Coated solid phase chips and preparation thereof
阅读说明：本技术 包覆的固相碎片及其制备 (Coated solid phase chips and preparation thereof ) 是由 D·艾格特-戈斯波斯 M·拉泰克 W·斯托克 N·罗特曼 于 2019-07-02 设计创作，主要内容包括：本发明涉及用于制备生物材料包覆的固相碎片的方法,涉及通过这样的方法制备的生物材料包覆的固相碎片,涉及在其上施加至少一个根据本发明的固相碎片的载体,并且涉及包含拉伸应力装置、提升头装置和移除装置的用于制备生物材料包覆的固相碎片的设备。(The invention relates to a method for producing biomaterial-coated solid-phase fragments, to biomaterial-coated solid-phase fragments produced by such a method, to a carrier on which at least one solid-phase fragment according to the invention is applied, and to a device for producing biomaterial-coated solid-phase fragments, comprising a tensile stress device, a lift head device and a removal device.)
1. A method for preparing a biomaterial-coated solid phase chip comprising:
-providing a biomaterial-coated solid support, wherein the solid support is fragmented and the fragments adhere to the membrane;
-creating a tensile stress on the membrane coated with the solid phase fragments;
-contacting the membrane with a poppet to pre-separate one or more solid phase fragments from the membrane; and
-removing one or more pre-separated solid phase fragments from the membrane.
2. The method of claim 1, wherein
(a) The biomaterial-coated surface (2) of the solid phase fragments occupies at least 10.25mm2Preferably at least 25mm2；
(b) At least one side (4,5) of the solid phase fragments has a length greater than 3.25mm, preferably greater than 5 mm; and/or
(c) The solid phase fragments are glass fragments.
3. The method according to claim 1 or 2, wherein the solid phase fragments have a thickness (3) between 0.05mm and 0.3mm, preferably between 0.1mm and 0.2 mm.
4. The method according to any one of claims 1-3, wherein the biomaterial is bound to the solid support via a linker molecule.
5. The method of claim 4, wherein the linker molecule
(a) Is a silicon-containing compound, preferably a silyl ether; or
(b) Comprising a structure selected from:
6. the method according to any one of claims 1-5, wherein at least 70%, preferably at least 85% of the surface (2) of the solid phase fragments is coated with a biological material.
7. The method according to any one of claims 1-6, wherein in the contacting step the lift head presses the membrane in the direction of the coated surface (2) of the solid phase pieces.
8. The method of claim 7, wherein the squeezing by the poppet does not damage, tear, or puncture the membrane.
9. The method according to any one of claims 1-8, wherein the tensile stress is generated by tensioning the membrane via a frame, preferably a circular frame.
10. The method of any of claims 1-9, wherein removing is accomplished via a removal device having suction cups.
11. The method of any one of claims 1-10, wherein the film is an adhesive film comprising:
(a) polyvinyl chloride (PVC); and/or
(b) An acrylic adhesive.
12. Biomaterial-coated solid phase fragments obtainable by the method according to any one of claims 1-11.
13. A support having applied thereto at least one solid phase fragment according to claim 12.
14. An apparatus for preparing biomaterial-coated solid-phase chips, comprising:
-a tensile stress device configured to generate a tensile stress on the membrane;
-a poppet device configured to pre-separate solid phase fragments from the membrane; and
-a removal device configured to remove solid phase debris from the membrane.
15. The apparatus of claim 14, further comprising at least one control device.
Technical Field
The invention relates to a method for producing biomaterial-coated solid-phase fragments, to biomaterial-coated solid-phase fragments produced by such a method, to a carrier on which at least one solid-phase fragment according to the invention is applied, and to a device for producing biomaterial-coated solid-phase fragments, comprising a tensile stress device, a lift head device and a removal device.
