy is not emitted as a single pulse, but as a sequence of pulses emitted in quick succession, which together form a pulse packet, the so-called pulse train. Such a pulse packet usually has slightly more energy than a single pulse in the conventional single shot mode. However, the pulses of the burst itself contain significantly less energy than a single pulse. With regard to the pulses within the burst, it can be provided that the pulse energy can be flexibly adjusted, in particular the pulse energy can either be kept essentially constant or the pulse energy can be increased or the pulse energy can be reduced.

[0101] A suitable laser source can be, for example, a neodymium-doped yttrium aluminum garnet laser with a wavelength of 1064 nanometers. This laser source, for example, generates a raw beam with a (1 / e2) diameter of 12 mm, and as an optical device, a biconvex lens with a focal length of 16 mm can be used. To generate the raw beam, a suitable beam-shaping optical device can be used, such as a Galilean telescope.

[0102] In particular, the laser source can be operated with a repetition rate between 1 kHz and 1000 kHz, preferably between 2 kHz and 100 kHz, particularly preferably between 3 kHz and 200 kHz.

[0103] In this case, the repetition rate and / or the scanning speed can be selected such that a desired spacing between adjacent filament-shaped lesions is achieved.

[0104] Suitable pulse durations of the laser pulses may lie in particular in the range of less than 100 picoseconds, advantageously less than 20 picoseconds.

[0105] The typical power of the laser source is particularly advantageously in the range of 20 to 300 W. The laser energy is substantially lower than the ablation energy of the substrate. According to an advantageous further development of the invention, a pulse energy of a pulse train of more than 400 microjoules is used to achieve filament-shaped lesions, and a total pulse train energy of more than 500 microjoules is advantageously used.

[0106] When operating an ultrashort pulse laser in burst mode, the repetition rate is the repetition rate of the output of the burst. The pulse duration is essentially independent of whether the laser is operated in single pulse mode or burst mode. The pulses within the burst typically have a pulse length similar to the pulses in single pulse mode. The burst frequency can be in the range of 15 MHz to 90 MHz, preferably in the range of 20 MHz to 85 MHz, and for example 50 MHz, and the number of pulses in the burst can be between 1 and 10 pulses, for example 6 pulses.

[0107] An advantageous method is a method in which the elongated focal region comprises a region with highest energy and a following widening portion, wherein the elongated focal region is positioned such that the widening portion is located in the surface region of the glass element base body.

[0108] In this way, narrowing microchannels can be produced in a particularly advantageous manner. The filament-shaped pre-damage is produced, as it were, by a damaged region which, in a subsequent etching step, leads to a widening at the channel inlet.

[0109] A particularly advantageous method provides that the filament-shaped defects do not reach at least one surface of the glass element base body and that an opening toward this surface is achieved by a subsequent etching step.

[0110] This can likewise be achieved by selecting the focal position and therefore the intensity of the laser beam. Assuming that the generation of filament-shaped damages is a nonlinear effect, a local beam intensity must be achieved in order to generate damages in the glass. This means that if the intensity is below this critical threshold, there are actually no damages in the glass or at least not enough damages to form filaments. Only the subsequent etching process causes the filament-shaped damages to widen and, so to speak, to create a gap (Durchbruch) through the relevant surface of the glass element matrix. The inventors have recognized that a matrix of channel outlets of microchannels can be generated particularly precisely in this way. In particular, in this case, the diameter of the channel outlets varies only slightly and / or at most small deviations from the ideal circular diameter of the channel outlets occur.

[0111] In the simplest case, the center point of the channel inlet can be located above the center point of the channel outlet. The microchannel now passes through the glass element vertically. However, it is also possible to stagger the center point of the channel inlet and the center point of the channel outlet so that the microchannel passes through the glass element at an angle that deviates from the orthogonal line through the surface of the glass element. Particularly suitable is an angle that deviates from the orthogonal line up to about 10°, i.e., an angle in the range of 0.1° to 10°. In particular, it is possible to make the passage angle of the microchannel not equal everywhere in the glass element. Local adjustment of the passage angle allows the channel matrix of the glass element to be adapted to the requirements.

[0112] The focal position is mainly used to support the widening of the channel opening. As mentioned above, the subsequent etching process widens the damaged area in the glass material. The larger the damaged area, the greater the widening.

