DE10136008B4 - Method for analyzing macromolecules and method for producing an analysis device - Google Patents

Abstract

A method of analyzing macromolecules with the aid of a microarray consisting of a plurality of spots (16) in a known arrangement, comprising a plurality of first macromolecules substantially equal to each other for a spot, the microarray resting on a solid surface (1). , preferably a solid-state chip, is located at the
a) at least one liquid drop (3) containing second macromolecules, is brought to the solid surface (1), wherein the liquid amount of a drop (3) is so small that only one spot (16) or a group of a few spots of the microarray at a time when at least one liquid drop (3) comes into contact,
b) after a predetermined time has elapsed, the at least one liquid drop (3) is moved by means of the momentum transfer of at least one surface acoustic wave (12, 14) to a next spot (16) or a further group of fewer spots,
c) repetition of steps a) and b) until the desired spots of the microarray with the at least one ...

Description

The The invention relates to methods for the analysis of macromolecules Help of a microarray and a method for producing a Analysis device for Macromolecules.

A Method for rapid analysis of macromolecules involves the use of a so-called microarray on a chip, similar as it is also known from the semiconductor industry. In such a microarray are known first possibly different macromolecules at different Positions arranged in a matrix form. These macromolecules become also called "probe molecules." A liquid with macromolecules ("Sample molecules") is about the Rinsed microarray, with at least one type of probe molecules on the microarray specific binding occurs (hybridization). Will then the liquid removed from the surface, remain primarily only at the sites of specific binding the sample molecules to be examined back. With the help of a spatially resolved measurement, z. B. a fluorescence measurement, it can be determined to which Sites to be examined macromolecules are present. From the known position of the individual probe molecules in the matrix form of the microarray can So it can be determined with what kind of macromolecules the to be examined macromolecules have entered into a specific binding.

Such Microarrays are z. B. for the study of macromolecules such Proteins, antigens or antibodies used. In particular, microarrays are also being investigated of DNA, e.g. As in DNA screening used.

The Duration of a corresponding analysis experiment is essential Part determined by the diffusion of the sample molecules to the probe molecules and may take some time. Is z. B. the concentration of the macromolecule to be examined in the liquid it may take a long time for it to be specific Binding partner found on the array.

One Another problem with the reaction (hybridization) between the located in the sample liquid macromolecules and the one on the surface located probe molecules lies in the fact that the to be examined macromolecules can be present in the liquid in a very wide range, the down to a few molecules. Nevertheless must be sufficient liquid be present to cover the entire array surface, so that all probe molecules also can be achieved. Thus, if necessary, a very low concentration of the sample molecules in the Liquid. However, such a low concentration causes a reaction equilibrium, that according to the law of mass action for chemical reactions very much strongly on the side of the educts. Sample molecules thus remain in the liquid solved, even if a matching probe molecule on the surface is still free would. To overcome this problem would have the Amount of starting material, d. H. the sample molecules in the Sample liquid, elevated be what is lacking a sufficient amount of starting material often can not be reached.

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

In Usually, the microarray after hybridization after a washed special protocol and then spatially resolved fluorescence measured. With special techniques is also a measurement of the fluorescence signal in situ during hybridization possible, for additional information about to gain the reaction kinetics. One possibility is with an optic of such shallow depth of field to measure the fluorescence signal, that only the area of the interface between the liquid film and the chip surface to Signal contributes. There are the second macromolecules mainly in the already bound Status. The fluorescence signal second macromolecules, the in the overlying liquid film in the unbound state, will be with such optics not detected. Similarly, with appropriately designed waveguides and evanescent Waves, lateral to the boundary between liquid and surface be passed, the fluorescence bound on the microarray surface molecules (L.L. Duveneck and A.P. Abel, Review on Fluorescence-based Planar Waveguide Biosensors, Proc. SPIE, Vol. 3858 (1999) pages 59 ff.).

The probe molecules are usually applied today by a printing technique (spotting). Such a printing technique is z. See, for example, VG Cheung et al., Nature Genetics Supplement, Volume 21, January 1999, pages 15 et seq. A solution with the desired probe molecule will either contact brought or contacted on the chip surface. Alternatively, the probe molecules can also z. B. photolithographically synthesized base by base on the chip. Both in spotting and in photolithographic production, a quantitative statement about the number and density of the probe molecules on a spot is difficult. This means that z. B. in a detection of the hybridized sample molecules z. B. by fluorescence only limited quantitative conclusions about the number of sample molecules are possible. The comparability of data obtained on different microarrays with nominally identical spots is considerably limited.

at a known method for the production of microarrays is the surface a solid state chip first homogeneously functionalized, so that z. B. by the action of light or by the action of an electrical Feldes the previously also functionalized probe molecules at the Bind surface. The chip is flooded homogeneously with a solution of the probe molecules (eg of the order of magnitude an amount of 100 μl). By local illumination, photoactive probe molecules bind locally to the desired Put. For electroactive probe molecules, which under the action of an electric Field at the surface bind, an electric field is generated locally, which triggers the binding. The spatial resolution comes in these methods so by the modulation of the electrical Field or the light intensity conditions. This is especially true for high spot density chips an expensive and expensive process. B. the modulation of the electric field necessary for a spatially resolved bond the probe molecules on the surface to ensure, must be in a layer below the chip surface a field of capacitors be appropriate to the later Spots locally correspond. Such a method is described by M.J. Heller, A.H. Ranger and E. Tu in Electrophoresis, 2000, 21, pages 157ff. described.

