DE10136008A1 - Analysis of macromolecules uses a micro-array of spots for a first macromolecule, with an applied liquid droplet with a second macromolecule moved between spots by an acoustic wave, and the reactions are studied - Google Patents

Abstract

For the analysis of macromolecules using a micro-array, a number of spots (16) are in a known layout, with a number of first macromolecules which are generally identical for a spot. The micro-array is on a solid surface (1) and preferably a solid chip. At least one liquid droplet (3), containing a second macro-molecule, is applied to the solid surface, which is so small that it comes into contact with only one spot or a group of a few spots in the micro-array. After a given time span, the liquid droplet is impelled to the next spot/spot group, by preferably a pulsed acoustic wave (14) from an interdigital transducer (5-8). The process is repeated until it has made contact with the required number of spots, and the reactions are studied.

Description

The invention relates to methods for analyzing macromolecules using a Microarrays and a method for producing an analysis device for Macromolecules.

One method for rapid analysis of macromolecules involves use a so-called microarray on a chip, similar to the one from the Semiconductor industry is known. In such a microarray, known first different types of macromolecules at different locations in a matrix form arranged. These macromolecules are also referred to as "probe molecules". A liquid with macromolecules ("sample molecules") is placed over the microarray rinsed with at least one type of probe on the microarray a specific bond is formed (hybridization). Then the liquid will come back removed from the surface, mainly remain only in the places of specific binding back to the sample molecules to be examined. With the help of a spatially resolved measurement, e.g. B. a fluorescence measurement can be determined which sites are to be examined macromolecules. From the known position of the individual probe molecules in the matrix form of the microarray can be determined with what kind of macromolecules the investigating macromolecules have entered into a specific bond.

Such microarrays are e.g. B. for the investigation of macromolecules such as Proteins, antigens or antibodies used. In particular Microarrays also for the analysis of DNA, e.g. B. in DNA screening, used.

The duration of a corresponding analysis experiment is essential Partly determined by the diffusion of the sample molecules to the probe molecules and can therefore take some time. Is z. B. the concentration of the Examining macromolecules in the liquid only slightly, so it can be very take a long time to find its specific binding partner on the array Has.

Another problem with the reaction (hybridization) between those in the Sample liquid and the macromolecules on the surface located probe molecules is that the macromolecules to be examined in a very wide range of amounts in the liquid can be up to down to a few molecules. Still must be sufficient Liquid should be present to cover the entire array surface, so that all Probe molecules can also be achieved. This may result in a very small one Concentration of the sample molecules in the liquid. Such a low one Concentration, however, causes a reaction equilibrium, which after Mass action law for chemical reactions is very much on the side of the educts. Sample molecules therefore remain dissolved in the liquid, even if a suitable one Probe molecule on the surface would still be free. To overcome this The amount of starting material, i.e. H. of the sample molecules in the Sample liquid, increased, due to the lack of a sufficient amount Source material is often not available.

The microarrays used are miniaturized arrays, which in the case of e.g. B. DNA microarrays include individual areas (spots) with probe molecules each with a diameter of about 100 microns. Many different DNA probes (spots) z. B. applied in a checkerboard pattern on the surface and simultaneously used for hybridization reactions. The densities that can be achieved on such a microarray today are, for. B. for DNA (deoxyribonucleic acid) up to about 250,000 different types of molecules per cm ² (M. Schena (ed.), "DNA Microarrays", Oxford University Press, 1999).

After hybridization, the microarray is usually washed according to a special protocol and the fluorescence is then measured in a spatially resolved manner. Using special techniques, it is also possible to measure the fluorescence signal in situ during hybridization in order to obtain additional information about the reaction kinetics. One possibility is to measure the fluorescence signal with optics of such a shallow depth of field that only the area of the interface between the liquid film and the chip surface contributes to the signal. There the second macromolecules are mainly in the already bound state. The fluorescence signal of two macromolecules which are in the unbound state in the overlying liquid film is not detected with such optics. Likewise, with appropriately designed waveguides and evanescent waves that are guided laterally to the interface between liquid and surface, the fluorescence on the molecules bound to the microarray surface can be examined (GL Duveneck and AP Abel, Review on Fluorescence-based Planar Waveguide Biosensors, Proc. SPIE , Vol. 3858 ( 1999 ) pages 59 ff.).

Today, the probe molecules are mostly applied using a printing technique (spotting). Such a printing technique is used e.g. B. from VG Cheung et al., Nature Genetics Supplement, Volume 21 , January 1999, pages 15 ff. A solution with the desired probe molecule is brought onto the chip surface either in a contactless or contacting manner. Alternatively, the probe molecules can also be e.g. B. can be synthesized photolithographically base by base on the chip. It is difficult to make a quantitative statement about the number and density of the probe molecules on a spot both in spotting and in photolithographic production. That means that e.g. B. when detecting the hybridized sample molecules z. B. only limited quantitative conclusions about the number of sample molecules are possible by fluorescence. The comparability of data obtained on different microarrays with nominally identical spots is considerably restricted.

