DE10224568A1 - Microarray device - Google Patents

Abstract

The present invention relates to a microarray device based on a porous material, preferably a microporous, polymeric membrane, which has a multiplicity of porous regions (zones) arranged in a high density according to a predetermined pattern, which can be controlled individually, between the porous ones Areas, depending on the requirement, a material exchange is possible or not possible.

Description

The invention relates to a microarray device. More specifically, the invention relates to a microarray device based a porous Material, for example a microporous, polymeric membrane, the a variety of high density according to a predetermined pattern arranged porous or microporous Has areas (zones) that can be controlled individually, wherein between the porous or microporous Areas, depending on requirements, a mass transfer possible or Not. possible is.

Microarrays are excellent Aids, a great one Number of different molecules to test against an unknown substance.

A microarray generally consists of a small, specific area, which is divided from the beginning into numerous even smaller areas in the range from 1000 to 100,000 of these areas per cm ² or can be divided later. These approximately 1,000 to approximately 100,000 even smaller areas (zones), ie specific points on the 1 cm ² microarray, can be controlled or addressed individually and independently of one another. In normal use, this means that a small amount of liquid, which can contain one or more reagents, can be deposited or applied to each of these specific points. The reagent (s) in each of the small amounts of liquid can all be different and under normal circumstances there should be no exchange of material or information from one point to another. In this way, each point can have a specific reagent bound to its surface. A reaction can then be carried out on the entire microarray. Due to the different reagents on the surfaces of the individual points, this reaction can lead to different results on these points and the results or

Signals can be sent from any single point independently be delivered.

The ones currently in use Microarrays are made on the basis of glass or silicon dioxide. For example, nucleic acid arrays such as DNA arrays (or Biochips) produced in such a way that the nucleic acid oligomers, such as DNA oligomers and RNA oligomers either photochemically (Affymetrix) positioned on a solid phase matrix or mechanically arranged become. The placement in microns on the target is done for example with the help of contact printers, such as type printers or dot matrix printers or inkjet printers that work piezoelectrically or in solenoid technology. The target can be a glass substrate, for example.

Typical, currently applied or deposited substances are nucleic acids (such as oligonucleotides, so-called ESTs, i.e. Expressed sequence tags, cDNAs), proteins or peptides, cells or cell fragments, tissues etc., i.e. almost everyone Types of biological molecules or cells, it can but also other chemicals, e.g. for test procedures for environmental protection, be deposited.

Analyzes using microarrays are described, for example, in Ross et al. (2000) Nature Genet. 24, 227-235, and Weinstein et al. (1997) Science 275, 343-349. In These studies were followed by ESTs (expressed sequence tags), i.e. short sections of cDNA to identify cDNA libraries or genomic sequence tags to characterize complex Genes or applied by gene homologists of other species as arrays. in the next Step was the application of mRNA samples from a cell line or from a cancer cell sample. In general, mRNA fragments can be made with this method in the large scale be compared. Moreover can many samples are examined at the same time.

With physical deposition or deposition of the target or target molecules on the substrate important that the Substances (target or target molecules) as individual points the substrate are formed. This is typically done by Selection of a suitable distance between the separated points on the surface reached.

For the size of the point on the surface, the surface tension and the nature of the solution to be deposited or deposited are of essential importance. If the surface tension is low and the substrate is hydrophilic, a quantity of solution of 1 nl will spread to a point with a diameter of more than 200 μm. In order to prevent the formation of large-area dots and to obtain a high density microarray, the surface is optimized, for example by silanization, so that it has hydrophobic properties. Oligonucleotides in particular are deposited on silanized glass to produce high density microarrays. In the below mentioned 1 a drop deposited on a hydrophobic surface is shown as an example, which after drying results in a point with a diameter that can be less than 100 μm. In the below mentioned 2 is shown schematically that a drop of solution that is deposited on a hydrophilic surface can lead to a dried spot with a diameter of much larger than 200 microns.