US 5,653,939 describes methods and apparatus for identifying molecules in a sample substance using a panel with different molecules bound thereto. The sample liquid is applied to the field and a signal is evaluated which depends on the binding of the molecules in the liquid with the molecules in the field.

A method for coating surfaces in which coating molecules containing reactive groups are covalently bonded to a surface by illumination with light is disclosed in US Pat DE 198 27 900 A1 described. The use of an optional adjustable exposure pattern to produce an exposure matrix is disclosed in U.S. Patent No. 5,376,866 DE 199 40 752 A1 disclosed.

WO 00/16082 describes an analytical method in which a solid surface is used, the wetting properties of which differ from the surrounding solid surface in such a way that a liquid preferably remains on it. WO 94/05414 and WO 00/10011 describe the use of sound waves for the distribution and mixing of liquids, while the distribution of liquids with surface acoustic waves in Patent Abstracts of Japan JP 10327590 A is described.

task The present invention is to provide methods by which an accurate and reproducible analysis of macromolecules is possible which are also limited raw material used. The production appropriate analysis device should be simple and reproducible to be possible.

Regarding the Method for analyzing macromolecules is the task by a method with the features of claim 1 or a method solved with the features of claim 6. Regarding a method for Preparation of a Macromolecule Analyzer the object by a method having the features of the claim 15 solved. under claims are each directed to preferred embodiments.

at a method according to the invention for the analysis of macromolecules with the help of a microarray consisting of several spots with probe molecules in known Arrangement is a microarray on a solid surface, preferably a chip, used. At least one drop of liquid with to be examined macromolecules is brought to the solid surface, the amount of liquid a drop chosen so small she will essentially just a spot or a group of a few Spots of the microarray touched at a time. Conveniently, should be there from a drop of liquid no more than 10% of all spots of a microarray are touched. On this way you can the reaction conditions (eg temperature or salt concentration) dependent on the touched Spots are optimized. The smaller the number of spots, the from a drop of liquid touched at a time the more specific is the setting of reaction conditions dependent on possible from the first macromolecules contained in a spot. In Preferably, the method is carried out such that only one Spot is touched by a drop of liquid at a time So that the Reaction conditions for each individual spot can be set individually.

To Expiration of a predetermined time, the z. B. by the typical reaction time in the liquid contained macromolecules with the macromolecules, as probes are located on a spot, is determined, with the help of Momentum transfer a surface sound wave the liquid drop to another Spot moves. Here again a predetermined time is waited until the typical reaction time has passed. In this way, the desired Spots or all spots of the used microarray with the liquid in sequential contact to be brought. Of course This process can also be parallelized by adding several drops simultaneously over different ones Spots are moved.

in the Connection can the with the liquid drop in touch have been examined, whether or in which Scope a reaction with the macromolecules in the liquid drop took place.

To can z. B. a spatially resolved Fluorescence measurement serve when the macromolecules to be examined fluorescently labeled are. Other detection methods, eg. B. using radioactive Sample molecules, are also usable.

The Examination of the liquid drop in touch spot on whether and / or to what extent one specific reaction with the material of the liquid drop occurred has, can be done at the end of the entire process d. H. if the liquid drop with all you want Spots in contact was.

As well it is with the method according to the invention possible, the investigation of whether second macromolecules with a spot or a Group fewer spots have responded already while the liquid with another spot or with a different group of less spots in touch is there to possibly lead there to the hybridization of the second macromolecules. While the Liquid so is in contact with the second spot or the second group of spots, already the first spot or the first group of spots can be evaluated.

Finally is it is also possible with appropriate process control, for. b. the fluorescence of the bound second macromolecules in situ to eat. This can z. As an optics shallow depth of field or appropriately arranged waveguides and evanescent waves used that are just the interface between liquid and chip surface to capture.

In the method according to the invention, the liquid containing the sample molecules is not applied to the entire surface of the microarray, but only to separate individual spots. While in classical microarray experiments liquid volumes of about 10-100 ul are necessary to cover a total sample area of 1 cm ² , the necessary amount of liquid can be reduced with the inventive method to a few nl, if z. B. spots are used with an extension of 100 microns. The small sample drop is moved step by step from spot to spot. The diffusion length is not in the order of 1 cm, but only in the order of 100 microns. At the points of the spots themselves then takes place a fast and efficient hybridization reaction.

by virtue of the small sample liquid quantity can the concentration of the starting materials even with the same amount on starting material much higher be there, instead of about 10-100 μl z. B. only about 10 nl needed become. That's how it works the reaction equilibrium on the side of the hybridized products push so that one much more accurate measurement even with little material to be examined possible is.