In a known method for producing microarrays, the Surface of a solid-state chip first functionalized homogeneously, so that, for. B. by Exposure to light or by exposure to an electric field the previous also bind functionalized probe molecules to the surface. The chip is homogeneously flooded with a solution of the probe molecules (e.g. in on the order of 100 µl). Tie through local lighting photoactive probe molecules locally at the desired locations. With electroactive Probe molecules that are exposed to an electric field Bind surface, an electrical field is generated locally, which triggers the bond. The With these methods, spatial resolution comes from the modulation of the electrical field or the light intensity. Especially with chips with high Spot density is a complex and expensive process. B. the Modulation of the electric field necessary to achieve a spatially resolved binding of the To ensure probe molecules on the surface must be in one layer a field of capacitors is attached below the chip surface, which the correspond to later spots locally. Such a procedure is described by M. J. Heller, A. H. Forster and E. Tu in Electrophoresis, 2000, 21, pages 157 ff.

The object of the present invention is to specify methods with which a accurate and reproducible analysis of macromolecules is also possible limited sample material can be used. The production of corresponding Analysis devices should be simple and reproducible.

With regard to the method for the analysis of macromolecules, the task is accomplished by a method with the features of claim 1 or a method with the Features of claim 6 solved. Regarding a manufacturing method The task of an analysis device for macromolecules is achieved by a method solved with the features of claim 15. Subclaims are in each case preferred configurations directed.

In a method according to the invention for the analysis of macromolecules with Using a microarray consisting of multiple spots with probe molecules in known arrangement, a microarray on a solid surface, preferably a chip used. At least a drop of liquid with too investigating macromolecules is placed on the solid surface, the Liquid amount of a drop is chosen so small that it essentially only one spot or a group of a few spots of the microarray touched at a time. Conveniently, one should Liquid drops should not be touched more than 10% of all spots in a microarray. To this The reaction conditions (e.g. temperature or Salt concentration) depending on the touched spots. The lower the number is the spots that are touched by a drop of liquid at a time the more specific the setting of reaction conditions depending on possible from the first macromolecules contained in a spot. In preferred The process is carried out in such a way that only one spot at a time is touched by a drop of liquid so that the reaction conditions for each individual spot can be set individually.

After a predetermined time, the z. B. by the typical response time of the macromolecules contained in the liquid with the macromolecules that act as Probe molecules located on a spot are determined using the Impulse transmission of a surface sound wave of the liquid drops to one next spot moves. A predetermined time is again waited here until the typical response time has passed. This way you can get the ones you want Spots or all spots of the microarray used with the liquid sequentially be brought into contact. Of course, this process can also can be parallelized by placing several drops simultaneously over different spots be moved.

Subsequently, those that come into contact with the liquid drop can Spots are then examined whether or to what extent a reaction with the macromolecules in the liquid drop.

For this, e.g. B. serve a spatially resolved fluorescence measurement if the investigating macromolecules are fluorescence-labeled. Other Detection methods, e.g. B. using radioactive sample molecules are also used.

Examination of those in contact with the liquid drop Spots as to whether and / or to what extent a specific reaction with the Material of the liquid drop has taken place at the end of the whole Process are carried out, d. H. when the liquid drop with everyone desired spots was in touch.

It is also possible with the method according to the invention to carry out the examination, whether second macromolecules react with one spot or a group of fewer spots have to perform while the liquid is on another spot or has fewer spots in contact with another group in order to To perform hybridization of the second macromolecule. So while the liquid is in contact with the second spot or the second group of spots the first spot or the first group of spots is already evaluated.

Finally, it is also possible with appropriate process management, for. b. the Measure the fluorescence of the bound second macromolecules in situ. This can z. B. optics of shallow depth of field or appropriately arranged waveguides and evanescent waves are used that only cover the interface between Detect liquid and chip surface.

In the method according to the invention, the liquid with the sample molecules is not applied to the entire surface of the microarray, but only to individual spots which are separated from one another. While in classic microarray experiments liquid volumes of about 10-100 µl are necessary to cover a total sample area of 1 cm ² , the amount of liquid required can be reduced to a few nl with the method according to the invention if, for. B. spots with an extension of 100 microns can be used. The small sample drop is gradually moved from spot to spot. The diffusion length is not in the order of 1 cm, but only in the order of 100 µm. A quick and efficient hybridization reaction then takes place at the spots themselves.

Due to the small amount of sample liquid, the concentration of the Starting materials can be much higher even with the same amount of starting material, because instead of about 10-100 ul z. B. only about 10 nl are needed. That's how it works Push the reaction equilibrium to the side of the hybridized products so that a much more accurate measurement even with little material to be examined is possible.

If necessary, the time that is waited before the liquid drop is removed from a spot again, shortened by the fact that the Drops of liquid with the help of the impulse transfer of a surface sound wave moved, that is "stirred". In return, a surface acoustic wave is reduced Intensity used as that for transporting the liquid drop, so that the Liquid drops do not move away from the spot.

The method according to the invention allows a freely programmable way of Sample liquid over the surface. With appropriately arranged spots An experimental strategy can be followed for different probe molecules in order to first approach those spots for which one is more positive Result expected. The reaction conditions can be individual for different spots can be chosen differently. So z. B. the temperature with the help of resistance heaters can be set differently. Here, for. B. the Chip are heated homogeneously because the spatial resolution is limited by the Expansion of the drop is guaranteed.

Likewise, e.g. B. the nature of the liquid to be examined by a Spot to be changed to the next. While in a spot z. B. the Salt concentration in the liquid has a first value, it can at a second spot changed to a second value by adding the appropriate buffer solution become. The buffer solution can also use the pulse transmission Surface sound wave are moved in the direction of the corresponding spot.