[0113] The shape of the elongated focus is utilized to set the characteristics of the widening of the channel entrance. The focus is designed so that the area with the highest energy is adjacent to the widening area when viewed along the beam axis. The elongated focus area is positioned so that the widening portion is located in the surface area of ​​the glass element substrate.

[0114] During a later etching, the widening becomes a widened channel inlet, in particular with a funnel-shaped widening. The etching step is advantageously carried out such that the microchannel has a diameter of 5 to 200 μm, preferably 7 to 130 μm, particularly preferably 10 to 100 μm, at least in the region of the channel inlet.

[0115] A variant of the method provides that the filament-shaped defects do not reach at least one surface of the glass element base body and are opened toward this surface by a subsequent etching step.

[0116] This is because it is advantageous to also provide the geometry of the channel outlet, which can be located in particular in the cylindrical region of the microchannel, as accurately as possible. In order to set the geometry of the channel outlet, it has been found that the filament-shaped damages should advantageously not pass through the surface of the glass element on which the channel outlet is located. The filament-shaped damages thus end below this surface. This is also set by a corresponding focus position. The opening of the microchannel, i.e. the channel outlet, is produced in this case by a subsequent etching step.

[0117] The surface of the glass element base body can be processed by grinding and polishing, in particular before and / or after etching. It has proven to be particularly advantageous to grind and polish after the etching step, since this allows particularly flat surfaces to be achieved around the channel inlet and / or channel outlet.

[0118] In an advantageous method, hemispherical dome-shaped recesses are formed on the inner wall of the channel by etching.

[0119] This is particularly true when a slow etching process is performed, especially when an etching rate of less than 15 μm per hour is intended.

[0120] It is assumed that the dome-shaped recess is caused by a structure that occurs when a filament-shaped damage is introduced. A liquid etching solution is particularly advantageous as an etching medium. According to this embodiment, the etching is therefore carried out in a wet-chemical manner. This can also be advantageous in order to remove glass components from the surface during etching.

[0121] Both acidic and alkaline solutions can be used as etching solutions. As acidic etching media, particularly suitable are HF, HCl, H2SO4, ammonium bifluoride, HNO3 solutions or mixtures of these acids. KOH- or NaOH alkaline solutions are preferably used as basic etching media. Higher stripping rates can usually be achieved with acidic etching solutions. However, alkaline solutions are preferred, in particular because only slow stripping is required anyway.

[0122] Etching is advantageously carried out in a temperature range of 40° C. to 150° C., particularly advantageously in a temperature range of 50° C. to 120° C. In general, silicate glasses with a low alkali content are particularly suitable for the structuring according to the invention. Too high an alkali content makes etching difficult. Therefore, according to a further development of the invention, it is provided that the glass of the glass element is a silicate glass with an alkali metal oxide content of less than 17 wt %.

[0123] The described glass elements with microchannels can advantageously be used for the examination of biological material, in particular human or animal or plant cells and / or cell components, in particular proteins and / or DNA.

[0124] As an alternative to spatially resolved diagnostics, glass elements can be used for single-molecule diagnostics or single-cell diagnostics due to their large number of blind holes. Here, a sample, in particular a liquid sample, is introduced into the blind hole, for example by coating via a scraper process or via the above-mentioned process. As a result, depending on the concentration selected, no or only a few or exactly one single molecule or cell to be examined enters the blind hole. In this case, the molecules and / or cells to be examined can be amplified in a targeted manner in the individual blind holes in order to subsequently perform an analysis, in particular a fluorescence analysis.

[0125] Particularly advantageous embodiments provide that, in combination with a microfluidic cell that can manipulate individual blind holes or groups of blind holes in a targeted manner, a specific test is performed for each such blind hole or group of blind holes. This is particularly advantageous for medical diagnostics, in order to determine the heterogeneity of a sample or to perform a plurality of different tests in a small space (high-complexity diagnostics). BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The invention is described in more detail below with reference to the accompanying drawings. The drawings and the associated embodiments represent a number of examples, but the invention is not limited to these examples. In the drawings, the same reference numerals respectively denote the same or corresponding elements. Reference numerals shown in one drawing may also apply to other drawings, even if they are not shown. The drawings show schematic examples of embodiments, the proportions need not correspond to the proportions or dimensions of the objects according to the invention. In the drawings:

[0127] Figure 1a to Figure 1d Different exemplary embodiments of glass elements with widened microchannels are shown in cross-sectional views.