When drying, the sample containing the reagent collects at the outer edge of the point. This results later when larger amounts of another substance are fired at this point should become a sensitivity problem, since the reagent of the sample is practically only at the edge of the point, but not or hardly at all in the middle of the point. Because of this density or concentration problem, a silanized glass is typically chosen as the target for nucleic acid microarrays, such as DNA microarrays.

However, it is with conventional ones Microarrays, if any, very difficult possible a higher one Concentration of a reagent with the help of separated solution droplets at a point on the microarray that the reagent is evenly distributed over the entire point is distributed and without a mass transfer between the solution droplets takes place or the droplets at least partially converge. For this reason, i.a. porous Membranes as material for the Microarray surface used, however, it is practically not possible to use microarrays with a droplet or spot distance of less than 200 μm create because porous Membranes have the property of absorbing liquids, so that so far none narrow, geographical delimitation could be achieved.

As a result, it was previously not possible to design a grid surface to create, e.g. with glass as a substrate is possible to make a reagent droplet on a very small surface deposit. Furthermore, the amount of the amount of reagent deposited is so far very limited.

It is therefore an object of the present invention a microarray device to provide a variety of according to a predetermined pattern arranged, individually controllable points, the Points one if possible high concentration of a desired Can absorb substance and whereby an exchange of material or information between the individual Points does not take place or, if desired, the possibility an exchange between individual points can be provided.

This task is carried out by the in the claims marked object solved. The The invention is based on the knowledge that a microarray device with the properties mentioned in the task can be provided that a porous Material, e.g. a microporous polymeric membrane, is treated such that the pore structure of the porous material at predetermined locations, for example a predetermined grid pattern from intersecting lines, is changed so that none More pores between the untreated areas (zones) of the porous material exist or, if desired, the porosity at the treated, predetermined locations relative to the untreated areas a desired one Dimension reduced becomes.

The porous material can be self-supporting be or on a carrier be applied. It can also be formed on a support by for example a polymer casting solution a plastic film or plate or on an inorganic support, such as coated a glass or ceramic plate and then a porous membrane from the casting solution in in a manner known per se, e.g. with the help of the evaporation process or the precipitation bath process, will be produced.

As a self-supporting porous material an asymmetric polymeric membrane may be mentioned as an example has a pore structure in which pores penetrate from a surface extend the membrane to the other surface, with the diameter of the pores from one surface to the opposite surface decreased so that on this opposite surface only pores with a much smaller diameter or at all there are no more pores. In the latter case, the microarray device of the present invention are made by the only Pore structure present in the membrane to a certain depth is changed at the predetermined points so that none connecting channels between the untreated areas exist more or at least the porosity in desired Dimension reduced is. The part of the membrane which does not have pores functions here practical as a carrier for the formed, separate porous areas (zones). At a Continuous pore membranes are on the opposite Side existing pores with a smaller diameter to a desired one Seal depth appropriately.

Treatment of the porous material to change The pore structure at the predetermined locations can be different Way to be done e.g. by applying fine cutting lines, by milling, engraving, Punching, by destroying the pore structure by using embossing or printing etc. For training the use of a laser is particularly fine and exact structures suitable. With the help of a laser beam, the finest non-porous lines can be created and areas by melting (in the case of thermoplastic material) or burning away (in the case of both thermoplastic and not fusible material) are generated in the porous material. This allows a predetermined pattern to be burned into the porous material be that the Pore structure in the areas hit by the laser beam gets destroyed, a non-porous, flatter one corresponding to the predetermined pattern Forms line structure or a structure in which the relevant Don't pose originally porous Material is more available.

Thus, for example, a “non-porous” area can be an area from which the originally porous material is made entirely has been removed. Such a state can be achieved, for example, if a porous material has been applied to a support and then the porous material has been completely removed at the predetermined locations, ie down to the underlying support, so that completely separate areas of porous material on the Carrier stay behind.

It is preferably the porous Material for the production of the microarray devices according to the invention a microporous Material, preferably a microporous polymer-based membrane.

