WO2003012389A2

WO2003012389A2 - Method for analysing macromolecules, analysis device and a method for producing an analysis device

Info

Publication number: WO2003012389A2
Application number: PCT/EP2002/007644
Authority: WO
Inventors: Jürgen SCRIBA; Christoph Gauer
Original assignee: Advalytix Ag
Priority date: 2001-07-24
Filing date: 2002-07-09
Publication date: 2003-02-13
Also published as: DE10136008A1; AU2002328867A1; EP1412533A2; DE10136008B4; WO2003012389A3

Abstract

The invention relates to a method for analysing macromolecules using a microarray, which consists of several spots in a known arrangement. Said spots comprise a plurality of first macromolecules that differ at least partially. The microarray is located on the surface of a solid body, preferably a chip. According to the method, at least one drop of liquid containing macromolecules is applied to the surface of the solid body, the quantity of liquid being so small that only one spot or a small group of spots of the microarray comes or come into contact with the drop of liquid. After a predetermined time has elapsed, the drop of liquid is displaced onto a subsequent spot or an additional small group of spots with the help of the transmission pulse of a surface sound wave. The spots that come into contact with the drop of liquid are then analysed to identify whether a reaction with the material of said drop has occurred. The invention also relates to a method for producing an analysis device for macromolecules. According to said method, at least one drop of liquid containing at least one type of macromolecule is applied to the surface of a solid body, preferably a chip, whereby the macromolecules have an active group that is activated during a modification of a corresponding external parameter, in order to allow bonding to the surface or to molecules that have already been bonded. The drop is displaced to a desired location on the surface with the help of the transmission pulse of one or more surface sound waves. The external parameter is modified in a homogeneous, non-local manner, permitting the bonding of the macromolecules to form the spot of a microarray, in which macromolecules from the drop of liquid are bonded to the surface. The invention also relates to an analysis device for carrying out the inventive analysis method.

Description

Method for analyzing macromolecules, analysis device and method for

Manufacture of an analyzer

The invention relates to methods for analyzing macromolecules with the aid of a microarray and a method for producing an analysis device for macromolecules.

One method for the rapid analysis of macromolecules involves the use of a so-called microarray on a chip, similar to that known from the semiconductor industry. In such a microarray, known first, possibly different, macromolecules are arranged at different locations in a matrix form. These macromolecules are also referred to as “probe molecules”. A liquid with macromolecules (“sample molecules”) is rinsed over the microarray, which forms a specific bond with at least one type of probe molecule on the microarray (hybridization). If the liquid is then removed from the surface again, the sample molecules to be examined remain only at the points of the specific binding. With the help of a spatially resolved measurement, e.g. B. a fluorescence measurement, it can be determined at which locations to be examined macromolecules are present. From the known position of the individual probe molecules in the matrix form of the microarray it can thus be determined with which type of macromolecules the macromolecules to be examined have entered into a specific bond.

Such microarrays are e.g. B. used to study macromolecules such as proteins, antigens or antibodies. In particular, microarrays are also used to examine DNA, e.g. B. used in DNA screening.

The duration of a corresponding analysis experiment is largely determined by the diffusion of the sample molecules to the probe molecules and can therefore take some time. Is z. For example, if the concentration of the macromolecule to be examined in the liquid is only low, it can take a long time before it has found its specific binding partner on the array.

Another problem with the reaction (hybridization) between the macromolecules in the sample liquid and the probe molecules on the surface is that the macromolecules to be investigated can be present in the liquid in a very wide range, which extends down to a few molecules , However, sufficient liquid must be available to cover the entire array surface so that all probe molecules can also be reached. This may result in a very low concentration of the sample molecules in the liquid. However, such a low concentration results in a reaction equilibrium which, according to the law of mass action for chemical reactions, is very much on the side of the starting materials. Sample molecules therefore remain dissolved in the liquid, even if a suitable probe molecule would still be free on the surface. In order to overcome this problem, the amount of the starting material, ie the sample molecules in the sample liquid, would have to be increased, which is often not possible due to a lack of a sufficient amount of starting material. The microarrays used are miniaturized arrays, which in the case of e.g. B. of DNA microarrays include individual areas (spots) with probe molecules each with a diameter of about 100 microns. There are 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).