[0128] Figure 2 A partial cross-sectional view of a glass element having widened microchannels is shown.

[0129] Figure 3 An electron micrograph of the wall of a microchannel is shown.

[0130] Figure 4a and Figure 4b A top view and a bottom view of the glass element are shown.

[0131] Figure 5 Micrographs of sections through glass elements are shown.

[0132] Figure 6 A system for examining biological material in a spatially resolved manner is shown.

[0133] Figure 7 The application of a coloring substance on the upper side of a glass element is shown.

[0134] Figure 8 The application of a coloring substance in a blind hole of a glass element is shown.

[0135] Fig. 9 An apparatus for laser processing of a glass element in preparation for subsequent etching is shown.

[0136] Fig.10a Smooth channel walls are shown in .

[0137] Fig.10b A structured channel wall with a roughness Ra is shown in FIG. DETAILED DESCRIPTION

[0138] Figure 1a An embodiment of a glass element (1) with a plurality of microchannels (2) is shown. In this embodiment, the microchannels connect the upper side (O) of the glass element with its lower side (U) and pass through the upper side and the lower side. As mentioned above, the upper side is usually the surface or side of the glass element facing away from the sample or on which the analysis is performed. The lower side is usually the surface or side of the glass element facing the sample and therefore the biological material to be examined. In the terminology of this description, the channel inlet (21) is located in the region of the lower side (U) and the channel outlet (22) is located in the region of the upper side (O).

[0139] exist Figure 1a In this example, the microchannel narrows in the direction from the bottom (U) to the top (O), in this example continuously. In general, at least local widening is referred to as funnel-shaped in this description. In the example shown, the narrowing is continuous. The contour of the microchannel (2) can also be referred to as truncated cone-shaped. The narrowed area extends over the entire thickness (s) of the glass element.

[0140] The thickness or material thickness (s) of the glass element after it has been processed can, as described, in particular be 0.1 mm to 3 mm. The glass element (1) shown has essentially planar parallel surfaces (O) and (U). The same generally applies to the side surfaces. The glass element (1) is advantageously a glass slide.

[0141] exist Figure 1b A similar embodiment is shown in , in which the narrowing of the microchannel is more pronounced. The narrowing of the microchannel (2) in the direction of the upper side (O) and / or the at least partial funnel-shaped configuration in the present invention result in the diameter (dU) of the channel inlet (21) on the lower side (U) of the glass element (1) being greater than the diameter (dO) of the channel outlet (22) on the upper side (O).

[0142] Figure 1cAnother embodiment is shown, in which the profile of the microchannel consists of a narrowed truncated cone-shaped area in the area of ​​the lower side (U) and a cylindrical area in the area of ​​the upper side (O). The overall shape can also be described as funnel-shaped. As mentioned above, the center point of the channel outlet (22) determines the pitch (P). The goal is to have the pitch as small as possible, because with a smaller pitch, the microchannel density increases. The pitch can be between 3 and 70 μm as described. As shown in this example, the center point of the channel inlet (21) is substantially below the center point of the channel outlet (22). That is: both center points are substantially located on the axis that passes vertically through the surfaces (O) and (U) of the glass element.

[0143] Figure 1d An embodiment is shown in which the microchannel (2) tapers from the bottom (U) and from the top (O) in the direction of the main body of the glass material. In the shape shown here as an example, this results in an hourglass shape or an X shape. In principle, this consists of two truncated cones that are inverted one above the other and that touch at their narrow sides.

[0144] Figure 2 Shown according to Figure 1b 1 is a partial cross-sectional view of a glass element (1). The opening angle α is shown, which is to be understood as the angle between the channel walls of the widening region of the microchannel (2). Advantageously, the opening angle α is 0.1° to 30°, particularly advantageously 2° to 18°, wherein the upper and lower limits of the interval are of course to be understood as being interchangeable.