In the context of the present invention, the term “microarray device” is understood to mean a device which has about 5 to about 1,000,000, preferably about 20 to about 100,000, porous zones per 1 cm ² area, which are separated from one another by. non-porous areas or areas with reduced porosity are separated.

Furthermore, the term "microporous material" or "microporous Membrane "to understand a material or membrane that Pores with an average diameter of approximately 0.001 to approximately 100 μm, preferably from approx. 0.01 to approx. 30 μm ,

The separation of the porous or microporous Structures offers the possibility the selective activation of porous or microporous sections. Can continue the microarrays according to the invention because of the big Surface also as Storage locations for Sample libraries can be used. Furthermore, the porous structures as a matrix for Microarrays can be used on unstructured membranes.

The invention is described below Reference to the figures and the example described in more detail. Show:

1 : deposited drop of a solution on a hydrophobic substrate

2 : deposited drop of a solution on a hydrophilic substrate

3 Example of a surface of a microarray device according to the invention with a raster-shaped line structure, for example burned in by a laser (top view and side view)

4 : schematic three-dimensional view of individual column-like points of the surface of the microarray device according to the invention obtained by, for example, burning with a laser

5 : schematic representation of the control of individual points on the surface of the microarray device according to the invention with a solution drop containing a reagent

6 Example dimensions of a point on the surface of the microarray device according to the invention obtained, for example, by burning with a laser from a microporous membrane

7 : Example of a design and arrangement of points in the surface of the microarray device according to the invention

8th Example of applying a test sample to the points of a microarray device according to the invention.

The microarray devices according to the invention have numerous zones with a porous or microporous structure thereon, which are separated from one another by the line pattern generated, for example, with the laser. The zones can have any desired geometric shape, for example a square, rectangular, round, oval, triangular, etc., and more than one geometric shape can also be present on the microarray. This makes it possible to implement any desired arrangement of zones on the microarray, for example one as in FIGS 7 and 8th shown arrangement of triangular surfaces 1 . 2 and 3 in close proximity, the one like in 8th shown application of a test sample on the closest points of the triangular areas allowed. Normally, the zones are separated from one another, for example by laser treatment of the microporous membrane, in such a way that there is no exchange of material or information (so-called cross talk) between them. However, it can also be achieved by suitable control of the laser beam that zones are not completely separated from one another, but rather a limited mass transfer between the remaining pores, which have not been eliminated by melting or burning away the membrane material, can take place.

The formation of the non-porous areas or Areas with reduced porosity, with the help of which the porous zones can be separated from each other in a variety of ways and is not particularly limited. Rather, it is particularly dependent on the type and dimensions of the desired nonporous Areas or areas with reduced porosity and of the type used porous Material.

The non-porous areas or areas with reduced porosity can for example mechanically by applying cutting lines, by milling, engraving, Punching, by pressing together, if necessary at elevated temperature, be produced by punching etc. However, the porous material can also by physical means, for example by melting the material at the predetermined locations or by chemical means, for example by etching or by targeted chemical reaction, if necessary by adding of substances that can react with the matrix material, so changed be that none Pores remain behind or the porosity is reduced.

Especially for the production of fine and complex structures has become the application of laser technology proven to be advantageous.

The microarray devices according to the invention have a porous or microporous surface which has been divided into tiny, three-dimensional, porous or microporous zones by appropriate treatment, for example by laser treatment. A microporous membrane e.g. B. has a very large ratio between the inner and outer surface. A typical membrane used in microfiltration has an inner surface, ie a surface of the walls of the pores, of approximately 100 to 400 cm ² , based on a square centimeter of the outer surface of the membrane. This means that a membrane can bind many times more specific reagent on its surface than a smooth surface (eg a glass substrate, a plastic film or a silicon dioxide surface). Thus, according to the invention, significantly higher concentrations of specific reagents or larger amounts of, for example, peptide, protein or DNA per surface unit of the microarray can be provided, the reagent also being uniformly distributed over the entire microporous structure and thus at any point in the zone for a reaction Available.