Background
Many test systems for clinical and medical diagnostics, veterinary medicine, pharmaceutical and toxicology research, food and environmental analytics, and for general biological and biochemical analysis are based on the use of solid phase bound biological agents (solid phase tests). A typical example is an immunoassay system, without which many areas of modern diagnostic and analytical chemistry cannot be imagined today. Immunoassays are techniques for measuring the presence of a substance by means of an immune response. Each immune response is characterized by an interaction between an antibody and an antigen. The result of the interaction is an antibody-antigen complex. Antibodies or corresponding antigens can be detected by in vitro assays. The detection of antibodies presupposes the addition of the relevant antigen to the test system and vice versa. Antibodies (immunoglobulins) are a specific type of protein. The antigen may be a biological or organic molecule, but may also be other chemical compounds. Most immunoassays require the separation of bound and free antibodies and antigens (heterogeneous immunoassays or solid phase immunoassays). The separation is accomplished by a reaction partner (antibody or antigen) bound to a solid phase. For example, when an antibody is coupled to a solid phase, unbound antigen can simply be separated from antibody-bound antigen. A solid phase bound biological agent is referred to if an antibody or antigen in the form of a biomolecule (e.g.protein, nucleic acid, polysaccharide, lectin) or as a component of a biological substance (tissue section, microorganism, cell) is bound to a solid phase.
Solid phase tests are generally easier to perform, more sensitive and simpler to automate than liquid phase tests, in which the reaction partners and reaction products are present in solution.
A particular form of diagnostic solid phase assay is the so-called indirect immunofluorescence assay (IIFT). For the identification of autoantibodies or infectious antibodies in, for example, patient samples, cells, tissue sections or purified, biochemically characterized substances are used as antigen matrix on microscope slides. In the first incubation step, in the case of a positive sample, the antibody to be detected from the diluted patient serum binds itself to the solid phase-bound antigen. In the second culturing step, the antibody is visualized using a fluorescently labeled anti-human antibody. The antibodies bound within stromal cells or stromal tissue are recognized under a fluorescence microscope and optionally their specific localization.
Indirect immunofluorescence assays allow for high specificity, i.e., positive and negative samples to produce large signal differences. Furthermore, depending on the localization of the respective antigen, a typical fluorescence pattern is shown for each bound antibody, and this can be used to identify the patient's autoantibodies. Furthermore, the entire antigen spectrum of the starting substrate is available, whereby many antibodies are captured and a high hit rate can be achieved. In addition, indirect immunofluorescence testing is the method of choice when the test antigen for the enzyme immunoassay cannot be processed for analysis.
The BIOCHIP technique includes an activation step in which cultured cells or tissue slices are applied to a physically or chemically activated coverslip.
EP 0117262B 1 discloses the BIOCHIP mentioned above.
EP 1718948B 1 describes the above-mentioned IIFT and corresponding BIOCHIP preparation method. The preparation method uses a needle that penetrates into an adhesive film on which the biomaterial-coated cover glass fragments are located. The needles penetrate the membrane so that the coverslip fragments can be removed from the membrane by the suction cups. However, larger (greater than 3.25mm side length) BIOCHIPs cannot be prepared using this preparation method.
It is also desirable to be able to produce relatively large BIOCHIPs for different applications, such as pathological examination of patient tissue or simultaneous examination of multiple different tissue types. Therefore, there is a need for a method that allows for the reproducible preparation of larger biomaterial-coated coverslips, and for such coated coverslips.
Disclosure of Invention
The preparation method, the biomaterial-coated solid phase fragments, the carriers comprising these and their preparation equipment (which includes larger BIOCHIP than known in the prior art) are described below and are the subject of the present invention.
The inventors of the present invention have unexpectedly found that the use of needles in the separation step of the process for the preparation of solid phase fragments coated with biological material can be replaced by a pre-separation step using a lift head. With this modification, significantly larger solid phase/coverslip fragments can be prepared. In particular, in the method according to the invention, a tensile stress is generated on the membrane coated with the solid phase fragments. Thereafter, the membrane is contacted with a poppet to pre-separate one or more solid phase fragments from the membrane.
In a first aspect, the present invention therefore relates to a method for preparing biomaterial-coated solid-phase fragments, comprising:
-providing a biomaterial-coated solid support, wherein the solid support is fragmented and the fragments adhere to the membrane;
-creating a tensile stress on the membrane coated with the solid phase fragments;
-contacting the membrane with a poppet to pre-separate one or more solid phase fragments from the membrane; and
-removing one or more pre-separated solid phase fragments from the membrane.