[0145] As described in detail above, the narrowed microchannels can resolve conflicting goals: on the one hand, the smallest possible pitch is pursued in order to increase the resolution of the inspection and to pursue high detection sensitivity and therefore a large diameter of the channel entrance, but on the other hand, the mechanical stability of the glass element must be ensured in order to be able to use it properly. In particular, sufficient material is retained in the unnarrowed area to ensure mechanical stability.

[0146] Figure 3 An electron micrograph of the wall of a microchannel (2) of a glass element (1) produced by the inventors according to the invention is shown, more specifically as Figure 1c 4. A section of the region (40) of the microchannel shown in FIG. As already mentioned, the transverse dimension or diameter of the channel (2) is less than 200 micrometers, in this example about 14 micrometers. Without being limited to the examples described here, diameters in the range of 5 micrometers to 200 micrometers are preferred.

[0147] The dome-shaped or rounded cap-shaped recesses (7) can be clearly seen, which are produced by etching filamentary damages which are introduced into the glass element by means of an ultrashort pulse laser beam. The depth of the dome-shaped recesses (7) is typically less than 5 μm and the lateral dimensions are typically 5 to 20 μm.

[0148] Due to the etching process of the filamentary damage, the dome-shaped recesses (7) are adjacent to each other, wherein the concave roundings (Rundungen) of the recesses (7) that abut against each other form ridges (70). It can also be seen that the ridges (70) form polygonal boundary lines (71) of the recesses (7) when viewed from above. Here, the average number of corners (72) of the boundary line (71) of the recess (7) is preferably also less than eight, preferably less than seven. In most dome-shaped The latter feature only appears when the area occupied by the concave portion is convex in the mathematical sense.

[0149] Figure 3 The ridge (70) of the channel 2 shown in FIG. 2 is very narrow, with no visible regions in which the concave curvature of the recess (7) transitions into one another via convexly curved regions on the ridge (70). Therefore, according to a further development of the invention, the structure of the channel (2) can also be such that the area share of the convexly shaped regions in the channel (2) is less than 5%, preferably less than 2%.

[0150] Figure 4a A schematic diagram of the upper side (O) and the lower side (U) of the glass element (1) is shown. The diameters of the channel inlet (21) and the channel outlet (22) are substantially the same. Figure 1d Such a pattern is contemplated in embodiments of the present invention.

[0151] Figure 4b A similar view is shown, but the diameter of the channel inlet (21) is larger than the diameter of the channel outlet (22). Figure 1a to Figure 1c It can be seen in the implementation method of.

[0152] As in Figure 4a and Figure 4b As shown in , the channel inlets and channel outlets form a matrix. Since the positions of the channel inlets can be assigned to the positions of the channel outlets, if the positions of the channel outlets are known, events can be determined in a spatially resolved manner on one side of the channel inlet and therefore on the sample side, for example determining whether DNA or a specific biological material is present.

[0153] Figure 5 A photograph of a cross section of a glass element (1) produced by the inventors, taken through an optical microscope, is shown, which glass element substantially corresponds to Figure 1cThe embodiment shown in . The cylindrical area of ​​the microchannel (2) adjoins the funnel-shaped widening area. The diameter of the channel inlet (21) on the lower side of the glass element (1) is greater than the diameter of the channel outlet (22) on the upper side. In this embodiment, Schott glass D263 is used. The invention is not limited to a specific glass type. Other embodiments are made of borosilicate glass and / or alkali-free glass. In particular, borosilicate glass is commonly used in laboratories due to its chemical resistance. As already described, glasses that exhibit low intrinsic fluorescence in the range of the visible spectrum are particularly advantageous.

[0154] Figure 6 An exemplary system (10) for determining biological material (60) in a spatially resolved manner is shown. The biological material to be examined is located on a sample carrier (62). A glass element (1) with the described microchannels (2) is in contact with the biological material (60). Of course, it is also possible to bring the glass element (1) into contact with the biological material without the biological material being prepared on a carrier. For example, the glass element can be in direct contact with, for example, a tissue or cell culture.