Possibly The time that is waited for before the drop of liquid returns from a spot is removed, be shortened by the fact that the liquid drop with the help of impulse transfer a surface sound wave is moved, that is "stirred" Surface acoustic wave lower intensity used as the transport of the liquid drop, so that the liquid drop does not move away from the spot.

The inventive method allows a freely programmable path of the sample liquid over the Surface. With appropriately arranged spots from different probe molecules can while a trial strategy are followed, so first those To approach spots for which one rather expects a positive result. Individually for different Spots can the reaction conditions are chosen differently. So z. B. the Temperature adjusted differently with the help of resistance heaters become. It can be z. B. the chip are heated homogeneously, since the spatial resolution by the limited extent of the drop is ensured.

Likewise, z. B. the nature of the liquid to be examined from one spot to the next to be changed. While at a spot z. For example, if the salt concentration in the liquid has a first value, it may be changed to a second value at a second spot by adding appropriate buffer solution. The buffer solution can also with the help of Impulsübertrages a surface acoustic 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. is freed from the liquid in a separate washing step before It is examined whether and to what extent a reaction of the second macromolecules in the liquid with the first macromolecules the spots took place. In the method according to the invention becomes the liquid drop with the help of the surface sound wave moved from the corresponding spot, so that immediately after an investigation whether bound second macromolecules remained at the spot are.

The Evaluation can happen parallel to the hybridization. For example from a fluorescently labeled sample molecule type in the sample liquid a drop is moved to a first spot. After the typical Hybridization time, the drop is moved to the next spot and parallel on first spot spatially resolved the fluorescence measured. At the second spot can z. B. to change the Reaction conditions change the temperature or a low one Volume of buffer solution be added to ensure the stringency of the reaction.

at a preferred embodiment of the method according to the invention is the amount of bound to the respective spot second macromolecules from the damping and / or speed change a surface sound wave determined by the presence of the second macromolecules. A surface sound wave is in the direction of the spot with the second bound to the first macromolecules macromolecules Posted. By the mass on the surface, which hybridized by the second macromolecules is formed, the surface acoustic wave in their intensity muted or changed their speed. With a detector for the surface sound wave can its intensity measured before and after hybridization of the second macromolecules and from the determined damping or speed change be deduced the number of bound second macromolecules. Possibly a calibration measurement with known parameters can be performed.

at another embodiment of the method according to the invention is at least a drop of liquid with second macromolecules on the solid surface with brought to the microarray, the amount of liquid in a drop so small is that in the essentially just a spot or a group of few spots of the Microarrays at a time with the liquid drop comes into contact. With the help of impulse transfer of two substantially mutually opposing surface acoustic waves, sent in the direction of the liquid drop in the temporal change become, the drop is moved back and forth. The intensity of the surface sound waves is chosen as that the Not completely dripping Will get removed.

During the Reciprocation is examined locally at the spot, whether and in which dimensions second macromolecules at the first macromolecules the spot or the group have hybridized fewer spots. To is z. B. again the fluorescence of fluorescently labeled second macromolecules examined. By the reciprocation of the drop, the time dependency the reaction can be determined. So is a statement about the Reaction rate or reaction kinetics possible. With increasing duration, i. increasingly frequent reciprocation of the drop over the spot of the microarray, more second macromolecules are bound, whereby z. B. the intensity the fluorescence of the bound second macromolecules on the respective spot increased becomes. If the fluorescence is only measured; when the liquid not on the spot or the group of spots is prevented that the still in the liquid unbound second macromolecules located on the fluorescence measurement participate and distort them would.

Of course, the inventive method combined to give an integral statement about the amount of hybridized macromolecules at a spot and over to obtain the reaction kinetics.

In the analytical method according to the invention, the liquid drop is moved by means of the momentum transfer of surface acoustic waves. On the surface of the chip on which the microarray is located, a surface acoustic wave is generated. The momentum transfer is then achieved either by the mechanical deformation of the solid surface or by the force of the accompanying electric fields on charged or polarizable matter in the liquid. The strength of the force on the liquid can be adjusted in a wide range over the amplitude of the surface acoustic wave. The surface acoustic wave can be generated in pulses with pulses of different lengths or continuously. Surface acoustic waves can be applied to piezoelectric substrates or substrates with piezoelectric regions, eg. As piezoelectric coatings produce. The movement by means of the momentum transfer of surface acoustic waves is particularly precise. In addition, no channels or other guiding mechanisms or pumping devices, such. B. micromechanics necessary to move the liquid on the surface.

The Surface acoustic wave can with the help of at least one surface wave generating device to be generated. Advantageously, this is a known per se Interdigital transducer used. In the simplest version has such an interdigital transducer two electrodes, the fingers in one another to grab. By applying a high-frequency alternating field, z. B. in the order of 10 to a few 100 MHz, is placed in a piezoelectric substrate or excited in a piezoelectric region of the substrate a surface acoustic wave, their wavelength itself as the quotient of the surface sound velocity and the frequency results. The propagation direction is perpendicular to the interlocking finger electrode structures. With help Such an interdigital transducer can be in a very simple manner a very defined surface acoustic wave produce. The production of such an interdigital transducer is with known lithographic processes and coating technologies economical and easy. Interdigital transducers can also, for. B. by irradiation an alternating electromagnetic field in one with the interdigital transducer connected antenna device to be controlled wirelessly.

