DE102004021904B4 - Method and device for generating an analysis arrangement with discrete, separate measurement ranges for biological, biochemical or chemical analysis - Google Patents

Abstract

A method for generating an analysis arrangement (3) with a plurality of discrete, separate measurement areas (16), which is designed for the detection of at least one analyte in a biological, biochemical or chemical detection method in samples to be brought into contact with the measurement areas (16) the steps a) providing at least one carrier (4), b) providing a supply of a reagent solution, and c) contactlessly transferring at least a certain amount of the reagent solution to the carrier to produce at least one of the plurality of discrete, separate measuring areas (16) or a previously identified area of the carrier (4) for the at least one discrete, separate measuring area (16) or onto a discrete measuring area (16) previously generated on the carrier (4) by irradiating the supply of the reagent solution with an electromagnetic energy beam.

Description

The present invention relates to a method and an apparatus for generating an analysis arrangement with a plurality of discrete and separate measurement ranges, which are suitable and designed to detect one or more compounds as analytes in one or more samples, after their application in said measuring ranges or Contacting with biological, biochemical or synthetic recognition elements previously immobilized in said measuring ranges, in a biological, biochemical or chemical (and optionally also physical) detection method. In particular, the present invention relates to such a method in the form of a "spotting" method, in which the samples to be analyzed are applied to the individual measuring ranges of the analytical device or to generate the individual measuring ranges on the analytical device with the aid of "spotting" technology. and a corresponding device, which may also be referred to as a "spotter".

For the purposes of the present invention, discrete, separate measuring ranges are to be defined by two- or three-dimensional regions on a carrier which assume immobilized binding partners there, for the detection of one or more analytes in one or more samples in a binding or affinity assay. The discrete, separate measurement areas may, but need not, be identified prior to the attachment of the binding partners on said substrate, for example in the form of a subdivision of the substrate in the geometry of the measurement areas corresponding discrete, separate areas. Preferably, the discrete, separate measurement areas are generated with the step of applying the binding partner to be immobilized on the carrier.

Mutually complementary binding partners are characterized by a certain affinity between them. Bonds between complementary binding partners (such as analytes and their biological, biochemical or synthetic recognition elements) can be reversible or irreversible.

Different such measuring ranges may include, for example, biological, biochemical or synthetic recognition elements of a single species or form or several different species or forms as immobilized binding partners, with which then in a subsequent step of the detection method one or more of the corresponding analytes (which are specific to the corresponding recognition elements recognized and bound) samples to be examined are brought into contact. One or two-dimensional arrangements of a plurality of such arrays of measurement areas of this first form are also referred to as "capture" arrays. The biological, biochemical or synthetic recognition elements immobilized in the measurement regions are preferably in an enriched, purified form, ie. H. in a higher concentration compared to their original occurrence in their production medium.

The discrete, separate measurement ranges may also be generated by applying the samples to be assayed for their ingredients themselves, without or after performing one or more purification steps such as fractionation of the underlying sample material. In this case, analytes contained in the applied samples should be regarded as immobilized binding partners. The sample material may comprise, for example, whole or disrupted cells, cell components, tissue sections, other biological materials or chemical materials. In the case of lysates as deposited samples, one- or two-dimensional arrays of such measurement regions of this second form are referred to as "lysate" arrays, the analytes of which then typically contain, after contacting with specific biological, biochemical or synthetic recognition elements in a step of an assay with so-called "Inverted assay architecture" can be determined.

For generating arrays of the first and second forms, a variety of methods and devices are known.

Known application methods include so-called "ink jet spotting," in which a piezoelectric effect similar to that used in the operation of an ink jet printer is used to apply the samples to a support to produce the desired measurement ranges.

In addition, mechanical "spotting" methods (for example by means of a spring, pin or capillary) are known in which a mechanical contact between a liquid storage device (eg pins or capillaries) containing the liquids to be deposited on the carrier with it the carrier to be immobilized compounds, and the surface of the carrier takes place. Such mechanical "spotting" methods are associated with several principal disadvantages. In the case of solid substrates with pressure- or scratch-sensitive surfaces where low surface roughness and surface freedom from mechanical damage are of crucial importance to the quality and feasibility of a surface to be carried out Detection method is (as for example in optical waveguides, glass or plastic plates or membranes as carriers), there is a high risk of quality degradation. Furthermore, there is a risk of a continuous change in the arrangements brought into contact with the support surface (eg capillary tips or pen tips), similar to a wear process, resulting in a corresponding continuous change in the geometry of the "spots" and the amounts of liquid applied therein can lead. This trend can tentatively be counteracted by continually adjusting the application conditions to the wearer's surface (eg, by adjusting the pressure force), but this involves considerable additional effort.