[0155] The microchannel (2) may contain a gel, in particular a gel as described. Figure 6 The gel is not shown in the figure. At least one DNA or RNA sensitive dye (610) is applied to the upper side of the glass element (1) in the area of ​​the channel outlet (22). If the biological material (60) passes through the microchannel (2), in particular with the aid of the gel, the biological material comes into contact with the dye (610) on the upper side. The typical coloring of the dye can therefore indicate whether a specific DNA chain is present or whether a specific DNA chain is present, depending on the dye. As described, the dye is usually irradiated with excitation light and the typical fluorescence of the dye is then measured with the aid of an evaluation device. The evaluation device is not shown in this figure for the sake of clarity. As also described, the evaluation device can in particular be an electronic camera.

[0156] Advantageously, different dyeing substances (610) can be attached to different channel outlets (22). Different DNA or DNA chains can then be identified when analyzing biological material.

[0157] The top view of the system (10) shown shows a pattern from which it is possible to infer in a spatially resolved manner whether a specific biological material, in particular DNA, is present. All statements made regarding DNA apply analogously to RNA.

[0158] The transport of the biological material (60) into or through the microchannel (2) can be supported by the presence of a transport device (65). Figure 6In the example of , the transport device is a device that generally uses an electric potential to transport biological material. A capacitor device is shown, in which a voltage source (V) is connected to electrodes (651, 652). In this example, the electrodes are arranged on the underside of the sample carrier (62) and on a non-conductive plate (653), in particular a glass plate or a plastic plate. The electrodes thus form a flat plate capacitor, with the biological material (62) and the glass element (1) being located between the plates of the flat plate capacitor. When a voltage is applied, an electric field is generated, which pulls or pushes the biological material and / or the medium surrounding it in the direction of the electrode (651).

[0159] The non-conductive plate (653) can also serve as a closure for the microchannel (2) when it is in contact with the upper side (O) of the glass element. This creates a blind hole, so to speak, and the staining substance is not removed from the surface of the glass element by the advancing biological material.

[0160] The electrodes ( 651 , 652 ) can expediently be applied by coating, in particular by sputtering.

[0161] Other configurations are also possible and are included in the present invention. In particular, it is also possible to use pressure and / or negative pressure instead of electric power to transport biomaterials.

[0162] Figure 7 It shows that especially according to Figure 6 A top view of a glass element (1) with a sensitive dyeing substance (610, 611, 612, 613, 614) on the upper side (O) in a system of FIG. The illustrated pattern schematically shows possible inspection results.

[0163] For clarity, Figure 8 Once again, an embodiment of a glass element is shown which comprises a microchannel (2) in the form of a blind hole (25). The aforementioned dyeing substance (610, 611) is attached to the bottom of the blind hole. The electrode (651) of the electric transport device (65) can be simply applied to the surface of the glass element. In order to be able to detect the dyeing substance fluorescence through the glass material located above the bottom of the blind hole, the electrode is in this case in particular a transparent electrode, which can be an ITO coating, for example. Alternatively, the electrode can also be configured as a grid and / or a grid, in particular in the case of an empty space above the bottom of the corresponding blind hole (25).

[0164] Fig. 9An embodiment of a laser processing device (20) is shown, by means of which filament-shaped damages (32) can be introduced into a glass element substrate (1) in order to subsequently introduce microchannels at the location of the filament-shaped damages (32) during an etching process. The device (20) comprises an ultrashort pulse laser (300) with an upstream focusing optical device (230) and a positioning unit (170). The positioning unit (170) can be used to laterally position the point of incidence (73) of the laser beam (270) of the ultrashort pulse laser (300) on the surface (O) of the flat glass element (1) to be processed. In the example shown, the positioning unit (170) comprises an xy table, on which the underside (U) of the glass element (1) rests. Alternatively or additionally, however, the optical system can also be designed to be movable in order to move the laser beam (270) so that the point of incidence (32) of the laser beam (270) can be moved while the glass element (1) remains in place.

[0165] Fig.10a and Fig.10b The effect of the structure of the microchannel wall is schematically shown. Fig.10a The smooth channel wall is shown in Fig.10b , the channel walls have a roughness Ra. The channel walls have an opening angle. The structure can have the shape of a dome-shaped recess. EOF stands for electroosmotic flow. Electroosmotic flow is the movement of a liquid along a polar surface in an electric field due to electric charges. Fig.10a Compared with the surface with smaller roughness, Fig.10b A greater roughness of the polar surface causes a reduction in the velocity of the EOF and possibly a reversal of the direction of movement of the polar biomaterial in the glass element.