Around certain localized areas of a surface with the surface acoustic wave to paint, can z. B. multiple interdigital transducer used in the desired arrangement be selectively controlled.

at a particularly advantageous method is a laterally limited Sound path of the surface sound wave generated with the help of so-called tapered interdigital transducer. In such a tapered interdigital transducer changes the finger spacing of the interdigitated finger electrodes along the transducer axis. For a given frequency, the resonance condition, that the Frequency equal to the quotient of the surface sound velocity and the finger distance is met only in a laterally spatially limited area. With Such a tapered transducer can therefore be a selected area the surface by appropriate choice of the frequency targeted with a surface sound wave be sonicated. In this way, individual spots or select individual rows of spots.

Should according to the above preferred embodiment, the damping a surface sound wave evaluated by the mass of hybridized second macromolecules can be used as a surface acoustic wave detector also an interdigital transducer can be used.

The Analytical method according to the invention can performed on a chip be on which the probe molecules are directly tied. However, it is particularly advantageous if the liquid drop with the macromolecules to be examined in a preferred residence area the solid surface moves whose wetting properties are so different from its surroundings distinguish that the liquid drop preferably stays on it. The probe molecules are then placed on this preferred location area.

By the definition of such a preferred residence area can the area on the solid, on which the liquid should be determined. The liquid with the to be examined macromolecules holds itself usually only on the preferred lounge area, without that channels or trenches necessary, which would otherwise restrict the movement of the liquid. The surface tension of the liquid drop additionally causes that the Liquid the preferred residence area without the action of an external force can not leave.

The different wetting properties can z. B. by an appropriate Coating either the preferred residence area or whose surroundings are realized. For example, hydrophilic or hydrophobic regions are defined. Are those to be examined macromolecules z. B. in aqueous solution, the preferred residence area is chosen to be more hydrophilic than that surrounding solid surface. This can either by a hydrophilic coating of the preferred Residence area or achieved by a hydrophobic environment become. A hydrophobic environment may, for. B. by a silanized surface will be realized. Depending on the wetting properties of the liquid, in which the macromolecules to be examined are contained, the Solid surface surrounding the residence area also hydrophilic, lipophobic or lipophilic compared to the surface of the preferred residence area to get voted. The wetting properties can continue to be modulated by microstructuring, as in the case of so-called lotus effect is the case on different Roughness of the surface based and so causes different wetting properties. Such a roughness modulation can z. B. by microstructuring the corresponding surface areas be obtained, for. B. by chemical treatment or ion irradiation. The production of areas of different wetting properties is by using already known lithographic process and / or coating technologies easily and inexpensively.

The Geometry of the preferred location area may be at the appropriate Application adapted become. For example, the preferred location area may meander through guided the microarray so that the individual positions of the microarray along the preferred location area line up. Likewise, a crosswise arrangement may be provided at which the individual positions of the microarray in the crossing areas are located.

Becomes the inventive method on such a preferred area of residence carried out, are the requirements for accuracy in the direction and lateral Propagation of the surface acoustic wave less, as the movement of the liquid drop is determined by the shape of the residence area with.

at another embodiment will be defined according preferred Places of residence provided only in the area of the individual spots, without being connected. The lounge areas serve then the localization of the liquid drop in the area of a spot or a group of fewer spots. By radiation a surface sound wave sufficient intensity can the liquid nevertheless be transported from one common area to the next. The so defined areas of residence act as an "anchor" of localization of the liquid drop a spot or in a group less spots facilitated.

The inventive method can z. B. used advantageously for the analysis of oligonucleotides become. It is particularly suitable for DNA screening, wherein the oligonucleotides to be examined for. B. short DNA strands (Deoxyribonukleinsäuresträngen) exist. Likewise but different macromolecules with specific binding like different proteins, different antigens or different antibodies in the Microarray be provided or analyzed.

at a method according to the invention for preparing a macromolecule analyzing device at least one drop of liquid with at least one kind of macromolecules on a solid surface, preferably the surface of a Chips, wherein the macromolecules have an active group, the change a corresponding external parameter is activated so that they a bond with the solid surface or already on the surface bound macromolecules can go down. The drop is made with the help of impulse transfer one or more surface acoustic waves to a desired location on the surface emotional. Then the outer parameter becomes homogeneously and not locally altered so as to allow binding of the macromolecules. The drop of liquid If necessary, you can then use it several times to move to another location on the chip the surface acoustic waves moves and the parameter is changed again so as to successively To generate microarray. Of course, these processes can also be parallelized be by adding several drops of liquid at the same time over the Solid surface moves and by the change of the external parameter at the desired Place a reaction in the drop of liquid contained macromolecules with the surface allows becomes. With the so bound to the solid surface molecules Then a microarray of probe molecules is formed.

in the Difference to conventional Method for producing a microarray, it is not necessary that the outer parameter only changed locally. This represents a significant simplification of the process, because z. B. no spatially resolved Illumination or spatially resolved electric fields are necessary. Unlike traditional ones Process in which the chip with the liquid that bind to the macromolecules contains, be flooded must, is Furthermore Material consumption significantly reduced. To flood the chip homogeneously, typically 10-100 μl are needed while one with the method according to the invention with nanoliter (nl) or few microliter (μl) amounts can get along.