In the following, preferred embodiments of the method according to the invention are described, wherein (a) the biomaterial-coated surface (2) of the solid-phase fragments occupies at least 10.25mm2Preferably at least 25mm2(ii) a (b) At least one side (4,5) of the solid phase fragments has a length greater than 3.25mm, preferably greater than 5 mm; and/or (c) the solid phase fragments are glass fragments.
In a preferred embodiment, the solid phase pieces have a thickness (3) of between 0.05mm and 0.3mm, preferably between 0.1mm and 0.2 mm.
In a further preferred embodiment, the biomaterial is bound to the solid support via a linker molecule. The linker molecule may be (a) a silicon-containing compound, preferably a silyl ether; or (b) a structure selected from:
in a preferred embodiment of the method according to the invention, at least 70%, preferably at least 85%, of the surface (2) of the solid phase fragments is coated with a biological material.
Further, in a preferred embodiment, the method comprises: in the contacting step, the poppet presses the membrane in the direction of the coated surface (2) of the solid phase pieces. In a further preferred embodiment, the membrane is not damaged, torn or pierced by the pressure caused by the lifting head.
In a further preferred embodiment, the tensile stress is generated by tensioning the membrane by means of a frame, preferably a circular frame.
In a preferred embodiment, the removal is effected by means of a removal device with suction cups.
In a further preferred embodiment of the process according to the invention, the film is an adhesive film comprising (a) polyvinyl chloride (PVC) and/or (b) an acrylic adhesive.
In a second aspect, the present invention relates to a biomaterial-coated solid phase fragment obtainable by the method according to the present invention.
In a third aspect, the invention relates to a carrier on which at least one solid phase fragment according to the invention is applied.
In a fourth aspect, the present invention relates to an apparatus for preparing biomaterial-coated solid phase fragments, comprising:
-a tensile stress device configured to generate a tensile stress on the membrane;
-a poppet device configured to pre-separate solid phase fragments from the membrane; and
-a removal device configured to remove solid phase debris from the membrane.
In a preferred embodiment, the apparatus according to the invention further comprises at least one control device.
A further aspect of the invention relates to solid phase fragments coated with a biomaterial and wherein (a) the biomaterial-coated surface (2) of the solid phase fragments comprises at least 10.25mm2Preferably at least 25mm2(ii) a And/or (b) at least one side (4,5) of the solid phase fragments has a length of more than 3.25mm, preferably more than 5 mm. Preferably, the solid phase fragments are glass fragments. In the preferred embodimentIn this case, the thickness (3) of the solid phase fragments is between 0.05mm and 0.3mm, preferably between 0.1mm and 0.2 mm. In a further preferred embodiment, the biomaterial is bound to the solid support via a linker molecule. The linker molecule may be (a) a silicon-containing compound, preferably a silyl ether; or (b) a structure selected from:
in a preferred embodiment of the solid phase fragments according to the invention, at least 70%, preferably at least 85% of the surface (2) is coated with a biomaterial.
As used herein, the expression "biological material" refers to material isolated from or derived from a living organism. Without being limited thereto, examples of biological materials include proteins, nucleic acids, lipids, cells, tissues, tissue sections, organs, organ sections, cell-based constructs, or combinations thereof. In some aspects, the biological material may refer to mammalian cells. In other aspects, biological material may refer to immobilized cell cultures, platelets, bacteria, viruses, mammalian cell membranes, liposomes, enzymes, or combinations thereof. In other aspects, the biological material may refer to a germ cell comprising a sperm cell, a spermatocyte, an oocyte, an egg cell, an embryo, a blastocyst, or a combination thereof. In other aspects, the biological material can refer to whole blood, serum, red blood cells, white blood cells, platelets, cerebrospinal fluid, viruses, bacteria, algae, fungi, or combinations thereof. Biological materials also include recombinantly produced, optionally purified, proteins and antibodies, e.g., a homogeneous population of monoclonal antibodies or a heterogeneous population of polyclonal antibodies.