[0166] The focusing optics (230) now focus the laser beam (270) onto an elongated focus in the radiation direction, i.e. transversely, in particular perpendicularly, to the irradiated surface (O). Such a focus can be produced, for example, by means of a conical lens (so-called axicon) or by means of a lens with large spherical aberration and / or large chromatic aberration. The positioning unit (170) and the ultrashort pulse laser (300) are preferably controlled by a computer (150) equipped with a program. In this way, a predetermined pattern of filamentary damages (32) distributed laterally along the surface (O) can be produced, in particular by reading in position data, preferably from a file or via a network. The shape of the filamentary damages (32) can be adjusted by means of the position of the focus, so that the microchannels produced by the subsequent etching are locally widened.

[0167] According to one embodiment, the laser beam can use the following parameters: The wavelength of the laser beam is 1064 nm, which is typical for YAG lasers. The laser beam is generated with an original beam diameter of 12 mm and then focused by means of an optical device in the form of a biconvex lens with a focal length of 16 mm. The pulse duration of the ultrashort pulse laser is less than 20 ps, ​​preferably about 10 ps. The pulses are emitted in the form of a pulse train with more than 2, preferably more than 4 pulses. The pulse train frequency is 12 to 48 ns, preferably about 20 ns, the pulse energy is at least 200 microjoules, and the pulse train energy is correspondingly at least 400 microjoules.

[0168] After one or more, in particular, several filament-shaped damages (32) have been introduced, the glass element (1) is removed and placed in an etching bath, in which the glass is removed along the filament-shaped damages (32) in an etching process, so that a microchannel (2) is introduced into the glass element 1 at the location of each such damage (32).

[0169] Preferred is an alkaline etching bath with a pH value of >12, for example a KOH solution of >4 mol / l, preferably >5 mol / l, particularly preferably >6 mol / l but <30 mol / l. According to one embodiment of the invention, etching is carried out at an etching bath temperature of >70°C, preferably >80°C, particularly preferably >90°C, regardless of the etching medium used.

[0170] The glass element and system according to the invention have the advantage of being able to examine biological materials in a spatially resolved manner. The glass element can be efficiently manufactured and is composed of materials commonly used in laboratory environments. After use, the glass can be melted again, thereby enabling the reuse of raw materials.

Claims

1. A glass element (1) for receiving and / or conveying biological material (60), in particular human or animal or plant cells and / or cell components, in particular proteins and / or DNA and / or RNA, The glass element (1) has a plurality of microchannels (2), which preferably connect one surface (U, O) of the glass element (1) to an opposite surface (O, U) of the glass element (1) or terminate in a blind hole (25), in, The microchannel (2) narrows from the lower side (U) toward the upper side (O); Preferably, the microchannel (2) is at least partially funnel-shaped.

2. The glass element (1) according to claim 1, wherein The diameter (dU) of the channel inlet (21) of the microchannel on the lower side (U) is greater than the diameter (dO) of the channel outlet (22) or channel end (25) in the region of the upper side (O).

3. The glass element (1) according to any one of the preceding claims, wherein: The microchannel (2) has a diameter (dU, dO) of 5 μm to 200 μm, preferably 7 μm to 130 μm, particularly preferably 10 μm to 100 μm, at least in the region of the channel inlet (21) or the channel outlet (22) or the channel end (25) of the glass element (1); preferably, the opening angle (α) of the funnel-shaped region is 0.1° to 30°, particularly preferably 2° to 18°.

4. The glass element (1) according to any one of the preceding claims, wherein: The funnel-shaped region in the region of the channel inlet (21) adjoins a cylindrical or funnel-shaped region, in particular in the region of the channel outlet (22) and / or in the end of the blind hole (25).

5. The glass element (1) according to any one of the preceding claims, wherein: Microchannel density is at least 20 / mm 2 , especially at least 400 / mm 2 , especially at least 10000 / mm 2 , in particular up to a maximum of 16000 / mm 2 .