Become z. B. macromolecules used with a photoactive group is used as an external parameter the lighting is chosen. Depending on the nature of the photoactive group, either their intensity or their Frequency changed, to bind the photoactive macromolecules with the surface or already bound macromolecules to enable. Of the liquid drops will be to the desired Place with the help of the surface acoustic wave moved and then the lighting turned on or accordingly changed. Only where the liquid is is just the macromolecules with the surface become one Binding enter. That's how it works generate a spot of a microarray very precisely and locally.

Are z. As macromolecules used with an electroactive group, serving as an external parameter to be changed, the electric field. The liquid drop is moved by means of the surface acoustic waves to the desired location on the solid-state chip. Then, the electric field is changed so that a reaction of the macromolecules in the liquid can take place with the surface of the chip or already bound macromolecules. It is not necessary that the electrodes for applying the electric field, a lo kale have limited extent. The entire chip surface can serve as an electrode, since the spatial resolution of the reaction is ensured by the limited amount of liquid.

Of the Drops can also be macromolecules included, based on temperature changes react. Is the drop with the help of surface sound waves to the desired Location has been moved, the chip surface is heated, in this place the reaction of the macromolecules with the surface or to allow already bound macromolecules. The temperature increase needs not being local because of spatial resolution due to the limited extent of the drop guaranteed becomes.

The Use of surface sound waves for movement of liquid drops offers in the production process according to the invention the same advantages as in the above-described analysis methods according to the invention. For generating the surface sound waves can as well as in the analysis method according to the invention Interdigital transducer, z. B. so-called tapped Interdigitaltransducer, be used. The use of such interdigital transducers offers in the production process according to the invention the same advantages as in the analysis method according to the invention.

The Production method according to the invention can on the free surface a solid state chip respectively. However, it is particularly advantageous if the solid-state chip before application of the manufacturing method according to the invention is provided with a preferred residence area, its wetting properties differ from his surroundings in such a way that the liquid with the macromolecules to be bound preferably stays on it. As in the analysis method according to the invention let yourself so the requirement for the accuracy of surface acoustic wave radiation reduce, as the movement of the liquid mainly on limited the preferred location area.

moreover is an analysis device produced by such a manufacturing method can be advantageously used to perform an analysis method according to the invention, at also a preferred area of residence as above described advantageous can be used.

One preferred residence area can, as in the analysis method according to the invention be prepared described. There are different geometries of the common area possible.

As well as in the analysis methods of the invention can several preferred residence areas are used, the are arranged at those locations of the solid surface, where the spots of the microarray should be located with the the production process according to the invention will be produced. The preferred living areas at these Places facilitate the localization of the liquid with the macromolecules, the to be bound to the spots. By radiating a surface sound wave sufficiently high intensity can still the liquid drop with the macromolecules, which are to form the spots of the microarray of a preferred residence area to the next to be moved.

at a preferred embodiment of the manufacturing method according to the invention is the amount of macromolecules bound in one place Measurement of damping and / or speed change of at least one surface acoustic wave destined to be sent towards the bound macromolecules becomes. After one or more spots of the microarray with the inventive method have been made, a surface sound wave in the direction the spot or spots and after passing through the appropriate Area of the surface in their intensity and / or with respect to the speed change. By comparison the intensity and / or Speed with the value before applying the macromolecules in the microarray was measured, the attenuation or speed change the surface sound wave through the presence the macromolecules be determined. This can be deduced from the amount of bound macromolecules become. So can for every single spot of the produced microarray on the there be closed number of macromolecules. Becomes in such a way measured microarray for the analysis of macromolecules z. B. according to the analysis method according to the invention used, an accurate quantitative statement is possible. Possibly will be added made a calibration measurement.

With the method according to the invention for the preparation of an analysis device can be precise and reproducible way to produce a microarray, in particular advantageously used with the analysis method according to the invention can be. The devices for generating surface acoustic waves, Preferably, therefore, the interdigital transducer, can first for the production of the microarray be used and then used to perform analyzes. Also an optionally present preferred residence area may initially be advantageous used in the production of the microarray and then at the following Analyzes are used.

Both in the inventive analy As well as in the manufacturing method according to the invention, a small amount of liquid is moved by means of surface acoustic waves in order to be able to react locally, either to build up a microarray from individual spots or to react the liquid in the material with individual spots of the microarray. The use of the momentum transfer of surface acoustic waves allows each a gentle and defined movement.

Based The attached figures are embodiments of the inventive method in Detail explained. The figures are not necessarily to scale and only schematically. It shows

1 an arrangement for carrying out the method according to the invention for the analysis of macromolecules,

2 another arrangement for carrying out a method according to the invention for the analysis of macromolecules,

3 a further arrangement for carrying out a method according to the invention for the analysis of macromolecules, and

4 a step in the preparation of an analysis device according to the invention.