As used in this application, the expression "coating" shall mean applying the biological material to the surface of the solid phase fragments. Encapsulation includes binding by adhesion, binding by linker molecules, film coating and encrustation. The coating can be complete or partial, e.g., greater than 30% or more, greater than 40% or more, greater than 50% or more, greater than 60% or more, greater than 70% or more, greater than 75% or more, greater than 80% or more, greater than 85% or more, greater than 90% or more, greater than 95% or more, greater than 97% or more of the surface of the solid phase fragments. In a further preferred embodiment, the coating is preferably applied over substantially the entire surface of the solid phase fragments.
As used interchangeably herein, the expression "linker molecule" or "linker" refers to any molecular unit that causes a spatial association between two or more molecules, groups of molecules, and/or molecular units. Preferably, the linker molecule has a covalent bond to the solid phase fragment and has a further covalent bond to the biomaterial. Exemplary linkers that can be used in the solid phase fragments and methods of the present invention are polymers, such as polyethylene glycol (PEG) units, wherein the polymer chain may additionally comprise alkyl, alkenyl, alkynyl, ester groups, ether groups, amide groups, imide groups, sulfo groups, and/or phosphodiester groups. Particularly preferably, the linker molecule is a silicon (Si) -containing compound, even further preferably the base structure R1R2R3Si-O-R4In which R is1、R2And R3Is an unsubstituted or substituted organic radical, and R4Is an alkyl or aryl group.
As used herein, "solid phase fragments" refers to any solid phase material to which biomolecules can adhere. Exemplary solid substrates that can be used with the biomaterials and methods of the present disclosure include spheres, slides, wells, and chips that can be made from a variety of solid phase materials including glass, polymers, and silicon. Particularly preferably, the solid phase fragments are biomaterial-coated glass carriers. As used herein, the expression "solid support" refers to a solid coated substrate as described above but prior to its disruption. In this respect, the above-mentioned material properties of the solid phase fragments also apply to the solid phase support.
As used herein, the expression "fragmenting" relates to a solid support reduced to two or more parts (fragments). The person skilled in the art knows various techniques for fragmenting, in particular, coated glass carriers. These include breaking, cutting (by means of a cutter using a blade or water jet), laser and scoring,but is not limited thereto. The shape of the resulting sheet may be uniform or varied. In a preferred embodiment, the sheet is of uniform shape, in particular rectangular. In yet a further preferred embodiment, the sheet is square. Preferably, at least one surface area of the fragmented pieces is no greater than 1500mm2No greater than 1000mm2No more than 750mm2No more than 500mm2No more than 400mm2No greater than 300mm2No greater than 200mm2Or not more than 120mm2. In a further preferred embodiment, the length of the sides (4,5) of at least one side is not more than 150mm2No greater than 130mm2No greater than 100mm2Not more than 80mm2No greater than 50mm2Not more than 40mm2No more than 30mm2Or not more than 25mm2。
As used herein, the expression "film" refers to an adhesive film or an adhesive film. An adhesive film in the context of the present invention is a film that allows adhesion to glass by virtue of its adhesive force at its surface, which preferably has a thickness of between 0.001mm and 1.5 mm. In a preferred embodiment, the work of adhesion, i.e. the force that must be expended to separate the adhesive film and solid phase fragments, is at least 0.00001J/mm2At least 0.00001J/mm2At least 0.00001J/mm2At least 0.00001J/mm2At least 0.0001J/mm2At least 0.001J/mm2At least 0.01J/mm2At least 0.1J/mm2At least 1J/mm2At least 10J/mm2Or at least 100J/mm2。
As used herein, "adhesive film" is understood to mean a (plastic) film provided with an adhesive on one or both sides. Adhesives are natural or synthetic materials that can adhere to the site of topical application. Preferably, the adhesive can adhere to glass. Further preferably, suitable adhesives are selected from the group consisting of polyisobutylene, acrylates, silicones, polyisoolefins, polyether block amide copolymers, polybutadiene, styrene-butadiene (or isoprene) -styrene block copolymer rubbers, vinyl-based high molecular weight materials such as polyvinyl alkyl ethers, polyvinyl acetate, ethylene-vinyl acetate copolymersPartially saponified products of polyvinyl acetate, polyvinyl alcohol and polyvinyl pyrrolidone, polyurethanes, or combinations thereof. In a preferred embodiment, the adhesive is an acrylic adhesive. The acrylic adhesive includes crosslinked and uncrosslinked acrylic copolymers selected from the group consisting of polymethacrylates, such as butyl acrylate, ethylhexyl acrylate, vinyl acetate, (meth) acrylic acids, such as butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, nonyl (meth) acrylate, decyl (meth) acrylate, undecyl (meth) acrylate, dodecyl (meth) acrylate, and tridecyl (meth) acrylate, and copolymers of at least one of the above esters and other monomers copolymerizable therewith. Preferred acrylate polymers are under the trade name
The film material (of the adhesive and adhesive films) may be a thin, flexible film material and include polymeric films such as polyvinyl chloride (PVC), Polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), Polystyrene (PS), Polyamide (PA), similar polymers, and combinations thereof; and a metal film such as aluminum (Al). Preferably, a polyvinyl chloride (PVC) film is used.