As a rule, the microarray is washed after hybridization according to a special protocol and then the fluorescence is 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. The second macromolecules are mainly in the already bound state there. The fluorescence signal of two macromolecules, which are in the unbound state in the liquid film above them, is not detected with such optics. Likewise, with appropriately designed waveguides and evanescent waves that are guided laterally to the interface between the liquid and the 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 Genetic Supplement, Volume 21, January 1999, pages 15ff. described. 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. Both at Spotting as well as in photolithographic production is difficult to make a quantitative statement about the number and density of the probe molecules on a spot. 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 is first functionalized homogeneously, so that, for. B. by the action of light or by the action of an electric field bind the previously also functionalized probe molecules on the surface. For this purpose, the chip is homogeneously flooded with a solution of the probe molecules (e.g. in the order of magnitude of 100 μl). Local illumination binds photoactive probe molecules locally to the desired locations. In the case of electroactive probe molecules which bind to the surface under the action of an electrical field, an electrical field is generated locally which triggers the binding. With these methods, the spatial resolution therefore comes about through the modulation of the electric field or the light intensity. Especially with chips with a high spot density, this represents a complex and expensive process. B. the modulation of the electric field necessary to ensure a spatially resolved binding of the probe molecules to the surface, a field of capacitors must be attached in a layer below the chip surface, which correspond to the later spots locally. Such a procedure is used by M.J. Heller, A.H. Forster and E. Tu in Electrophoresis, 2000, 21, pages 157ff. described.

The object of the present invention is to provide methods and a device with which an accurate and reproducible analysis of macromolecules is possible, which can also be used with limited sample material. The production of corresponding analysis devices should be simple and reproducible. With regard to the method for analyzing macromolecules, the object is achieved by a method having the features of claim 1 or a method having the features of claim 6. With regard to a method for producing an analysis device for macromolecules, the object is achieved by a method having the features of claim 15. With regard to the analysis device, the object is achieved with an analysis device with the features of claim 25. Subclaims are each directed to preferred configurations.

In a method according to the invention for the analysis of macromolecules with the aid of a microarray, which consists of several spots with probe molecules in a known arrangement, a microarray is used on a solid surface, preferably a chip. At least one drop of liquid with the macromolecules to be examined is placed on the surface of the solid, the amount of liquid in a drop being so small that it essentially touches only one spot or a group of a few spots of the microarray at a time. Conveniently, no more than 10% of all spots of a microarray should be touched by a liquid drop. In this way, the reaction conditions (e.g. temperature or salt concentration) can be optimized depending on the spots touched. The smaller the number of spots that are touched by a drop of liquid at a time, the more specifically it is possible to set reaction conditions depending on the first macromolecules contained in a spot. The process is preferably carried out in such a way that only one spot is touched by a drop of liquid at a time, so that the reaction conditions can be set individually for each individual spot.

After a predetermined time, the z. B. is determined by the typical reaction time of the macromolecules contained in the liquid with the macromolecules, which are located as probe molecules on a spot, is moved to a next spot with the aid of the impulse transfer of a surface acoustic wave of the liquid drops. Here, a predetermined time is again waited until the typical reaction time has passed. In this way, the desired spots or all spots of the microarray used with the liquid can be sequenced be brought into contact. Of course, this process can also be parallelized by moving several drops simultaneously over different spots.

The spots that have come into contact with the liquid drop can then be examined for whether or to what extent a reaction with the macromolecules in the liquid drop has taken place.

For this, e.g. B. serve a spatially resolved fluorescence measurement if the macromolecules to be examined are fluorescence-labeled. Other detection methods, e.g. B. using radioactive sample molecules can also be used.

The examination of the spots that have come into contact with the liquid drop to determine whether and / or to what extent a specific reaction with the material of the liquid drop has taken place can be carried out at the end of the entire process, i. H. when the drop of liquid was in contact with all the desired spots.

It is also possible with the method according to the invention to carry out the investigation as to whether second macromolecules have reacted with one spot or a group of fewer spots while the liquid is in contact with another spot or with another group of fewer spots in order to find there possibly lead to hybridization of the second macromolecules. 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 can already be evaluated.

