DE10316689A1 - Three-dimensional chip - Google Patents

Abstract

Chip for examining biological material with a carrier unit made of porous material, on which the reference molecules used for the detection of the sample molecules are immobilized.

Description

The The invention relates to a chip for examining biological samples and methods of manufacturing these chips.

biochips find in recent Extensive application, for example in genetic analysis, mutation detection or the study of protein / protein interactions and - in general - at Diagnosis of diseases or the assessment of the corresponding genetic Disposition.

It Both gene chips and protein chips (microarrays) are known.

The Known chips consist of a carrier and the "target" molecules immobilized on the carrier (hereinafter also reference molecules). The reference molecules become dependent selected by the purpose of the investigation. The principle of the biochip consists of the interactions between the known reference molecule and a as yet unknown sample molecule - for example from a patient or other biological material and from this evidence conclusions for the purpose of the investigation to draw.

To the Detection of nucleic acids have to the sample molecules first hybridize with the reference molecules. Protein chips interact with protein chips, for example the interactions between an immobilized Antigen as a reference molecule and an antibody from the patient sample. The interactions that are carried out are often through Fluorescence or isotope labels made visible. The evaluation the chips require that the Position of the reference molecules is known on the chip.

It a variety of methods of chip production are known. This are often based either on the direct synthesis of the reference molecules on the carrier or the pre-synthesis of the reference molecules and their subsequent immobilization on the carrier. The need to determine the position of the reference molecules on the Determining the chip makes the known methods complex and expensive expensive.

biochips have the special advantage compared to other detection methods, that she allow the processing of a complex question. The definition of the samples on the carrier allows about that a largely automated evaluation of the results, that of the possibilities the information technology is supported. Nevertheless have the known biochips have not yet achieved the desired level of sensitivity and specificity.

task The present invention is therefore to provide a biochip which is an improvement over the Represents state of the art. Furthermore, methods are proposed the one if possible simple, fast and inexpensive Allow manufacturing of these chips.

Under sensitivity the smallest concentration in the present context Understood sample molecules, with the help of the chip according to the invention can still be detected.

This Task is solved by a device according to the main claim. The manufacturing processes are the subject of independent process claims.

The The present invention is based on the idea of a porous material as a carrier of the biochip and use the reference molecules on this porous material to immobilize. The manufacturing processes are based on the same to arrange these polymers so that they are in a three-dimensional Represent chip.

The chip according to the invention has due to the porous Materials have the advantage that for immobilization of the reference molecules an enlarged surface for disposal stands. The density of the reference molecules per carrier unit can thus be increased.

Furthermore, the use of porous material can make it possible to dispense with a further solid carrier substance in the sense of a support layer. This not only has advantages in terms of production technology and costs, but also enables all-round access of the sample molecules from the test sample to the reference molecules. In contrast, there is access to the sample molecules with known chips only possible on one side. The easier access improves the sensitivity of the chip.

As support material can one or more polymers can be selected from the following group: Polyethylene, polyvinyl chloride, polypropylene, polyethylene terephthalate (PET), polyamide, polyimide, polystyrene, polyvinylidene chloride (PVDC), Polycarbonate, cellophane, acetate or polysulfonate.

Table 1 provides an overview of the advantageous properties of these polymers.

The chip according to the invention can only consist of a layer of the carrier material. The necessary stability the layer can be achieved by choosing a suitable layer thickness become. However, different layers are advantageously combined. The layers can consist of the same or different polymers.

advantageously, become the reference molecules immobilized on the polymers before the layers are brought together. The reference molecules are immobilized on the support by means of the methods known to the person skilled in the art. The layers with each a quality of reference molecules are hereinafter referred to as "carrier units".

In The chip is a particularly advantageous embodiment from a variety of carrier units, the - checkerboard-like - in X- and Y direction and one above the other lie next to each other in the Z direction.

By the increase the number of carrier units used let yourself the number of different provided for the investigation reference molecules vary. Of course can individual carrier units also several times - e.g. for control purposes - occur.

In a preferred embodiment can the individual layers of the carrier units 0.01 to 1 micron. The area of the carrier units can be up to a few Square meters.

at some as porous support material used polymers, or with very thin layers, can it may be beneficial to the carrier units at least partially from proppants to surround. Also other proppants be advantageous on the top or bottom of the carrier. This can also from permeable or porous Material. As a rule, they are not equipped with reference molecules. Furthermore can the layers over Holding means, such as clips, connected to each other his.

The leave carrier according to the invention itself easily and inexpensively produce.

In a first alternative, the manufacturing process begins with to load individual polymer layers with reference molecules. The number the layers vary depending from the purpose of the investigation. The next step is the layers (Carrier units) laid on top of each other to form a first block and, for example, with a Microtome cut at right angles to the layer plane. This results in first sheet-like Cuts with "ribbons" from carrier units, each containing different reference molecules.

slice of different blocks can be stacked again to form a second block ("multilayer blocks"). This block too will be in thin layered Cut units at right angles to the plane of the layers.