As used interchangeably herein, the expression "tensile stress" or "mechanical stress" is denoted by the symbol "σ" and is, on an imaginary cross-section through the body, the component F in the i-directioniThe i direction being based on a component FiArea of action a (normal n). The stress can be calculated as follows:
in a preferred embodiment, the tensile stress on the membrane equipped with solid phase fragments is at least 0.00001N/mm2At least 0.00001N/mm2At least 0.00001N/mm2At least 0.00001N/mm2At least 0.0001N/mm2At least 0.001N/mm2At least 0.01N/mm2At least 0.1N/mm2At least 1N/mm2At least 10N/mm2Or at least 100N/mm2。
As used herein, "lift head" refers to a working tool that can be brought into contact with a membrane provided with solid phase debris and is configured such that, with its aid, the debris can be removed from the membrane by the method according to the invention. In this respect, the lift head has at least one side contactable with the membrane. The sides/faces of the poppet may have any shape but are preferably circular or oval. In other preferred embodiments, the sides/faces of the poppet have a circumference or diameter of 0.001mm to 6mm, 0.05mm to 5mm, 0.1mm to 4mm, 0.5mm to 3mm, 0.7mm to 2mm, or 1mm to 1.5 mm. In a further preferred embodiment, the lift head arrangement according to the invention has at least one lift head constructed as above.
As used herein, the expression "pre-separation" refers to partial separation of solid phase fragments from the membrane. In preferred embodiments, "pre-separation" means that at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, or at least 97% of the surface sides of the solid phase fragments are separated from the membrane. Without being bound by any particular theory, it is speculated that the pre-separation in the method according to the invention is produced by the cooperation of the tensile stress on the membrane with the contact and compression of the poppet in the direction of the clad surface. Due to such movement of the poppet under the solid phase debris, the membrane appears to deflect and separate from the areas of the debris not in contact with the poppet (see fig. 5).
As used interchangeably herein, the terms "remove" and "take-off" refer to the final separation of the solid phase fragments from the membrane. This means that the previously bound surface side of the solid phase fragments is 100% separated from the membrane. After removal, there is no direct or indirect connection between the solid phase fragments and the membrane. Such a step is preferably carried out by means of a device having suction cups.
The biomaterial-coated surface of the solid-phase fragments is denoted by reference numeral (2) in fig. 1. By an edge (4) and(5) the dimensions of the surface are determined. In a preferred embodiment, the biomaterial-coated surface (2) of the solid-phase fragments occupies at least 5mm2At least 10mm2At least 10.25mm2At least 15mm2At least 20mm2At least 25mm2At least 30mm2At least 40mm2At least 45mm2Or at least 50mm2。
The edges of the biomaterial-coated solid phase fragments are denoted by reference numerals (4) and (5) in fig. 1. Preferably, at least one side (4,5) of the solid phase pieces has a length of more than 1mm, more than 1.5mm, more than 2mm, more than 2.5mm, more than 3.25mm, more than 4mm, more than 4.5mm, more than 5mm, more than 7mm, more than 10mm, more than 15mm, more than 20mm or more than 22 mm. In an alternative embodiment, the two sides (4) and (5) are each greater than the aforementioned length.