Finally, if the process is carried out appropriately, it is also possible, for example, to measure the fluorescence of the bound second macromolecules in situ. For this, e.g. B. optics with shallow depth of field or appropriately arranged waveguides and evanescent waves can be used, which only detect the interface between the liquid and the 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 rather 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 μl, for. B. only about 10 nl are needed. The reaction equilibrium can thus be pushed to the side of the hybridized products, so that a much more precise measurement is possible even with little material to be examined.

If necessary, the time that is waited before the liquid drop is removed again from a spot can be shortened by moving the liquid drop within itself by means of the impulse transmission of a surface sound wave, that is to say “stirring” it. A surface sound wave of lower intensity is used for this. than that for transporting the liquid drop so that the liquid drop does not move away from the spot.

The method according to the invention allows a freely programmable path of the sample liquid over the surface. In the case of suitably arranged spots from different probe molecules, an experimental strategy can be followed in order to first approach those spots for which a positive result is expected. The reaction questions can be conditions can be selected differently. So z. B. the temperature can be set differently using resistance heaters. Here, for. B. the chip are heated homogeneously, since the spatial resolution is guaranteed by the limited expansion of the drop.

Likewise, e.g. B. the nature of the liquid to be examined can be changed from one spot to the next. While in a spot z. B. the salt concentration in the liquid has a first value, it can be changed to a second value at a second spot by adding appropriate buffer solution. The buffer solution can also be moved in the direction of the corresponding spot with the aid of the impulse transmission of a surface sound wave.

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 examining whether and to what extent a reaction of the second macromolecules in the liquid with the first macromolecules of the spots has taken place. In the method according to the invention, the liquid drop is moved away from the corresponding spot with the aid of the surface sound wave, so that an investigation can be carried out immediately afterwards to determine whether bound second macromolecules have remained on the spot.

The evaluation can take place parallel to the hybridization. For example, a drop is moved from a fluorescence-labeled sample molecule type in the sample liquid to a first spot. After the typical hybridization time, the drop is moved to the next spot and the fluorescence measured in parallel at the first spot. At the second spot z. For example, to change the reaction conditions, change the temperature or add a small volume of buffer solution to ensure the stringency of the reaction.

In a preferred embodiment of the method according to the invention, the amount of second macromolecules bound to the respective spot is determined by the damping and / or change in speed of a surface sound wave determines the presence of the second macromolecules. A surface sound wave is sent in the direction of the spot with the second macromolecules bound to the first macromolecule. The mass on the surface, which is formed by the hybridized second macromolecules, dampens the surface sound wave in its intensity or changes its speed. With a detector for the surface sound wave, its intensity can be measured before and after the hybridization of the second macromolecules, and the number of bound second macromolecules can be deduced from the damping or change in speed determined thereby. If necessary, a calibration measurement can be carried out with known parameters.

In another embodiment of the method according to the invention, at least one drop of liquid with second macromolecules is brought onto the surface of the solid body with the microarray, the amount of liquid in a drop being so small that essentially only one spot or a group of a few spots of the microarray at a time with the Drops of liquid come into contact. The drop is moved back and forth with the help of the impulse transmission of two essentially opposite surface sound waves, which are sent alternately in the direction of the liquid drop. The intensity of the surface sound waves is chosen so that the drop is not completely removed.

During the back-and-forth movement, it is examined locally at the spot whether and to what extent second macromolecules have hybridized fewer spots to the first macromolecules of the spot or group. For this, z. B. again examined the fluorescence of fluorescence-labeled second macromolecules. The time dependence of the reaction can be determined by the back and forth movement of the drop. A statement about the reaction rate or the reaction kinetics is possible. With increasing time, ie increasingly frequent back and forth movement of the drop over the spot of the microarray, more second macromolecules are bound. B. the intensity of the fluorescence of the bound second macromolecules at the respective spot is increased. Will the Fluorescence measured only when the liquid is not on the spot or the group of spots prevents the unbound second macromolecules still in the liquid from participating in the fluorescence measurement and falsifying it.

Of course, the methods according to the invention can be combined in order to obtain an integral statement about the amount of hybridized macromolecules at a spot and about the reaction kinetics.