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

On the surface are interdigital transducers 5 . 6 . 7 and 8th in taped version. The interdigital transducers consist of electrodes 9 with finger-like projections, which engage in pairs. Alternatively, other geometries of interdigital transducers are conceivable, as are known from the technology of surface acoustic wave filters. When applying an alternating field to the electrodes 9 of an interdigital transducer, a surface acoustic wave having a wavelength corresponding to the finger spacing of the electrodes and whose propagation direction is substantially perpendicular to the finger electrodes is generated. Due to the tapered design, in which the distance between the fingers is locally different, a laterally limited surface acoustic wave can be generated. The transducers each include a large number of interdigitated fingers, only a few of which are shown schematically and not to scale.

The number and arrangement of the interdigital transducer is adapted to the desired conditions. In the exemplary embodiment of a device for carrying out the analysis method according to the invention, a preferred residence area is located between the interdigital transducers 11 , Its wetting properties are so different from its surroundings that an aqueous solution preferably resides thereon. This is z. B. silanized the environment of the preferred residence area. Such a preferred residence area improves the localization of aqueous solution, but is not necessarily provided for carrying out the process of the invention. In the area of the preferred residence area 11 are spots 16 arranged from macromolecules. The individual spots 16 each include macromolecules for each spot 16 z. B. are different.

With such an arrangement, the analysis method of the present 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 field of spots 16 brought as it is in 1 is visible. The drop of liquid is of the order of a few nl to μl. A spot has z. B. an extension of 100 microns, if a drop with a few nl is used.

With the help of surface acoustic waves, using the interdigital transducers 5 . 6 . 7 and 8th are generated, an impulse is generated on the amount of liquid. For this purpose, the corresponding interdigital transducer via the electrodes 9 and corresponding terminal electrodes (not shown) applied an electromagnetic alternating field of several tens to several hundreds of MHz. Where the finger spacing is such that the resonant condition is substantially met, a surface acoustic wave is generated which propagates substantially perpendicular to the fingers of the interdigital transducer. This is exemplified in 1 through the arrow 14 indicated. The surface acoustic wave transmits an impulse to the amount of liquid 3 and moves the drop of liquid from one spot to another. With the help of in 1 shown four interdigital transducer 5 . 6 . 7 and 8th can the liquid drop 3 so defined to the place of a particular spot 16 bring.

After a residence time about the typical hybridization time in the liquid drop 3 existing macromolecules with the macromolecules present on the spot, the liquid drop 3 again with the help of Surface sound waves removed from the spot. It can be moved to another spot to allow for appropriate hybridization at that location. After the desired spots 16 with the liquid 3 is analyzed, at which spots a hybridization with the liquid present in the macromolecules has taken place. Are z. B. in the liquid fluorescently labeled macromolecules can be determined by a spatially resolved measurement of fluorescence at which spots a reaction has taken place. Is the type of macromolecules that are involved in each spot 16 In this way, it is possible to determine the nature of the macromolecules in the drop of liquid 3 back close.

It can also be carried out in situ measurement. After the liquid drop 3 has been applied to the first spot, he is z. B. using the surface acoustic wave 14 of the first interdigital transducer 7 moved a bit away from the spot. With the opposite interdigital transducer 6 a second surface acoustic wave is generated, alternating with the first surface acoustic wave 14 in the opposite direction to the drop 3 acts. Then the drop moves 3 backwards across the spot. Due to the temporal change of the two surface sound waves, so the drop moves back and forth. Every time you touch the drop 3 Macromolecules from the liquid react with the spot with macromolecules at the spot. By measuring z. B. the fluorescence at those times, at which the drop 3 is not located at the spot, so the temporal course of the reaction can be determined and made a statement about the reaction kinetics.

The Measurement of fluorescence or other detection parameter, such as z. As the electrical charge or mass, in a known manner with corresponding here not closer of interest and evaluation devices and evaluation programs.

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

There only a small amount of liquid is necessary, the concentration of the macromolecules to be examined in the Be liquid droplets higher, even if only a small amount of starting material is present on the macromolecules to be examined.

on the other hand can with conventional methods comparable concentration the sample amount much lower be, since only Nanolitermengen needed become.

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

By appropriate timing of the interdigital transducer and the use of interdigital transducers with even finger spacing is possible. If the drop of liquid reaches a corner of the meandering area of residence 17 , another interdigital transducer is used synchronously in time, which allows a movement in the bent direction.

The evaluation takes place as well as in an arrangement of 1 ,

The inventive method can also be used with an analysis device according to the 3 be performed. Here is the common area 19 shaped like a cross. Even with such an arrangement, the demands on the precision of the interdigital transducer are lower.

Of course they are also other geometries of the living areas conceivable, to the corresponding Adapted conditions are. The figures show the arrangement only schematically. Indeed There is a much larger number of spots on a chip, so that z. B. a DNA screening analysis possible is.