With the analysis method according to the invention it is no longer necessary that the Solid surface z. B. in a separate washing step from the liquid is released before it is examined whether and to what extent a reaction of the second macromolecules in the liquid with the first Macromolecules of the spots took place. In the invention The drop of liquid is moved by means of the surface sound wave from the appropriate spot moved so that immediately afterwards an investigation can take place whether bound second macromolecules remain at the spot are.

The evaluation can take place parallel to the hybridization. For example from one fluorescence-labeled sample molecule type in the sample liquid becomes a Drops moved to a first spot. After the typical hybridization time, the Drops moved to the next spot and resolved in parallel at the first spot measured the fluorescence. At the second spot z. B. to change the Reaction conditions change the temperature or a small volume Buffer solution can be added to ensure the stringency of the reaction.

In a preferred embodiment of the method according to the invention, the Amount of the second macromolecules bound to the respective spot from the Damping and / or change in speed of a surface sound wave determines the presence of the second macromolecules. A Surface sound wave is in the direction of the spot with the one on the first macromolecule bound second macromolecules. By the mass on the surface, the is formed by the hybridized second macromolecules, the Surface sound wave attenuated in intensity or its speed changed. With A detector for the surface sound wave can measure its intensity before and after the hybridization of the second macromolecules are measured and from the thereby determined damping or speed change to the number of bound second macromolecules are closed. Possibly a calibration measurement with known parameters can be carried out.

In another embodiment of the method according to the invention at least one drop of liquid with second macromolecules on the Solid surface brought with the microarray, the amount of liquid of a drop like this small is that essentially only one spot or a group of a few spots of the microarray is in contact with the liquid drop at a time comes. With the help of the momentum transfer of two essentially against each other directed surface sound waves, which alternate in the direction of the Drop of liquid are sent, the drop is moved back and forth. The The intensity of the surface sound waves is chosen so that the drop does not is completely removed.

During the back and forth movement, it is examined locally at the spot whether and in what degree of second macromolecules on the first macromolecules of the spot or the group hybridized fewer spots. For this, z. B. again Fluorescence of fluorescence-labeled second macromolecules was examined. Through the Float of the drop can change the time dependence of the reaction be determined. So is a statement about the reaction rate or Reaction kinetics possible. As time increases, i.e. H. increasingly more common Moving the droplet over the spot of the microarray becomes more second macromolecules bound, whereby z. B. the intensity of the fluorescence bound second macromolecules at the respective spot is increased. Will the Fluorescence is only measured when the liquid is not on the spot or the group of spots, it is prevented that the still in the Unbound second macromolecules located on the liquid Fluorescence measurement and would falsify it.

The methods according to the invention can of course be combined, for an integral statement about the amount of hybridized macromolecules to get a spot and about the reaction kinetics.

In the analysis method according to the invention, the liquid drop is removed with the help of the impulse transfer from surface sound waves. On the surface of the The chip on which the microarray is located becomes a surface sound wave generated. The momentum transfer is then either by mechanical deformation the solid surface or by the force of the accompanying electric fields on charged or polarizable matter in the liquid. The The strength of the force acting on the liquid can vary over a wide range The amplitude of the surface sound wave can be set. The Surface sound waves can be generated in pulses with pulses of different lengths or continuously become. Surface acoustic waves can be on piezoelectric substrates or Substrates with piezoelectric areas, e.g. B. piezoelectric Coatings. The movement with the help of the momentum transfer from Surface sound waves are particularly precise. In addition, there are no channels or others Guide mechanisms or pumping devices, such as. B. micromechanical Pumping, necessary to move the liquid on the surface.

The surface sound wave can with the help of at least one Surface wave generating device can be generated. Advantageously, this becomes an in itself known interdigital transducer used. The simplest version has one Such an interdigital transducer has two electrodes which interlock like fingers. By Creation of a high-frequency alternating field, e.g. B. in the order of 10 up to a few 100 MHz, is in a piezoelectric substrate or in a piezoelectric region of the substrate excited a surface sound wave, the Wavelength as a quotient of the surface sound velocity and the Frequency results. The direction of propagation is perpendicular to one another gripping finger electrode structures. With the help of such an interdigital transducer generate a very defined surface acoustic wave in a very simple manner. The manufacture of such an interdigital transducer is known lithographic processes and coating technologies inexpensive and simple. Interdigital transducers can also, e.g. B. by irradiation of a alternating electromagnetic field in a connected to the interdigital transducer Antenna device can be controlled wirelessly.

Around certain localized areas of a surface with the To sweep over surface sound wave can, for. B. several interdigital transducers in the desired arrangement can be used, which are controlled selectively.

In a particularly advantageous method, a laterally limited sound path is used the surface sound wave with the help of so-called tapered interdigital transducers generated. With such a tapered interdigital transducer, the Finger distance of the interlocking finger electrodes along the Transducerachse. For a given frequency the resonance condition is that the frequency equal to the quotient of the surface sound velocity and the Finger distance is only met in a laterally spatially limited area. With a such a tapered transducer can be a selected range of Targeted by choosing the appropriate frequency with a surface Surface sound wave. In this way, individual spots or select individual rows of spots.

According to the preferred embodiment described above, the damping of a Surface sound wave through the mass of the hybridized second macromolecules can also be evaluated as a surface acoustic wave detector Interdigital transducers can be used.