In the analysis method according to the invention, the liquid drop is moved with the aid of the impulse transmission of surface sound waves. A surface acoustic wave is generated on the surface of the chip on which the microarray is located. The momentum transfer is then achieved either by the mechanical deformation of the solid surface or by the force effect of the accompanying electric fields on charged or polarizable matter in the liquid. The strength of the force acting on the liquid can be set in a wide range via the amplitude of the surface sound wave. The surface sound wave can be generated in pulses with pulses of different lengths or continuously. 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 impulse transfer of surface sound waves is particularly precise. In addition, no channels or other guide mechanisms or pumping devices, such as. B. micromechanical pumps, necessary to move the liquid on the surface.

The surface acoustic wave can be generated with the aid of at least one surface wave generating device. An interdigital transducer known per se is advantageously used for this purpose. In the simplest version, such an interdigital transducer has two electrodes that interlock like fingers. By applying a high-frequency alternating field, e.g. B. in the order of 10 to a few 100 MHz, a surface acoustic wave is excited in a piezoelectric substrate or in a piezoelectric region of the substrate, the Wavelength is the quotient of the surface sound velocity and the frequency. The direction of propagation is perpendicular to the interdigitated finger electrode structures. With the help of such an interdigital transducer, a very defined surface sound wave can be generated in a very simple manner. The production of such an interdigital transducer is inexpensive and simple using known lithographic processes and coating technologies. Interdigital transducers can also, e.g. B. can be controlled wirelessly by irradiation of an alternating electromagnetic field in an antenna device connected to the interdigital transducer.

In order to sweep over certain localized areas of a surface with the surface acoustic wave, e.g. B. several interdigital transducers can be used in the desired arrangement, which are selectively controlled.

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

If, according to the preferred embodiment described above, the attenuation of a surface sound wave by the mass of the hybridized second macromolecules is to be evaluated, an interdigital transducer can also be used as the surface sound wave detector. The analysis method according to the invention can be carried out on a chip on which the probe molecules are directly bound. However, it is particularly advantageous if the liquid drop with the macromolecules to be examined is moved on a preferred location area of the solid surface, the wetting properties of which differ from its surroundings in such a way that the liquid drop is preferably on it. The probe molecules are then arranged on this preferred location area.

The area on the solid body on which the liquid is to move can be determined by defining such a preferred location area. The liquid with the macromolecules to be examined is usually only in the preferred location area, without the need for channels or trenches which would otherwise restrict the movement of the liquid. The surface tension of the liquid drop additionally means that the liquid cannot leave the preferred area of residence without the action of an external force.

The different wetting properties can e.g. B. can be realized by an appropriate coating either of the preferred lounge area or its surroundings. For example, hydrophilic or hydrophobic areas can be defined. Are the macromolecules to be examined e.g. B. contained in aqueous solution, the preferred location is chosen so that it is more hydrophilic than the surrounding solid surface. This can be achieved either by a hydrophilic coating of the preferred area or by 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 contained, the solid surface surrounding the area of residence can also be selected to be hydrophilic, lipophobic or lipophilic in comparison to the surface of the preferred area of residence. The wetting properties can also be modulated by microstructuring, as is the case with the so-called lotus effect, which is based on different roughness of the surface and so causes different wetting properties. Such roughness modulation can e.g. B. be obtained by microstructuring the corresponding surface areas, for. B. by chemical treatment or ion radiation. The production of areas with different wetting properties is simple and inexpensive by using already known lithographic processes and / or coating technologies.

The geometry of the preferred lounge area can be adapted to the corresponding application. For example, the preferred location area can be meandered through the microarray, so that the individual positions of the microarray line up along the preferred location area. Likewise, a crosswise arrangement can be provided, in which the individual positions of the microarray are located in the intersection areas.

If the method according to the invention is carried out on a preferred location area designed in this way, the requirements for the accuracy in the direction and lateral spread of the surface sound wave are lower, since the movement of the liquid drop is also determined by the shape of the location area.