In an embodiment which is not shown, preferred residence areas are provided only in the area of the individual spots without being connected to one another. Such residence areas are used to localize the liquid drop in the area of the individual spots. By radiating a surface acoustic wave of sufficient intensity, the liquid drop can nevertheless be moved from one location area to the next. The However, surface acoustic wave precision requirements are reduced because the preferred location areas at each spot help localize the drop in their area.

The inventive method can also without use of a preferred residence area 11 . 17 . 19 be carried out when the control of the interdigital transducer is precise enough. Instead of a taped interdigital transducer, it is also possible to use a number of different and individually controllable transducers, which always cover only a small area of the surface with a surface acoustic wave.

4 shows a step in the production process for a microarray according to the invention. On the solid state chip 1 is a preferred residence area 11 intended. A drop of liquid 2 will be on the preferred location area 11 z. B. applied by means of a pipetting robot. The amount of liquid is z. B. in the order of 1 nl to a few ul. The liquid drop contains macromolecules which are to serve as probe molecules and for this purpose are to be bound at predetermined locations. The drop of liquid is made by means of surface acoustic waves, which interact with the interdigital transducers 5 . 6 . 7 and 8th be brought to a desired location. Exemplary is a surface wave 13 shown with the interdigital transducer 7 is generated in a manner already described above for the analysis method according to the invention be. By the momentum transfer of the surface acoustic wave 13 on the liquid drop 2 this one is going in the direction 15 emotional. Is the liquid drop 2 arrived at the desired location, the sonication is stopped with the surface acoustic wave, so that the liquid drop 2 comes to rest. Thereafter, an external parameter is changed, to which the material in the liquid drop 2 responding. For example, the macromolecules contained in the liquid drop may be photoactive. In this case, an illumination source of suitable wavelength and intensity is switched on, which is above the chip 1 is arranged and the entire chip homogeneously illuminated. The surface chemistry of the chip or the preferred residence area 11 is chosen so that a binding of the macromolecules with the surface is made possible by the illumination of the photoactive group of the macromolecules. In this way, a spot is generated at a defined location of the surface, at which the macromolecules from the amount of liquid 2 tie. The amount of fluid 2 can then be moved further to create another spot at another point.

in the Connection can z. B. another amount of liquid be applied, in which other macromolecules are located, which should be tied in other places of the chip, so one Microarray to produce, in which different spots on different macromolecules are located.

It can also liquid drops with different macromolecules successively over the same Spot to be moved. This way you can create different sequences hanged together be desired probe molecules to create. The macromolecules from liquid drops, to a later Time over the surface be moved on which already macromolecules from earlier moved over the surface liquid drops If necessary, hybridize with the first macromolecules to possibly longer macromolecules to build.

Become macromolecules used, which have an electroactive group which when applied an electric field makes a bond with the surface, so instead of lighting a homogeneous electric field suitable strength, Orientation and polarity created.

Of the entire process can also be parallelized to the process speed in the production of a microarray.

In an analogous manner, the arrangements of 2 and 3 produce.

The Localization of liquid drops, the over to the formation of the microarray the surface can be sent through the appropriate preferred living areas supports become. So it is also possible to provide an arrangement of preferred residence areas on the solid surface, where the spots are after the manufacturing process should. Through such common areas that are not interconnected are, the localization of the liquid in the formation of the Microarrays supported, so that requirements to the precision the through the surface sound waves induced movement are reduced.

In the method according to the invention for producing a microarray, therefore, the external parameter, in the examples described, the illumination or the electric field, does not have to be generated locally, but may 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 with the liquid drop 2 in which the macromolecules to be bound are located.

Claims (23)