The analysis method according to the invention can be carried out on a chip on which the probe molecules are directly bound. It is particularly advantageous it, however, if the liquid drop with the sample to be examined Macromolecules moved on a preferred area of the solid surface whose wetting properties are so different from its surroundings differ that the liquid drop is preferably on it. The Probe molecules are then arranged on this preferred location area.

By defining such a preferred lounge area, the Area on the solid body on which the liquid is to move is determined become. The liquid with the macromolecules to be examined remains in the Usually only on the preferred lounge area, without channels or Trenches are necessary, which otherwise restrict the movement of the liquid would. The surface tension of the liquid drop additionally causes that the liquid the preferred location area without the influence of an external Strength cannot leave.

The different wetting properties can e.g. B. by a appropriate coating either of the preferred lounge area or its Environment can be realized. For example, hydrophilic or hydrophobic Areas to be defined. Are the macromolecules to be examined e.g. B. in contain aqueous solution, the preferred location is chosen so that it is more hydrophilic than the surrounding solid surface. This can either by a hydrophilic coating on the preferred area or can be achieved through a hydrophobic environment. A hydrophobic environment can e.g. B. can be realized by a silanized surface. Depending on the Wetting properties of the liquid in which the macromolecules to be examined are included, the solid surface surrounding the occupied area also hydrophilic, lipophobic or lipophilic compared to the surface of the preferred Stay area can be selected. The wetting properties can continue to be modulated by microstructuring, as is the case with the so-called The lotus effect is the case, which is based on different roughness of the surface and so causes different wetting properties. Such Roughness modulation can e.g. B. by microstructuring the corresponding Surface areas are obtained, e.g. B. by chemical treatment or Ion irradiation. The production of different areas Wetting properties is achieved by using already known lithographic processes and / or coating technologies simple and inexpensive.

The geometry of the preferred lounge area can match the corresponding one Application to be adjusted. For example, the preferred one Lounge area are meandering through the microarray, so that the individual Positions of the microarray are along the preferred area line up. A crosswise arrangement can also be provided, in which the individual positions of the microarray in the intersection areas.

If the method according to the invention is designed in such a way preferred lounge area, the requirements for accuracy in the direction and lateral propagation of the surface sound wave less because the movement of the drop of liquid through the shape of the area is determined with.

In another embodiment, correspondingly defined preferred ones Lounge areas only provided in the area of the individual spots, without each other to be connected. The lounge areas then serve the Localization of the liquid drop in the area of a spot or group fewer spots. Sufficient by irradiating a surface sound wave The liquid can nevertheless move from one area to another in intensity be transported. The areas defined in this way act like an "anchor", which is the location of the drop of liquid on a spot or on a group fewer spots easier.

The inventive method can, for. B. advantageous for the analysis of Oligonucleotides are used. Among other things, it is particularly suitable for DNA screening, with the oligonucleotides to be examined e.g. B. from short DNA strands (deoxyribonucleic acid strands) exist. You can also however different macromolecules with specific binding like different proteins, different antigens or different antibodies in the Microarray provided or analyzed.

In a method according to the invention for producing an analysis device for macromolecules at least one liquid drop with at least one kind of macromolecules on a solid surface, preferably the surface a chip, applied, the macromolecules having an active group, which is activated when a corresponding external parameter is changed, that they have a bond with the solid surface or already on the surface bound macromolecules can enter. The drop is made using the Impulse transmission of one or more surface sound waves to one desired location on the surface. The external parameter then becomes homogeneous and not changed locally so that the macromolecules can bind becomes. The drop of liquid can then be moved several times to another location of the Chips moved using the surface sound waves and the parameters again be changed in order to successively create a microarray. Of course these processes can also be parallelized by several Liquid drops are simultaneously moved across the solid surface and through the change in the external parameter at the desired location is a reaction of the in the macromolecules containing the liquid drops with the surface becomes. With the molecules bound to the solid surface in this way, a Microarray formed from probe molecules.

Unlike conventional methods of manufacturing a microarray it is not necessary that the external parameter is only changed locally. This poses represent a significant simplification of the process, since e.g. B. no spatially resolved Lighting or spatially resolved electrical fields are necessary. In contrast to conventional methods, in which the chip with the liquid that the to contains binding macromolecules, must also be flooded Material consumption significantly reduced. To flood the chip homogeneously, typically 10-100 ul are required, while with the invention Processes with nanoliter (nl) or a few microliter (µl) amounts get by can.

Are z. B. Macromolecules with a photoactive group is used as external parameter selected the lighting. Depending on the nature of the photoactive Group is either changed in intensity or frequency by one Binding of the photoactive macromolecules to the surface or already to allow bound macromolecules. The drop of liquid is transferred to the desired location with the help of the surface sound wave and then the Lighting switched on or changed accordingly. Only where the Liquid just located, the macromolecules will surface with one Enter into a bond. A spot of a microarray can be localized very precisely produce.

Are z. B. Macromolecules with an electroactive group serves as external field to be changed the electric field. The drop of liquid with the help of the surface sound waves to the desired location on the Solid state chip moves. Then the electrical field is changed so that a Reaction of the macromolecules in the liquid with the surface of the chip or already bound macromolecules can take place. It is not necessary that the electrodes for applying the electric field a local limited To have expansion. The entire chip surface can serve as an electrode, since the Local resolution of the reaction guaranteed by the limited amount of liquid is.