In another embodiment, correspondingly defined preferred lounge areas are provided only in the area of the individual spots, without being connected to one another. The location areas then serve to localize the drop of liquid in the area of a spot or a group of fewer spots. By irradiating a surface sound wave of sufficient intensity, the liquid can still be transported from one area to the next. The stay areas defined in this way act like an "anchor", which makes it easier to locate the drop of liquid at a spot or at a group of fewer spots.

The inventive method can, for. B. can be used advantageously for the analysis of oligonucleotides. Among other things, it is particularly suitable for DNA screening, with the oligonucleotides to be examined e.g. B. consist of short strands of DNA (deoxyribonucleic acid strands). However, different macromolecules with specific binding such as different proteins, different antigens or different antibodies can also be provided or analyzed in the microarray.

In a method according to the invention for producing an analysis device for macromolecules, at least one drop of liquid with at least one type of macromolecule is applied to a solid surface, preferably the surface of a chip, the macromolecules having an active group which is activated in this way when a corresponding external parameter changes that it can bond with the surface of the solid or with macromolecules already bound to the surface. The drop is moved to a desired location on the surface with the aid of the impulse transmission of one or more surface sound waves. The external parameter is then changed homogeneously and not locally in such a way that binding of the macromolecules is made possible. The liquid drop can then be moved several times to a different location of the chip with the aid of the surface sound waves and the parameter changed again in order to successively generate a microarray. Of course, these processes can also be parallelized by moving several liquid drops simultaneously over the surface of the solid and by changing the external parameter at the desired location to enable the macromolecules contained in the liquid drops to react with the surface. A microarray of probe molecules is then formed with the molecules bound to the surface of the solid.

In contrast to conventional methods for producing a microarray, it is not necessary for the external parameter to be changed only locally. This represents 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 must be flooded with the liquid which contains the macromolecules to be bound, this is also the case Material consumption significantly reduced. In order to flood the chip homogeneously, typically 10-100 μl are required, while the method according to the invention can be used with nanoliter (nl) or a few microliter (μl) amounts.

Are z. B. Macromolecules with a photoactive group, the lighting is chosen as an external parameter. Depending on the nature of the photoactive group, either its intensity or its frequency is changed in order to enable the photoactive macromolecules to bind to the surface or to macromolecules that have already been bound. The drop of liquid is moved to the desired location using the surface sound wave and then the lighting is switched on or changed accordingly. Only where the liquid is located will the macromolecules bind to the surface. In this way, a spot of a microarray can be generated very precisely and locally.

Are z. B. used macromolecules with an electroactive group, serves as an external parameter to be changed, the electric field. The drop of liquid is moved to the desired location on the solid-state chip with the aid of the surface sound waves. Then the electrical field is changed such 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 for the electrodes for the application of the electrical field to have a locally limited extent. The entire chip surface can serve as an electrode, since the spatial resolution of the reaction is guaranteed by the limited amount of liquid.

The drop can also contain macromolecules that respond to changes in temperature. If the drop has been moved to the desired location with the aid of surface acoustic waves, the chip surface is heated in order to enable the reaction of the macromolecules with the surface or already bound macromolecules at this location. The temperature increase need not be local, since a spatial resolution is guaranteed by the limited extent of the drop.

The use of surface acoustic waves for the movement of liquid drops offers the same advantages in the production method according to the invention as in the analysis methods according to the invention described above. To generate the surface sound waves, as in the analysis method according to the invention, interdigital transducers, e.g. B. so-called tapered interdigital transducers can be used. The use of such interdigital transducers in the production method according to the invention offers the same advantages as in the analysis methods according to the invention.

The production method according to the invention can take place on the free surface of a solid-state chip. However, it is particularly advantageous if the solid-state chip is provided with a preferred area of residence before use of the production method according to the invention, the wetting properties of which differ from its surroundings in such a way that the liquid with the macromolecules to be bound is preferably thereon. As with the analysis methods according to the invention, the requirement for the accuracy of the surface sound wave radiation can be reduced, since the movement of the liquid is mainly limited to the preferred location area.