The solid phase chip thickness is indicated by reference numeral 3 in fig. 1. Preferably, the solid phase fragments have a thickness between 0.001mm and 1.5mm, between 0.005mm and 1mm, between 0.01mm and 0.8mm, between 0.05mm and 0.6mm, between 0.06mm and 0.5mm, between 0.07mm and 0.4mm, between 0.08mm and 0.3mm and between 0.1mm and 0.2 mm. The values are the same for the thickness of the solid support.
As used herein, the expression "pressing in the direction of the coating surface" means that the poppet is placed on the membrane directly on the unassembled side under the solid phase fragments and the fragments are pressed upwards. Upon pressing, on the one hand, the position of the poppet changes, and on the other hand, the position of the membrane and the solid phase fragments adhering thereto changes relative to each other. On the one hand, this can be achieved by the membrane being held rigidly in its position, while the lifting head is guided in a movement from bottom to top. Alternatively, the poppet may be held rigidly in one position while the membrane and solid phase fragments adhered thereto are pressed over the poppet. In a third alternative, the poppet and the membrane may be moved towards each other by changing the position of both the poppet and the membrane. In a preferred embodiment of the invention, the stroke of the lift head in the direction (or reverse direction) is 0.1mm to 30mm, 0.3mm to 25mm, 0.5mm to 20mm, 0.8mm to 15mm, 1mm to 13mm, 3mm to 10mm, 4mm to 8mm or 5.5mm to 6.5 mm.
As used herein, "without damage, tearing, or puncturing" means that no damage is left after the poppet contacts and presses on the membrane. In the context of the present disclosure, no damage is stretching and discoloration of the film. In the context of the present disclosure, damage is the occurrence of holes or tears in the film, protruding fibers on the film surface and the occurrence of build-up, i.e. local accumulation of film material. Thus, the poppet deflects the membrane and solid phase fragments after contact and optionally leaves stretch and discoloration on the membrane.
As used herein, the expression "frame" refers to a clamping system to which the membrane can be fitted. Such systems are known, for example, as clamping devices from wafer production. Available clamping devices are commercially available, in particular from Minitron Elektronik GmbH (Ingolstadt, germany), Technovision, Inc. (Nakayama, japan), Ultron Systems, Inc. (moorpak, usa) and Thai-Hibex (pathmutani, thailand). In a preferred embodiment, the frame described herein is part of a tensile stress device according to the invention.
Removal devices with suction cups, as are suitable for use in the method according to the invention, are exemplarily described in paragraphs [0059] to [0061] and [0063] to [0066] in EP 1718948B 1, and are herein the subject of the present disclosure by reference.
As used herein, "support" refers to a solid to which solid phase fragments according to the present invention can be attached. The support has at least one flat face onto which one or more solid phase fragments can be applied. In a preferred embodiment, the support material consists of or comprises glass, polymer or silicon. The solid phase fragments and the support are preferably brought together by adhesive bonding of at least one surface, melting or by a physical connection system, for example a mortise and tenon joint. However, the combination of the carrier and the solid phase fragments is preferred. Furthermore, it is preferred that at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten solid phase fragments according to the invention are applied to the carrier. In a preferred embodiment, the carrier may have a diameter of at least 5X 25mm2At least 15X 25mm2At least 25X 25mm2At least 40X 25mm2Or at least 60X 25mm2The size of (c). For example, at least 5X 25mm in size2The carrier of (2) can be assembled with a total of 5 carriers each having a diameter of 5X 5mm2Solid phase fragments of size, 20 each having 2.5X 2.5mm2Solid phase fragments of size or 125 each having a size of 1X 1mm2Solid phase fragments of size.
As used herein, "apparatus for preparing solid phase fragments according to the present invention" refers to one or more workpieces comprising a tensile stress device, a lift head device, and a removal device as described herein. The apparatus may be wholly or partially separated from other apparatus and form a preparation system or all apparatus may be directly or indirectly connected to one another.
As used herein, the expression "antibody" refers to a protein from the class of globulins, which is formed in response to certain substances (so-called antigens) in vertebrates. Antibodies are components of the immune system. Antibodies are produced by a class of white blood cells, B lymphocytes. They can be distinguished according to different classes, namely immunoglobulin A, immunoglobulin D, immunoglobulin E, immunoglobulin G, immunoglobulin M, immunoglobulin W and immunoglobulin Y.