The drop can also contain macromolecules that respond to temperature changes react. Is the drop with the help of surface sound waves desired location has been moved, the chip surface is heated to at that location the reaction of the macromolecules with the surface or already bound To enable macromolecules. The temperature increase need not be local, because a spatial resolution guaranteed by the limited extent of the drop becomes.

The use of surface sound waves to move Liquid drops offer the same advantages in the production method according to the invention as in the analysis methods according to the invention described above. to Generation of the surface sound waves can be just as with the Analysis method according to the invention interdigital transducer, for. B. so-called taped Interdigital transducers can be used. The use of such interdigital transducers offers the same advantages as in the manufacturing method according to the invention in the analysis method according to the invention.

The manufacturing method according to the invention can on the free surface of a Solid state chips are made. However, it is particularly advantageous if the Solid state chip before using the manufacturing method according to the invention with a preferred lounge area is provided, its wetting properties differ from its surroundings in such a way that the liquid with the to bind macromolecules preferably thereon. Just like with the Analysis methods according to the invention can thus meet the requirement for accuracy reduce the surface sound wave radiation, since the movement of the Liquid is mainly limited to the preferred area.

In addition, one manufactured with such a manufacturing method Analysis device can be used advantageously to an analysis method according to the invention to carry out, in which also a preferred lounge area as already above described can be used advantageously.

A preferred lounge area can, as in the case of those according to the invention Analysis methods described are produced. There are different ones Geometries of the lounge area possible.

Just as with the analytical method according to the invention, several can preferred lounge areas are used in those places of Solid surface are arranged on which the spots of the microarray should be located, which is produced with the manufacturing method according to the invention becomes. The preferred lounge areas at these locations make it easier Localization of the liquid with the macromolecules bound to the spots should be. By irradiating a surface sound wave sufficiently high Intensity can nonetheless be the drop of liquid with the macromolecules that make up the Microarray spots are to be formed from a preferred lounge area be moved to the next one.

In a preferred embodiment of the invention Manufacturing process is the amount of macromolecules bound in one place by Measurement of the damping and / or speed change of at least one Surface sound wave determined in the direction of the bound macromolecules is sent. After one or more spots of the microarray with the have been produced according to the invention, a Surface sound wave sent in the direction of the spot or spots and after passing through the corresponding area of the surface in its intensity and / or with respect to Measure speed change. By comparing the intensity and / or Velocity with the value before the macromolecules were deposited in the microarray was measured, the damping or speed change of the Surface acoustic wave can be determined by the presence of the macromolecules. from that can be deduced from the amount of bound macromolecules. This means that for each individual spot of the microarray that is produced, the spot on there located number of macromolecules can be closed. Will one like that measured microarray for analysis of macromolecules e.g. B. according to Analysis methods used according to the invention is an accurate quantitative statement possible. If necessary, a calibration measurement is also carried out.

With the method according to the invention for producing an analysis device a microarray can be produced in a precise and reproducible manner, which in particular advantageously used with the analysis method according to the invention can be. The devices for generating the surface sound waves, preferably the interdigital transducer, can initially be used to manufacture the Microarrays are used and then used for analysis perform. A preferred lounge area, if any, can also be created initially advantageously used in the manufacture of the microarray and then in the following Analyzes are used.

Both in the analysis method according to the invention and in Manufacturing method according to the invention is a small amount of liquid using Surface sound waves moved to react locally, either to Structure of a microarray from individual spots or to react in the Liquid material with individual spots of the microarray. The use of the Impulse transmission of surface sound waves enables one each gentle and defined movement.

Embodiments of the invention are shown on the basis of the attached figures Procedure explained in detail. The figures are not necessary true to scale and only schematic. It shows

Fig. 1 shows an arrangement for performing the method according to the invention for the analysis of macromolecules

Fig. 2 shows another arrangement for carrying out a method according to the invention for the analysis of macromolecules

Fig. 3 shows a further arrangement for performing a method according to the invention for the analysis of macromolecules and

Fig. 4 shows a step in the manufacture of an analysis device according to the invention.

Fig. 1 shows a device for carrying out the analysis method according to the invention. 1 identifies the surface of a solid-state chip, similar to how it z. B. is also used for applications in semiconductor technology. The material of the substrate of this surface is piezoelectrically and advantageously coated with silicon dioxide. A silicon dioxide coating guarantees a well-defined and well-known surface chemistry.

Interdigital transducers 5 , 6 , 7 and 8 are located on the surface in a tapered version. The interdigital transducers consist of electrodes 9 with finger-like extensions which engage in pairs. Alternatively, other geometries of interdigital transducers are also conceivable, as are known from the technology of surface acoustic wave filters. When an alternating field is applied to the electrodes 9 of an interdigital transducer, a surface acoustic wave is generated with a wavelength which corresponds to the finger spacing of the electrodes and whose direction of propagation is essentially perpendicular to the finger electrodes. Due to the tapered design, in which the distance between the fingers differs locally, a laterally limited surface sound wave can be generated. The transducers each include a large number of interlocking fingers, only a few of which are shown schematically and not to scale.

The number and arrangement of the interdigital transducers is adapted to the desired circumstances. In the exemplary embodiment shown of a device for carrying out the analysis method according to the invention, there is a preferred lounge area 11 between the interdigital transducers. Its wetting properties differ from its surroundings in such a way that an aqueous solution is preferably on it. For this purpose, e.g. B. silanized the surroundings of the preferred lounge area. Such a preferred lounge area improves the localization of aqueous solution, but is not necessarily provided for carrying out the method according to the invention. Spots 16 made of macromolecules are arranged in the area of the preferred residence area 11 . The individual spots 16 each include macromolecules z for each spot sixteenth B. are different.