6. The glass element (1) according to any one of the preceding claims, wherein: The channel wall of the microchannel (2) comprises a structure with a plurality of rounded, dome-shaped depressions (7), which in particular have a depth of less than 5 μm.

7. The glass element (1) according to any one of the preceding claims, wherein: At least one DNA- or RNA-sensitive dye (610, 611, 612, 613, 614) is present in the region of the channel outlet (22) or at the bottom of the blind hole (25).

8. The glass element (1) according to any one of the preceding claims, wherein In the region of the channel outlet ( 22 ) or at the bottom of the blind hole ( 25 ) there are molecules for the polymerase chain reaction, in particular DNA, primers, nucleotides and / or the enzyme DNA polymerase.

9. A system (10) for examining biological material (60), in particular human or animal or plant cells and / or cell components, in particular proteins and / or DNA and / or RNA, The system comprises a glass element (1) having a plurality of microchannels (2), in particular a glass element according to any one of claims 1 to 8, in, The glass element (1) is used in an operating state to accommodate the biomaterial (60) in the microchannel (2) and / or to transport the biomaterial (60) from the lower side (U) of the glass element to the region of the upper side (O) of the glass element via the microchannel (2). The system has an evaluation device, by means of which the biological material can be examined in an operating state.

10. The system (10) according to claim 9, wherein: In the region of the channel outlet (22) or at the bottom of the blind hole (25) there is at least one DNA- or RNA-sensitive dye (610, 611, 612, 613, 614), the color information of which can be detected by the evaluation device, in particular in a spatially resolved manner.

11. The system (10) according to any one of claims 9 to 10, wherein: The glass element (1) is assigned a conveying device (65), by means of which, in an operating state, the biological material (60) to be examined is introduced into the microchannel (2) of the glass element (1) and / or the biological material (60) to be examined is conveyed through the microchannel (2), Preferably, the conveying device (65) comprises: a negative pressure unit, with which negative pressure is applied to the outlet side of the glass element in an operating state; and / or means for generating an electrical potential gradient, in particular across the thickness of the glass element, so that the biological material (60) to be examined can be introduced into the microchannel (2) and / or transported through the microchannel (2) by flow or by an electric field; and / or A doctor unit, with the aid of which the biological material to be examined is introduced into the blind hole by coating.

12. The system (10) according to any one of claims 9 to 11, wherein: In the operating state, the biological material in the microchannel (2) and / or the blind hole (25) is amplified, in particular in the region of the channel outlet (22) or at the bottom of the blind hole (25) molecules for the polymerase chain reaction are present.

13. The system (10) according to any one of claims 9 to 12, wherein: The evaluation device comprises a closed or nanoporous membrane on the outlet side of the microchannel (2), the membrane consisting in particular of a non-conductor or semiconductor or a combination of non-conductive, conductive and / or semiconductive components, in particular of glass, silicon, graphene and / or biomolecules, in particular lipid molecules or transmembrane proteins.

14. A method for producing a glass element (1) having a plurality of microchannels (2), the microchannels connecting one surface (U, O) of the glass element with an opposite surface (O, U) of the glass element, the glass element (1) being in particular a glass element (1) according to any one of claims 1 to 7, the method comprising the following method steps: - providing an ultrashort pulse laser (300), - providing a glass element substrate (1), - directing the pulsed laser beam (270) of the ultrashort pulse laser (300) onto the glass element substrate (1), wherein: - the wavelength of the laser beam (270) and the material of the glass element substrate (1) are matched to one another so that the workpiece is substantially transparent to the laser beam (270), and wherein, - focusing the laser beam (270) into a focal region (73) which is elongated in the beam direction, the focal region (73) being located at least partially within the glass element substrate (1), wherein - the intensity of the laser beam (270) and the extension of the focus are so great that the laser beam (270) leaves a filament-shaped damage (32) in the workpiece, and a plurality of such filament-shaped damages (32) are introduced, - Etching the filamentous damage (32) into a microchannel (2).

15. The method according to claim 14, wherein: The filament-shaped damage (32) does not reach at least one surface (O) of the glass element base body (1) and opens toward the surface (O) through a subsequent etching step.

16. The method according to any one of claims 14 to 15, wherein: By etching, a dome-shaped recess (7) is introduced into the channel inner wall of the microchannel (2).

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