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

A preferred lounge area can be created as already described in the analysis methods according to the invention. Different geometries of the lounge area are possible. As with the analysis methods according to the invention, several preferred location areas can also be used, which are arranged at those locations on the surface of the solid body where the spots of the microarray that are produced with the production method according to the invention are to be located. The preferred locations at these locations facilitate the localization of the liquid with the macromolecules to be bound to the spots. By irradiating a surface sound wave of sufficiently high intensity, the liquid drop with the macromolecules which are to form the spots of the microarray can nevertheless be moved from one preferred location area to the next.

In a preferred embodiment of the production method according to the invention, the amount of macromolecules bound at one location is determined by measuring the attenuation and / or change in speed of at least one surface sound wave which is sent in the direction of the bound macromolecules. After one or more spots of the microarray have been produced using the method according to the invention, a surface sound wave is sent in the direction of the spot or spots and after passing through the corresponding area of the surface, its intensity and / or the speed change is measured. By comparing the intensity and / or speed with the value that was measured in the microarray before the macromolecules were applied, the attenuation or change in speed of the surface sound wave can be determined by the presence of the macromolecules. The amount of bound macromolecules can be deduced from this. The number of macromolecules located there can thus be inferred for each individual spot of the microarray produced. If such a measured microarray for the analysis of macromolecules z. B. used according to the analysis method according to the invention, an accurate quantitative statement is 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 can be used particularly advantageously with the analysis method according to the invention. The devices for generating the surface sound waves, preferably the interdigital transducer, can first be used for the production of the microarray and then used to carry out analyzes. Any preferred stay area that may be present can also be advantageously used initially in the manufacture of the microarray and then used in the following analyzes.

Both in the analysis method according to the invention and in the production method according to the invention, a small amount of liquid is moved with the aid 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 material in the liquid with individual spots of the microarray. The use of the impulse transmission of surface sound waves enables a gentle and defined movement.

Refinements of the method according to the invention are explained in detail with reference to the attached figures. The figures are not necessarily to scale and are only schematic. It shows

FIG. 1 shows an arrangement for carrying out the method according to the invention for analyzing macromolecules,

FIG. 2 shows another arrangement for carrying out a method according to the invention for analyzing macromolecules,

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

Figure 4 shows a step in the manufacture of an analysis device according to the invention. Figure 1 shows a device for performing 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 in a tapered version on the surface. 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 wave filters. When an alternating field is applied to the electrodes 9 of an interdigital transducer, a surface sound wave is generated with a wavelength that 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 located thereon. 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 stay area 11. The individual spots 16 each comprise macromolecules, which for each spot 16 z. 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. brought into the area of the spots 16 with the aid of a pipetting robot, 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 an impulse 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 place 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 a corresponding hybridization at the location there. 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. Is the type of macromolecule that is attached the individual spots 16, known, can be inferred in this way from the type of macromolecules in the liquid drop 3.

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 acoustic wave is generated, which acts in alternation with the first surface acoustic wave 14 in the opposite direction on the drop 3. Then the drop 3 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 with appropriate measuring and evaluation devices and evaluation programs that are not of interest here.

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 macromolecules to be examined in the liquid drop can be higher, even if only a small amount of starting material is present on the macromolecules to be examined. On the other hand, if the concentration is comparable to that of conventional methods, the amount of sample can be much smaller since only nanoliter amounts are required.

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 which is located on this meandering location area 17 can be moved along the location area with the aid of 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 process 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 corresponding circumstances. The figures show the Arrangement only schematically. In fact, there is a much larger number of spots on a chip, so that e.g. B. a DNA screening analysis is possible.

In an embodiment not shown, preferred areas of residence are provided only in the area of the individual spots, without being connected to one another. Such location areas serve to localize the drop of liquid in the area of the individual spots. By irradiating a surface sound wave of sufficient intensity, the liquid drop can nevertheless be moved from one area to the next. However, the requirements for the precision of the surface wave radiation are reduced, since the preferred areas of residence at the individual spots support the localization of the drop in their 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, a number of different and individually controllable transducers can also be used, which always only cover a narrow area of the surface with a surface sound wave.