With the microarray devices according to the invention can now those are also provided that have a range of chemical different surfaces have, such as Surfaces with Ion exchange groups or functional groups, such as basic and / or acidic groups that have specific adsorption or formation of covalent bonds to various biomolecules allow. Because of the preferably microporous structure of the zones of the Microarray devices according to the invention readily absorb this one substance to be deposited without that hydrophilic groups must be introduced into the target. It also ensures that no Cross talk takes place between the zones, even if the zones are in high density are arranged on the microarray.

Because larger amounts of substance on deposited the microarray devices according to the invention can be is their detection with, for example, CCD camera systems (charged coupled device-camera systems). Because of the increased concentration of target substance resulting from the larger amount that can be absorbed, you get a better signal-to-noise ratio between the Signal and background or signal and noise.

With the help of precise control of the laser beam or with a photographic mask, any desired pattern can be burned into the membrane, for example as in 3 shown regular pattern of squares or rectangles. This means that even the most complicated structures and patterns can be realized on the surface of the microarray in one step.

It can be done as follows that first that predetermined patterns with the laser in the microporous membrane is burned in and then the substances) on the individual zones is or will be deposited. But it can also work in reverse order or branding the pattern and the separation of the substances take place simultaneously or working in parallel.

The microarray devices according to the invention can be used to implement microporous zones which can take up liquid samples in the nanoliter to microliter range, microporous zones with a base area in the micrometer range, for example a square base area of 80 μm side length, being able to be produced. The distance between the zones can be even smaller, for example 40 μm. When using, for example, microporous membranes with a thickness in the range from 10 μm to 500 μm, corresponding thicknesses of the zones can be achieved after the laser treatment, whereby the base area should naturally arise in a reasonable ratio to the thickness of the zones. For microporous membranes, a minimum side length of the zones of approximately one third of the membrane thickness is assumed. With a square base area of 80 μm side length of the zone, for example, a thickness of 140 μm can easily be realized, as shown in 6 is shown. If the microarray device according to the invention has a carrier, the thickness of the overall device, consisting of the porous or microporous material and the carrier, is in the range from 100 μm to 4 mm, preferably 200 μm to 3 mm and more preferably 300 μm to 2 mm.

As porous or microporous membranes that for the Manufacture of the microarray device In principle, all of the present invention can be used polymeric, porous or microporous Membranes in question, for example membranes based on polyamides (such as nylon), polyvinylidene fluoride (PVDF), polyether sulfones (PES), Polysulfones (PS), polycarbonates, polypropylene (PP), cellulose acetate, Cellulose nitrate, regenerated cellulose with chemically modified Surface etc. or mixtures thereof, membranes based on cellulose acetate, Cellulose nitrate or regenerated cellulose with chemically modified surface are preferred.

Regenerated cellulose membranes can be mentioned as examples of the above-mentioned chemically modified membranes, into which functional groups such as aldehyde, epoxy, sulfonic acid, carboxylic acid, quaternary ammonium and / or diethylammonium groups are introduced. Due to the functional groups or the introduced ionic charges, peptides, proteins or nucleic acids, for example DNA, are bound reversibly or covalently (for example in the case of aldehyde and epoxy-modified membranes). A further preactivation also allows selective partial re Generate active groups by, for example, oxidizing a regenerated cellulose membrane after structuring by oxidative agents (eg I ₂ ) to the corresponding aldehyde.

The microarray devices according to the invention can can be produced in the following manner, for example.

A pre-made microporous membrane is used either as such or on an inorganic or organic carrier laminated. As an organic carrier can basically all polymeric films can be used. Preferably the carrier is as a plate, in particular made of PVC. After that, for example with a laser the predetermined desired pattern of lines or surfaces into the microporous Branded membrane. The intensity of the laser beam can be adjusted in this way be that the Laser beam the microporous Structure of the membrane at the points hit by the laser beam Completely destroyed, whereby only hydrophobic, blackened Lanes remain down to the molecular level, which is any liquid transport prevent between the emerging zones. The intensity of the laser beam and / or the irradiation time can also be set that the microporous structure the membrane is only destroyed to a certain depth, so that one Connection of the zones formed in a predetermined, restricted manner for one Mass transport is maintained.