The result of this combination of different carrier units with a subsequent cut is a “multimatrix” which contains each carrier unit that was originally used in the respective blocks. This manufacturing principle is in the 1 shown.

at Knowledge of the layers used and knowledge of the cut as well the position of a can be reassembled each carrier unit Predetermine beyond doubt or even understand later.

Alternatively, the carrier according to the invention can also be produced using a second method. This is in 2 outlined:

This The essence of the process is to first mount the carrier units a proppant to arrange and on the opposite End of the carrier units covered with a sliding mask. This mask has openings through which the backing materials with reagents - for example also with the reference molecules - loaded can be. The openings can for example, be cylindrical or conical. The mask itself can over the known methods of lithography are generated.

After the in the 2 In the overview shown, a device can be used which comprises a mask made of a solid material with a thickness of 400 to 500 μm. It has conical openings, the upper diameter of which can be 100 μm and the lower 5 μm, for example. This mask is relative to the support means in the X and Y directions 4 . 2 displaced. Other dimensions are also possible.

In this embodiment, the support means consist of 50 to 100 μm thick plastic units with passages of 5 to 10 μm. The carrier material itself is in receptacles 3 arranged in the dar embodiment can be 50 microns thick. The receptacles for the carrier material provided in these support means also have a diameter of 5 to 10 μm. These recesses can consist entirely of the porous carrier material or can be filled with it.

A schematic representation of the recesses filled with different types of carrier materials (spherical, rod-shaped or gel-like) is shown in FIGS 4 and 5 recognizable.

to When the chip is loaded, the mask becomes relative in the X and Y directions to the support means emotional. This process is repeated until all of the desired support units contains the reagent. This creates initially a chip with different carrier units, that expand in the X and Y directions. So this chip is two-dimensional. To complete the chip, the mask can be used as well the opposite one Proppant, on which the carrier unit are arranged to be removed. Thus the backing material again accessible from both sides of the sample material.

The carriers in the recesses can be successively equipped with nucleic acids, for example, using the method according to the invention ( 6 ). For this purpose, oligonucleotides are placed over the support in a defined order. This "growth" of the reference molecules on the carrier is also in the 7 shown.

In a further embodiment, the chips produced in this way can be placed one on top of the other in the Z direction, so that the carrier units are aligned. The result is a three-dimensional chip ( 2 ).

The Evaluation of the carrier according to the invention sets ahead of the with the reference molecules interacting sample molecules inside the carrier unit are detectable.

The Examination of three-dimensional arrangements is possible, for example confocal laser scanning microscopy (LCSM) possible. This allows an evaluation also below the chip surface.

The use of confocal laser scanning microscopy for evaluating the chip according to the invention is described in 3 represented schematically:
The steps 1 to 4 shown show the recordings of the signals along the Z axis. Step 5 shows the movement along the Y axis.

in the Step 1, the laser beam passes through the light-refractive plate to the lens of the microscope and is thus on points with a Diameter from 5 to 10 μm focused. This stimulates the luminescence in the spot. As The result of this excitation is a second light beam through the Plate sent to the detector. The signal and its spatial Coordinates are documented. Then the laser moves and focuses on another spot.

Claims (8)

Chip for the investigation of biological material with a carrier unit made of porous Material on which those used to detect the sample molecules reference molecules are immobilized. Chip according to claim 1 characterized by at least two carrier units with different reference molecules in the X or Y axis lie together. Chip according to claim 2, characterized in that at least some of the carrier units lie together in the Z-axis. Chip according to one of the preceding claims, characterized characterized that the support units at least partially from proppants or holding devices are surrounded. Chip according to one of the preceding claims characterized that the support units include one of the following polymers: Polyethylene, polyvinyl chloride, Polypropylene, polyethylene terephthalate (PET), polyamide, polyimide, polystyrene, Polyvinylidene chloride (PVDC), polycarbonate, cellophane, acetate or Polysulfonate. Method for producing a chip according to one of the preceding claims, comprising the following steps: a. Immobilizing a group of sample molecules on a first carrier unit and immobilizing a second group of sample molecules on a second carrier unit, b. Arranging the carrier units to form a block with a common Z axis, c. Make cuts perpendicular to the block plane, d. Repeating steps a. till B. to provide a second block of support units and sections thereof, e. Combine the cuts from c. and d. to a block with a common Z axis, f. Repeating the step of b. Process for producing a chip according to a of claims 1 to 5 with the following steps: a) Arranging carrier units made of porous Material on a proppant b) Cover the carrier units at their the proppant opposite End with a mask with at least one opening that opens a passage to one carrier unit located below the mask can enable. c) Move the mask so that the opening is over a support unit lies. d) loading the carrier unit with reference molecules. e) Investigation of biological molecules of a three-dimensional carrier characterized by the use of confocal laser scanning microscopy Use of confocal laser scanning microscopy for Evaluation of a three-dimensional chip.