With such an arrangement, the analysis method according to the invention is carried out as follows. A drop of liquid 3 with macromolecules to be examined is z. B. with the help of a pipetting robot in the area of spots 16 , as can be seen in FIG. 1. The liquid drop is of the order of a few nl to µl. A spot has B. an expansion of 100 microns when a drop with a few nl is used.

With the help of surface sound waves, which are generated with the interdigital transducers 5 , 6 , 7 and 8 , a pulse is generated on the amount of liquid. For this purpose, an electromagnetic alternating field of a few 10 to a few 100 MHz is applied to the corresponding interdigital transducer via the electrodes 9 and corresponding connecting electrodes, not shown. Where the finger spacing is such that the resonance condition is essentially fulfilled, a surface acoustic wave is generated which propagates essentially perpendicular to the fingers of the interdigital transducer. This is indicated by the arrow 14 in FIG. 1 as an example. The surface sound wave transmits a pulse to the liquid quantity 3 and thus moves the liquid drop from one spot to another. With the help of the four interdigital transducers 5 , 6 , 7 and 8 shown in FIG. 1, the liquid drop 3 can thus be brought to the location of a specific spot 16 in a defined manner.

After a residence time, which corresponds approximately to the typical hybridization time of the macromolecules present in the liquid drop 3 with the macromolecules present on the spot, the liquid drop 3 is again removed from the spot with the aid of surface acoustic waves. It can be moved to another spot to enable appropriate hybridization at that location. After the desired spots 16 have come into contact with the liquid 3 , it is analyzed at which spots a hybridization with the macromolecules present in the liquid has taken place. Are z. B. fluorescence-marked macromolecules in the liquid can be determined by a spatially resolved measurement of the fluorescence, at which spots a reaction has taken place. If the type of macromolecules located on the individual spots 16 is known, the type of macromolecules in the liquid drop 3 can be inferred in this way.

An in situ measurement can also be carried out. After the liquid drop 3 has been applied to the first spot, it is z. B. with the help of the surface sound wave 14 of the first interdigital transducer 7 moved a little away from the spot. With the opposite interdigital transducer 6 , a second surface sound wave is generated, which acts in alternation with the first surface sound wave 14 in the opposite direction on the drop 3 . The drop 3 then moves backwards over the spot. Due to the temporal change of the two surface sound waves, the drop moves back and forth. Each time the drop 3 touches the spot, macromolecules from the liquid react with macromolecules at the spot. By measuring z. B. the fluorescence at those times when the drop 3 is currently not at the spot, the time course of the reaction can be determined and a statement can be made about the reaction kinetics.

The measurement of fluorescence or another detection parameter, such as. B. the electrical charge or the mass is carried out in a known manner corresponding measuring and evaluation devices not of interest here and Evaluation programs.

In the described method according to the invention, only a very small amount of liquid 3 is necessary, so that a reliable and reproducible measurement is possible even with a limited amount of material to be examined. It is not necessary to flood all spots 16 with the liquid at the same time.

Since only a small amount of liquid is necessary, the concentration of the can investigating macromolecules in the liquid drop may be higher even if there is only little starting material on the macromolecules to be examined.

On the other hand, it can be compared with conventional methods Concentration the sample amount can be much lower, since only nanoliter amounts are required become.

Fig. 2 shows the geometry of another arrangement for carrying out the analysis method according to the invention. The spots 16 are arranged here along a meandering area 17 . A drop of liquid located on this meandering area 17 can be moved along the area using the interdigital transducers so that it touches all spots sequentially. This arrangement of the location area lowers the requirements for the accuracy of the lateral resolution of a single interdigital transducer. In the method step shown in FIG. 2, a surface sound wave 12 is generated with the aid of the interdigital transducer 8 , so that a drop of liquid can be moved upward in the part of the area 17 on the left in the figure. For the sake of clarity, the liquid drop itself is not shown.

Appropriate timing of the interdigital transducers also enables the use of interdigital transducers with an even finger spacing. When the liquid drop reaches a corner of the meandering area 17 , another interdigital transducer is used synchronously, which enables movement in the bent direction.

The evaluation is carried out in the same way as for an arrangement in FIG. 1.

The method according to the invention can also be carried out with an analysis device according to FIG. 3. Here the lounge area 19 is cruciform. Even with such an arrangement, the requirements for the precision of the interdigital transducers are lower.

Of course, other geometries of the lounge areas are also conceivable, which are adapted to the relevant circumstances. The figures show the Arrangement only schematically. In fact there is a lot larger number of spots on a chip, so that, for. B. a DNA screening analysis is possible.

In one embodiment, not shown, preferred areas are only provided in the area of the individual spots without being connected to one another. Such areas are used to localize the drop of liquid in the Area of individual spots. By irradiating a surface sound wave the liquid drop of one can nevertheless have sufficient intensity Lounge area to be moved to the next. The precision requirements of the However, surface wave radiation is reduced as the preferred ones Localization areas at the individual spots the localization of the drop in their Support area.