As used herein, the expression "antigen" preferably refers to a substance to which antibodies and certain lymphocyte receptors can self-specifically bind. The antigen may be a protein, but may also be a glycoprotein, carbohydrate, lipid or other substance. In the context of the present invention, the antigen is preferably a protein or a post-translationally modified protein.
In general, "at least 1" as used herein means 1, 2, 3, 4,5, 6, 7, 8, 9 or more, where data optionally refers to the type of substance in question and not the absolute number of molecules.
Depending on the particular implementation requirements, the control means can be implemented as hardware and/or software for exemplary embodiments of the present invention. The control device mentioned here can be realized here as at least one control device or by a plurality of control devices cooperating. Implementation can be performed using a digital storage medium, such as a floppy disk, a DVD, a blu-ray disk, a CD, a ROM, a PROM, an EPROM, an EEPROM or FLASH memory, a hard disk, or some other magnetic or optical memory (which stores electronically readable control signals that interact with or cause certain methods to be performed).
A Programmable hardware component may be formed as a control device by means of a processor, a Central Processing Unit (CPU), a computer System, an Application-Specific Integrated Circuit (ASIC), an Integrated Circuit (IC), a System On Chip (SOC), a Programmable logic device, or a Field Programmable Gate Array (FPGA) having a microprocessor.
Although some aspects are described in connection with the solid phase fragments according to the invention, it is clear that said aspects are also a description of the corresponding (preparation) methods and vice versa.
To prepare the method according to the invention, the biomaterial-coated solid support can be prepared as follows: biological substances such as those mentioned above are first applied to a carrier material, such as paper-thin (0.15mm) glass, for example a cover slip. In general, the biological activity of the biological substance used for coating and called matrix remains unchanged. If necessary, the biomaterial may be covalently bonded to the carrier via a linker molecule.
The biological substance-coated carrier material, for example a cover slip, is fixed on a plastic film provided with an adhesive, for example in the form of a film ring, with which the biological substance is not in contact. The membrane ring may be a tensile stress means (12), which in a preferred embodiment is associated with a control means (15) and may be controlled by a control signal SIG2, for example in order to electronically initiate the removal process on the device according to the invention (see fig. 2).
With the substance layer, i.e., substrate, side down, the coverslip is placed in a recess or template of the device, with the uncoated coverslip edge lying flat. The template serves as a defined orientation of the cover slip in position relative to the membrane and the rotation angle. The coverslip is attached to the membrane by adhesion, with the uncoated side of the coverslip meeting the membrane located in the membrane ring.
Then, the support material (cover glass) was divided into millimeter-sized pieces (solid-phase pieces) together with the biological substance (substrate) using an apparatus equipped with diamond (crusher or crushing device) without cutting the film.
After the fragmenting, the substrate-equipped cover glass on the film ring is additionally rolled between the flexible substrate and a pressure roller in order to fragment the not yet fragmented substrate fragments. However, the matrix fragments produced here are so dense that their edges rub against one another. To prevent this, the matrix fragments may be separated by concentric stretching of the film in a ring of film. For this purpose, the membrane clamped between two concentric rings is stretched in all directions, for example by pressing into the same ring instead of a larger ring. Clean cut pieces are then obtained, with precise dimensions and precise distribution of the substrate pieces.
In order to remove solid phase fragments according to the invention, the membrane ring is placed on a membrane holder of an assembly machine which is movable in at least one direction. The membrane support is removed entirely in the center.
The assembly machine is loaded with analysis plates, i.e. for example printed slides, which can be supplied to the machine on a transfer tray. The transfer tray may have movable sides that secure and thus better position the slides.
The assembly machine is equipped with a conveying device consisting of two movable conveying carriages moving on rails. On a transport carriage, vacuum-supported suction cups are mounted, which can be moved in at least two, preferably three, spatial directions and are driven by stepping motors. The suction cup is pneumatically supported and rotatable. In addition, the suction cup is part of a removal device (14), which in a preferred embodiment is associated with a control device (15) and can be controlled by a control signal SIG1, for example in order to electronically control the movement of the suction cup or of the entire removal device in the movement directions R1 and R2 (see fig. 2).