The invention is illustrated by the following example explained further.

example

A microporous membrane (nitrocellulose, pore size 0.2 μm) with a thickness of approximately 140 μm is laminated on a carrier film (PVC). Then a laser (Nd YAG) is used to burn in a predetermined pattern (array) of square dots with a side length of 80 μm in such a way that lines with a width of 40 μm are formed between the dots. The microarray device obtained has approximately 6900 completely separate square microporous points (partial zones) with a base area of approximately 6400 μm ² and a thickness of approximately 140 μm.

Claims (26)

Microarray device comprising zones made of a porous material, which are arranged according to a predetermined pattern, the zones through non-porous Areas or areas with reduced porosity are separated from one another. The microarray device of claim 1, wherein the zones are out a porous Material on a support are upset. The microarray device of claim 2, wherein the carrier is a Plastic film, a plastic film or a plastic plate is. Microarray device according to one of claims 1 to 3, wherein the zones from a microporous Material are formed. Microarray device according to one or more of claims 1 to 4, the zones being formed from a membrane. The microarray device of claim 5, wherein the membrane is a microporous Membrane is. Microarray device according to 6, wherein the microporous membrane a microporous Is polymer membrane. The microarray device of claim 7, wherein the membrane is on Based on polyamides, polyvinylidene fluoride, polyether sulfones, polysulfones, Polycarbonates, polypropylene, cellulose acetate, cellulose nitrate or regenerated cellulose with a chemically modified surface or Mixtures of it is formed. Microarray device according to one or more of claims 1 to 8, the porous Zones one side length in the range of approximately 40 μm up to about 100 μm exhibit. Microarray device according to one or more of claims 1 to 9, the porous zones a distance of about 20 μm up to about 60 μm from each other to have. Microarray device according to one or more of claims 1 to 10, the porous zones through non-porous Areas are separated from each other. Microarray device according to one or more of claims 1 to 11, wherein the microarray device has a thickness of approximately 10 μm to approximately 500 μm. Microarray device according to one or more of claims 2 to 12, with the carrier as a plate, preferably made of PVC, is formed. A method of manufacturing a microarray device, comprising the steps of: - providing a porous material, and - forming zones in the porous material which are separated by non-porous regions or regions with ver reduced porosity. The method of claim 14, wherein the porous material before or after the Form the zones by using non-porous areas or areas reduced porosity are separated from each other, is applied to a carrier. The method of claim 15, wherein the carrier is a plastic film, a plastic film or is a plastic plate. A method according to any one of claims 14 to 16, wherein the forming the separated zones is done with a laser. A method according to one or more of claims 14 to 17, wherein the porous material a microporous Material is. A method according to one or more of claims 14 to 18, wherein the porous material is a membrane. Method according to one or more of claims 14 to 19, wherein the membrane a microporous Membrane is. The method of claim 20, wherein the microporous membrane a microporous Is polymer membrane. 22. The method of claim 21, wherein the microporous polymer membrane by applying a polymer casting solution to a support and Form the membrane from the casting solution in the evaporation process or precipitation bath process was generated. The method of claim 22, wherein the microporous membrane based on polyamides, polyvinylidene fluoride, polyether sulfones, Polysulfones, polycarbonates, polypropylene, cellulose acetate, cellulose nitrate or regenerated cellulose with a chemically modified surface or Mixtures of it were formed. Method according to one or more of claims 14 to 23, wherein the zones in the porous material are formed so that they one side length in the range of approximately 40 μm up to about 100 μm exhibit. The method according to one or more of claims 14 to 24, wherein the non-porous areas or the areas with reduced porosity are formed so that the zones in the porous Material are separated from each other so that the distance is in the range of about 20 μm up to about 60 μm lies. Method according to one or more of claims 14 to 25, wherein the training of the zones in the porous Material carried out in this way is that the Zones by non-porous Areas completely are separated from each other.