The method according to the invention can also be carried out without the use of a preferred location area 11 , 17 , 19 if the control of the interdigital transducers is precise enough. Instead of tapered interdigital transducers, it is also possible to use a number of different and individually controllable transducers, which always cover only a narrow area of the surface with a surface sound wave.

Fig. 4 shows a step in the inventive method for manufacturing a microarray. A preferred residence area 11 is provided on the solid-state chip 1 . A drop of liquid 2 is, for the preferred residence region. 11 B. applied with the aid of a pipetting robot. The amount of liquid is e.g. B. in the order of 1 nl to a few µl. The liquid drop contains macromolecules which are intended to serve as probe molecules and are to be bound at predetermined locations for this purpose. For this purpose, the liquid drop is brought to a desired location with the aid of surface acoustic waves, which are generated with the interdigital transducers 5 , 6 , 7 and 8 . A surface wave 13 is shown as an example, which is generated with the interdigital transducer 7 in a manner which has already been described above for the analysis method according to the invention. The impulse transmission of the surface sound wave 13 onto the liquid drop 2 moves it in the direction 15 . When the liquid drop 2 has arrived at the desired location, the sound is stopped with the surface sound wave, so that the liquid drop 2 comes to rest. An external parameter to which the material in the liquid drop 2 reacts is then changed. For example, the macromolecules contained in the liquid drop can be photoactive. In this case, an illumination source of suitable wavelength and intensity is switched on, which is arranged above the chip 1 and illuminates the entire chip homogeneously. The surface chemistry of the chip or the preferred location area 11 is selected so that the macromolecules are bonded to the surface by the illumination of the photoactive group of the macromolecules. In this way, a spot is generated at a defined location on the surface, at which the macromolecules from the amount of liquid 2 bind. The amount of liquid 2 can then be moved on to generate another spot at another location.

Following z. B. a further amount of liquid can be applied in the there are other macromolecules attached to other locations on the chip in order to create a microarray in which different Spots of different macromolecules are located.

There can also be liquid drops with different macromolecules are moved successively over the same spot. That way you can different sequences are attached to each other to create desired probe molecules produce. The macromolecules from liquid droplets leading to a later one Point of time can be moved across the surface on which macromolecules are already made liquid droplets previously moved over the surface are bound may hybridize with the first macromolecules to form longer macromolecules, if necessary.

Are macromolecules used that have an electroactive group that when an electrical field is applied, a bond is formed with the surface, so instead of lighting, a homogeneous electric field of suitable strength, Orientation and polarity.

The whole process can also be parallelized to that Increase process speed in the production of a microarray.

The arrangements of FIGS. 2 and 3 can also be generated in an analogous manner.

The location of the liquid droplets required to form the microarray across the Surface can be sent by the appropriate preferred Lounge areas are supported. So it is also possible to arrange from to provide preferred areas of stay on the solid surface which the spots should be after the manufacturing process. Through such Localization areas that are not connected become the localization assists the liquid in the formation of the microarray, so that requirements the precision of the movement induced by the surface sound waves are reduced.

In the method according to the invention for producing a microarray, the external parameter, in the examples described the lighting or the electrical field, does not have to be generated locally, but can be homogeneous over the entire chip surface. The spatially resolved binding is ensured by the fact that only a very small part of the surface is connected to the liquid drop 2 in which the macromolecules to be bound are located.

Claims (24)