Method for analyzing macromolecules using a microarray consisting of several spots ( 16 ) in a known arrangement comprising a plurality of first macromolecules which are substantially equal to each other for a spot, the microarray being located on a solid surface ( 1 ), preferably a solid-state chip, in which a) at least one drop of liquid ( 3 ), which contains second macromolecules, on the solid surface ( 1 ), the amount of liquid in a drop ( 3 ) is so small that only one spot ( 16 ) or a group of a few spots of the microarray at a time with the at least one drop of liquid ( 3 b) after expiration of a predetermined time the at least one liquid drop ( 3 ) by means of the momentum transfer of at least one surface acoustic wave ( 12 . 14 ) to a next spot ( 16 ) or a further group of fewer spots is moved, c) repetition of steps a) and b) until the desired spots of the microarray with the at least one liquid drop ( 3 d) examination of the at least one drop of liquid ( 3 ), whether and / or to what extent a specific reaction with the material of the at least one liquid drop ( 3 ) has taken place. Method for the analysis of macromolecules Claim 1, wherein the step d) individually after the respective implementation of the Step a) or after the respective implementation of step b) is performed. Method for the analysis of macromolecules Claim 1, wherein the step d) after step c) is performed. Method for analyzing macromolecules according to one of Claims 1 to 3, in which a surface acoustic wave is directed in the direction of one or more spots ( 16 ) and from their damping and / or speed change on the amount of at each spot ( 16 ) bound second macromolecules is closed after the liquid drop ( 3 ) has been removed. Method according to one of claims 1 to 4, in which step a) comprises the following step a1): a1) movement of the liquid drop by means of the momentum transfer of at least two mutually directed surface acoustic waves, thus alternatingly onto the at least one liquid drop ( 3 ), during which a time-dependent examination of the spot or of the group of fewer spots is carried out as to whether and / or to what extent a specific reaction with the material of the at least one liquid drop ( 3 ) has taken place. Method for analyzing macromolecules using a microarray consisting of several spots ( 16 ) in a known arrangement comprising a plurality of first macromolecules which are substantially equal to each other for a spot, the microarray being located on a solid surface ( 1 ), preferably a solid-state chip, in which a) at least one drop of liquid ( 3 ), which contains second macromolecules, on the solid surface ( 1 ), the amount of liquid in a drop ( 3 ) is so small that only one spot ( 16 ) or a group of a few spots of the microarray at a time with the at least one drop of liquid ( 1 ) a1) the at least one liquid drop ( 3 ) with the aid of the momentum transfer of at least two mutually facing surface acoustic waves, the change in the direction of the at least one liquid drop ( 3 ), and during which time-dependent investigation is carried out as to whether and / or to what extent a specific reaction with the material of the at least one liquid drop ( 3 ) has taken place. Method for the analysis of macromolecules according to one of Claims 1 to 6, in which not more than 10%, preferably only one of the spots ( 16 ) of the microarray at a time with the liquid drop ( 3 ) come into contact. Method according to one of claims 1 to 7, wherein the second macromolecules are fluorescent and for investigation in step d) or step a1) the fluorescence is evaluated. Method of analyzing macromolecules according to one of Claims 1 to 8, in which the drop of liquid ( 3 ) using the surface acoustic wave ( 12 . 14 ) on a preferred residence area ( 11 . 17 . 19 ) of the solid surface ( 1 ) whose wetting properties differ from its surroundings in such a way that the drop of liquid ( 3 ) preferably stays on it. A method of analyzing macromolecules according to claim 9, wherein the preferred location area ( 17 ) meandering. A method of analyzing macromolecules according to claim 9, wherein the preferred location area ( 19 ) is cruciform. A method for the analysis of macromolecules according to any one of claims 1 to 8, wherein the Flüs drop of liquid ( 3 ) with the help of surface sound waves ( 12 . 14 ) on a solid surface ( 1 ), which has a number of preferred residence areas whose wetting properties are so different from their surroundings that the drop of liquid ( 3 ) preferably, and at the locations of the spots ( 16 ) are arranged. Method for the analysis of macromolecules one of the claims 1 to 12, in which the plurality of first macromolecules different oligonucleotides and / or different proteins and / or different antigens and / or different antibodies comprises whose location on the microarray is known. Method for analyzing macromolecules according to one of Claims 1 to 13, in which the at least one surface acoustic wave ( 12 . 14 ) with at least one, preferably tapered, interdigital transducer ( 5 . 6 . 7 . 8th ) is produced. 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 drop of liquid ( 2 ) with at least one kind of macromolecules on a solid surface ( 1 ), preferably a chip, wherein the macromolecules have an active group, which is activated upon change of a corresponding external parameter in order to bind to the surface ( 1 ) or with molecules already bound to the surface, b) the at least one droplet ( 2 ) by means of the momentum transfer of one or more surface sound waves ( 13 ) to a desired location on the surface ( 1 c) the external parameter is changed in at least a portion of the surface homogeneously and non-locally so as to allow binding of the macromolecules. Method for producing an analysis device according to claim 15, wherein the active group is a photoactive group and the outer parameter the lighting, in particular their intensity or frequency include. Method for producing an analysis device according to claim 15, wherein the active group is an electroactive group and the outer parameter include an electric field. Method for producing an analysis device according to claim 15, in which macromolecules are used, the active group with temperature increase responds, and the outer parameter the Temperature of the surface includes. Method for producing an analysis device according to one of Claims 15 to 18, in which the surface acoustic wave is detected by means of at least one, preferably tapered, interdigital transducer ( 5 . 6 . 7 . 8th ) is produced. Method for producing an analysis device according to one of Claims 15 to 19, in which an array of a plurality of spots ( 16 ), on which macromolecules with the surface ( 1 ), the liquid drop ( 2 ) by means of the momentum transfer of one or more surface acoustic waves over the solid surface ( 1 ) and the outer parameter is always used to allow bonding to the surface ( 1 ) is changed when the liquid drop ( 2 ) has reached a predetermined position. Process for producing an analysis device according to one of Claims 15 to 20, in which the solid-state surface ( 1 ) at least one preferred location ( 11 ), the wetting properties of which differ from its surroundings in such a way that the at least one drop of liquid ( 2 ) preferably stays on it. Method for producing an analysis device according to one of the claims 15 to 21, in which the macromolecules different oligonucleotides and / or different proteins and / or different antigens and / or different antibodies include. Method for producing an analysis device according to one of Claims 15 to 22, in which, after carrying out steps a), b) and c), the amount of macromolecules bound to a location is determined by measuring the damping and / or speed change of at least one surface acoustic wave, which is sent in the direction of the bound macromolecules after the liquid drop ( 2 ) has been removed from the location.