FIG. 4 shows a step in the production method for a microarray according to the invention. A preferred residence area 11 is provided on the solid-state chip 1. A drop of liquid 2 is z. 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 sound 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. is written. The impulse transmission of the surface sound wave 13 to the liquid drop 2 moves it in the direction 15. When the liquid drop 2 has arrived at the desired location, the sonication is stopped with the surface sound wave, so that the liquid drop 2 comes to rest. Thereupon an external parameter is changed, to which the material in the liquid drop 2 reacts. 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 such that the macromolecules are bound by the illumination of the photoactive group of the macromolecules to the surface. 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 which there are other macromolecules which are to be bound at other locations on the chip, in order to produce a microarray in which different macromolecules are located at different spots.

Liquid drops with different macromolecules can also be moved successively over the same spot. In this way, different sequences can be attached to one another in order to generate desired probe molecules. The macromolecules from liquid drops that are later moved over the surface, on which macromolecules from liquid drops that have previously moved over the surface are already bound, hybridize with the first macromolecules to form longer macromolecules, if necessary.

If macromolecules are used which have an electroactive group which binds to the surface when an electric field is applied, so instead of lighting, a homogeneous electric field of suitable strength, orientation and polarity is created.

The entire process can also be parallelized to increase the process speed when manufacturing a microarray.

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

The location of the liquid droplets that are sent over the surface to form the microarray can be supported by the corresponding preferred location areas. It is thus also possible to provide an arrangement of preferred areas of residence on the surface of the solid body at which the spots are to be located according to the manufacturing process. Such localization areas, which are not connected to one another, support the localization of the liquid in the formation of the microarray, so that requirements for 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

Method for analyzing macromolecules, analysis device and method for producing an analysis device

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 multiplicity 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, in which

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 of the liquid drops (3) with the aid of the pulse transmission of at least one surface sound wave (12, 14) is moved to a next spot (16) or another group of fewer spots,

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 have 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) is carried out individually after the respective implementation of step a) or after the respective implementation of step b).

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):

a1) 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 group - pe fewer spots is 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 multiplicity 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, in which

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.

a1) 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 the fluorescence is evaluated for examination in step d) or step a1).

9. 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 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 with the aid of the surface sound waves (12, 14) on a solid surface (1) which has a number of preferred areas where 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 plurality of first macromolecules comprises different oligonucleotides and / or different proteins and / or different antigens and / or different antibodies, the position of which 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, wherein the active group comprises a photoactive group and the external parameter comprises the illumination, in particular its intensity or frequency.

17. A method of manufacturing an analyzer according to claim 15, wherein the active group comprises an electroactive group and the external parameter comprises an electric field.

18. A method for producing an analysis device according to claim 15, in which macromolecules are used, the active group of which reacts when the temperature rises, and the external parameter comprises the temperature of the surface.

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 bond 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) by means of one or more surface sound waves (13) and by changing the external parameter In order to enable the binding of the macromolecules in the respective liquid drop (2), an arrangement of macromolecules is generated on the surface of the solid (1).

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 area (11) whose wetting properties 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 claims 15 to 22, wherein the macromolecules comprise different oligonucleotides and / or different proteins and / or different antigens and / or different antibodies.

24. A method for producing an analysis device according to any 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 damping 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.

25. Analysis device for manipulating small amounts of liquid with

a multiplicity of separate areas of residence on a surface, the wetting properties of the areas of residence differing from their surrounding surface in such a way that the liquid preferably resides thereon, and

- At least one surface wave generation device for generating a surface sound wave, the surface sound wave generation device or the surface sound wave generation devices being provided in a number and / or arrangement such that a surface sound wave with a pulse component in the direction of the connecting line between the two stay areas generates for every two adjacent location areas can be.

26. Analysis device according to claim 25, wherein the surface comprises the surface of a piezoelectric solid substrate or a substrate with a piezoelectric coating.

27. Analysis device according to one of claims 25 or 26, wherein the at least one surface sound wave generating device comprises a preferably tapered interdigital transducer.

28. Analysis device according to claim 25, in which a plurality of individually controllable interdigital transducers are provided.

29. Analysis device according to one of claims 25 to 28, in which the surface surrounding the occupied areas is hydrophobic in comparison to the occupied areas.

30. Analysis device according to claim 29, wherein the surface surrounding the occupied areas is silanized.

31. Analysis device according to one of claims 25 to 30, wherein the surface is coated with silicon dioxide.