1. A method for analyzing macromolecules with the aid of a microarray which consists of a plurality of spots ( 16 ) in a known arrangement which comprise a plurality of first macromolecules which are essentially identical to one another for a spot, the microarray being located on a solid surface ( 1 ), preferably a solid-state chip, is located in the a) at least one liquid drop ( 3 ) containing second macromolecules is brought onto the solid surface ( 1 ), the amount of liquid of a drop ( 3 ) being so small that essentially only one spot ( 16 ) or a group of a few spots the microarray comes into contact with the liquid drop ( 1 ) at a time, b) after a predetermined time the liquid drop ( 3 ) is moved to a next spot ( 16 ) or a further group of fewer spots with the aid of the impulse transmission of at least one surface sound wave ( 12 , 14 ), c) repetition of steps a) and b) until the desired spots of the microarray were in contact with the liquid drop ( 3 ). d) Examination of the spots that come into contact with the liquid drop ( 3 ) to determine whether and / or to what extent a specific reaction with the material of the liquid drop ( 3 ) has taken place. 2. A method for analyzing macromolecules according to claim 1, in which step d) individually after the respective implementation of step a) or after the the respective implementation of step b) is carried out. 3. A method for analyzing macromolecules according to claim 1, in which step d) is carried out after step c). 4. A method for analyzing macromolecules according to one of claims 1 to 3, in which a surface sound wave is sent in the direction of one or more spots ( 16 ), and from their damping and / or speed change to the amount of those bound to the respective spot ( 16 ) second macromolecule is closed after the liquid drop ( 3 ) has been removed. 5. The method according to any one of claims 1 to 4, wherein step a) comprises the following step a1): 1. Movement of the liquid drop with the aid of the impulse transmission of at least two essentially opposing surface sound waves, which are alternately sent onto the liquid drop ( 3 ) in such a way that the drop is moved back and forth, and in the meantime a time-dependent examination of the spot or the group fewer spots are then carried out on whether and / or to what extent a specific reaction with the material of the liquid drop ( 3 ) has taken place. 6. A method for analyzing macromolecules with the aid of a microarray which consists of a plurality of spots ( 16 ) in a known arrangement which comprise a plurality of first macromolecules which are essentially identical to one another for a spot, the microarray being located on a solid surface ( 1 ), preferably a solid-state chip, is located in the a) at least one liquid drop ( 3 ) containing second macromolecules is brought onto the solid surface ( 1 ), the amount of liquid of a drop ( 3 ) being so small that essentially only one spot ( 16 ) or a group of a few spots of the microarray comes into contact with the liquid drop ( 1 ) at a time. b) the liquid drop ( 3 ) is moved back and forth with the aid of the impulse transmission of at least two essentially opposite surface sound waves, which are alternately sent in the direction of the liquid drop ( 3 ), and during which time is examined whether and / or in which Measured a specific reaction with the material of the liquid drop ( 3 ) has taken place. 7. A method for analyzing macromolecules according to one of claims 1 to 6, in which no more than 10%, preferably essentially only one of the spots ( 16 ) of the microarray come into contact with the liquid drop ( 3 ) at a time. 8. The method according to any one of claims 1 to 7, wherein the second Macromolecules are fluorescent and can be examined in step d) or Step a1) the fluorescence is evaluated. 9. A method for analyzing macromolecules according to one of claims 1 to 8, in which the liquid drop ( 3 ) is moved with the aid of the surface acoustic wave ( 12 , 14 ) on a preferred location area ( 11 , 17 , 19 ) of the solid surface ( 1 ), whose wetting properties differ from its surroundings in such a way that the liquid drop ( 3 ) is preferably on it. 10. A method for analyzing macromolecules according to claim 9, wherein the preferred location area ( 17 ) is meandering. 11. A method for analyzing macromolecules according to claim 9, wherein the preferred location area ( 19 ) is cruciform. 12. A method for analyzing macromolecules according to one of claims 1 to 8, in which the liquid drop ( 3 ) is moved by means of the surface sound waves ( 12 , 14 ) on a solid surface ( 1 ) which has a number of preferred areas of residence, the wetting properties differ from their surroundings in such a way that the liquid drop ( 3 ) is preferably on it and which are arranged at the locations of the spots ( 16 ). 13. A method for analyzing macromolecules according to one of claims 1 to 12, in which the large number of first macromolecules are different Oligonucleotides and / or different proteins and / or different antigens and / or different antibodies, their position on the microarray is known. 14. A method for analyzing macromolecules according to one of claims 1 to 13, in which the at least one surface sound wave ( 12 , 14 ) is generated with at least one, preferably tapered, interdigital transducer ( 5 , 6 , 7 , 8 ). 15. A method for producing an analysis device for macromolecules, in particular for carrying out the method according to one of claims 1 to 14, in which a) at least one liquid drop ( 2 ) with at least one type of macromolecule is applied to a solid surface ( 1 ), preferably a chip, the macromolecules having an active group which is activated when a corresponding external parameter changes in order to bind to it enable the surface ( 1 ) or with molecules already bound to the surface, b) the drop ( 2 ) is moved to a desired location on the surface ( 1 ) with the aid of the impulse transmission of one or more surface sound waves ( 13 ), c) the external parameter in at least one area of the surface is changed homogeneously and not locally in such a way that binding of the macromolecules is made possible. 16. A method for producing an analysis device according to claim 15, in which the active group a photoactive group and the outer parameter the lighting, especially its intensity or frequency. 17. A method for producing an analysis device according to claim 15, in which the active group an electroactive group and the external parameter include an electric field. 18. A method for producing an analysis device according to claim 15, in the macromolecules are used, the active group at Temperature increase responds, and the external parameter is the temperature of the Surface includes. 19. A method for producing an analysis device according to one of claims 15 to 18, in which the surface sound wave is generated with the aid of at least one, preferably tapered, interdigital transducer ( 5 , 6 , 7 , 8 ). 20. A method for producing an analysis device according to one of claims 15 to 19, in which to form an arrangement of a plurality of spots ( 16 ) on which macromolecules are connected to the surface ( 1 ), the liquid drop ( 2 ) with the aid of the Impulse transmission of one or more surface sound waves is moved over the solid surface ( 1 ) and the external parameter is changed to enable the binding to the surface ( 1 ) whenever the liquid drop ( 2 ) has reached a predetermined position. 21. A method for producing an analysis device according to one of claims 15 to 19, in which a plurality of liquid drops ( 2 ) are moved to predetermined positions for them on the solid surface ( 1 ) with the aid of one or more surface acoustic waves ( 13 ) and by changing the external parameter to enable the binding of the macromolecules in the respective liquid drop ( 2 ), an arrangement of macromolecules on the solid surface ( 1 ) is generated. 22. A method for producing an analysis device according to any one of claims 15 to 21, wherein the solid surface ( 1 ) comprises at least one preferred residence area ( 11 ), the wetting properties of which differ from its surroundings in such a way that the at least one liquid drop ( 2 ) is preferred on it. 23. A method for producing an analysis device according to one of the Claims 15 to 22, in which the macromolecules have different oligonucleotides and / or different proteins and / or different antigens and / or comprise different antibodies. 24. A method for producing an analysis device according to one of claims 15 to 23, in which, after carrying out steps a), b) and c), the amount of macromolecules bound at one location is determined by measuring the attenuation and / or speed change of at least one surface sound wave which is sent towards the bound macromolecules after the liquid drop ( 2 ) has been removed from the location.