Located below the membrane ring with the membrane fitted with matrix fragments mounted in the assembly machine is a piston-like device (removal device (13)) with a lift head supporting the separation of the matrix fragments from the membrane. The lift head slowly lifts about 4-8mm from the rest position (see fig. 5), touching the membrane from below and causing a pre-separation. The final removal is controlled from above by suction of the substrate fragments by the suction cups. The lift head is part of a lift head arrangement (13), which in a preferred embodiment is associated with the control device (15) and can be controlled by a control signal SIG3, for example in order to electronically control the movement of the lift head or the entire lift head arrangement (13) in the movement directions R3 and R4 (see fig. 2).
The second transport carriage located on the transport device of the assembly machine serves as a so-called "adhesive carriage". It is equipped with a device containing a removable adhesive needle and connected to the adhesive container by a hose. Prior to application of the matrix fragments to the slide, the adhesive needle meters or distributes the UV adhesive in a volume measured proportionally to the area of the matrix fragments onto a selected mounting location or surface of the slide, with the adhesive needle in contact with or in close proximity to the slide. Immediately thereafter, the matrix fragments were placed on the slide at the locations wetted by the adhesive. Additional matrix fragments are then accurately placed on the slide in the same manner. Alternatively, the adhesive can also be metered in a contactless manner. Such systems are known in the prior art and are high-performance injection valves for contactless microdosing of low to medium-viscosity media, which are sold, for example, by Nordson corporation (Westlake, usa) in the form of "P-Jet CT injection valves".
The assembly machine (synonymously "apparatus for preparing biomaterial-coated solid-phase fragments) may contain means for in-process control and immediate error correction serving as the work step performed.
The prepared carrier comprising the solid phase fragments according to the invention can then be subjected, for example, to various staining techniques (e.g., hematoxylin-eosin staining) for pathological examination. Alternatively, for example, the IIFT test as described by Damoiseaux et al (Damoiseaux, j. et al (2009) Journal of Immunological Methods, vol 348, pp 1-2, pp 67-73) can be performed on the vectors according to the invention.
Drawings
FIG. 1 schematically shows the structure of a biomaterial-coated solid phase chip (1).
Fig. 2 schematically shows the structure of an apparatus for preparing biomaterial-coated solid-phase chips (1) comprising a tensile stress device (12), a lift head device (13), a removal device (14) and a control device (15).
Fig. 3 shows various embodiments of a poppet.
Fig. 4 shows the arrangement of the poppet head on the membrane containing the solid phase fragments (1), the solid phase fragments (1) being pre-separated from the membrane and the top view of the membrane after removal of one solid phase fragment (1). (A) The membrane is in contact with a piston-shaped lifting head below one solid phase fragment (1). (B) A solid phase fraction (1) is pre-separated by means of a piston-shaped lifting head. (C) A solid phase chip (1) is pre-separated by a lift head device (13) comprising a ball point pen, the tip of which acts as a lift head. (D) After removal of one solid phase fragment (1), a membrane comprising fragmented solid phase carriers.
Figure 5 shows schematically the pre-separation of solid phase fragments (1) from the membrane by means of a poppet. (A) The solid phase fragments (1) adhere to the membrane before contacting the poppet. The solid-phase fragments (1) are in full contact with the membrane with their uncoated side. (B) By creating a tensile stress on the membrane and contacting the poppet, the solid phase fragments (1) are separated from the membrane around the contact site. The solid-phase fragments (1) are thus only partially in contact with the membrane on their uncoated side.
Fig. 6 shows in tabular form the maximum solid phase fragment size that can be prepared by the method according to EP 1718948B 1 and the method according to the invention.
Examples
Materials and instruments:
-various lift heads: the ball-point pen comprises a ball-point pen refill, a cylindrical head screw, a 3D printer head, a positioning pin and a pipettor tip;
-a vice for receiving a lifting head;
membranes stretched in a membrane ring and equipped with solid phase fragments of various formats
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