These risks due to mechanical contact can be at least partially avoided if the delivery of a volume of liquid from a capillary occurs by breaking the meniscus of a liquid contained therein upon contact between the capillary and the support surface. A corresponding method for producing a microarray of analyte-specific test areas on a solid support is described, for example, in US Pat EP 0 804 731 B1 described. According to this document, it is proposed to press the capillary with the liquid contained therein at a defined location on the solid support with such a force that the meniscus in the capillary channel is broken and a selected volume of the liquid present in the capillary channel (eg a reagent solution). is applied to the solid support. However, this approach disadvantageously requires a very accurate adjustment of the distance between the solid support and the capillary.

Advantageous to the method described above are droplet deposition methods in which droplets of the liquid to be applied travels some free passage through the air and no contact between the support surface and the container (or ends) containing the liquid prior to the deposition step is required. An example of this is in U.S. Patent No. 5,763,170 It is proposed to form an array of different biological molecules on a support surface in a gas environment to form individual droplets of a liquid containing a plurality of different biological molecules, each liquid droplet having on average at least one biological molecule, and wherein the liquid droplets for generating the desired measurement ranges are applied in the form of an array on a solid support under sterile conditions.

A crucial problem of all previously described deposition methods is that while they are suitable, discrete measurement ranges of relatively small diameter (eg, less than 100 microns, minimally currently about 80 microns) with application of individual, relatively small amounts of liquid (for example, in the order of picolitres to Nanoliters volume per measuring range), but all the devices used for this purpose require relatively complex arrangements of guide lines for the liquids to be applied and the required storage volumes of the respective liquids to be applied are relatively large (typically of the order of microliters to milliliters). In the case of the devices and methods known from the prior art, a large number of losses of substances contained in the liquid quantities to be transferred, substances to be subjected to analytical detection as well as system-related distortions of the following analysis results can additionally disadvantageously occur. In the storage vessels and guide lines, substance loss can occur Adsorption come on their surfaces. If different substances contained in a sample are adsorbed to a different degree, it can also lead to a falsification of the concentration ratios of different substances in addition to a falsification of the absolute amounts to be determined. In the case of removal of the samples to be transferred or amounts of reagent from small storage vessels, there may be drying losses of solvent liquid with the disadvantageous result of an incorrect determination of excessive concentrations. In addition, in the case of cells as the sample material to be transferred, typically, lysis of these cells is required, which is also associated with a potential loss of cell material. These disadvantages of the conventional arrangements and methods result in typically that the transfer efficiency to a carrier to produce an array with discrete, separate measurement ranges is less than 1%.

In addition, the conventional devices and methods suffer from a number of other system-related limitations and disadvantages: transfer of undissolved or solid material from a sample material is typically not possible, but only liquids or substances dissolved therein can be transferred. In addition, for the transfer from the stock (to reagent solution or sample material) relatively large stocks (in terms of volumes in the order of microliters to milliliters) necessary so that no information about the different ingredients and their (concentration) ratio, for example, in individual cells can be won. Arrays of discrete, Therefore, separate measurement areas may typically contain only averaged information over a relatively large initial set, for example, across a plurality of cells, and provide no information about the composition of individual cells or of a few cells of existing areas of a cell assemblage (eg, tissue). Thus, the high detection sensitivity of novel analysis devices, as given for example by micro-arrays, in particular on planar thin-film waveguides as carriers, can be exploited in any adequate manner, but it is a large part of the spatially resolved, origin-related information of the material to be examined, which with In principle, the above-mentioned analytical arrangements could be obtained or further processed, lost due to the inadequately large necessary initial quantities for the aforementioned conventional "spotting" methods and "spotting" arrangements.

In addition, the amount of sample transferred by conventional "spotting" methods and "spotting" arrangements is generally difficult to quantify, and the drying process associated with the limitation to liquid samples, after their transfer to discrete measurement areas on a support, can lead to quality variations of the generated measuring ranges, for example, to fluctuations or inhomogeneous distribution of the applied binding partners in the measuring ranges or to different sizes, difficult to control dimensions of the measuring ranges, d. H. Fluctuations in "spot morphology" or "spot diameter".

For the application of samples from a very small storage volume, for example of the order of individual cells or of only very few cells (for example <1000 cells), the previously described known application methods are not suitable for the many reasons mentioned.

From the US 2004/0053326 A1 A laser-assisted microdissection method is known in which regions of a nitrocellulose transfer layer are ablated on selected cells by means of a laser, in order subsequently to enable a transfer of these cells by means of electrophoresis or capillary action.

According to the document WO 00/23182 A2 Laser irradiation is carried out for the purpose of improving the adhesion of individual sample molecules at different measuring ranges with the aid of a binding material, wherein a photoconductive layer is irradiated by means of a laser beam in order to make this layer electrically conductive and then with the aid of the thus closed circuit individual sample molecules in the Accumulate near desired measuring ranges.

In the US 6,139,831 A It is proposed to irradiate a substrate with the aid of an electromagnetic energy beam, wherein a layer is melted by the energy irradiation and then a gas bubble is formed, which enables the transmission of the desired particles when the energy irradiation is switched off.

The publication DE 199 40 752 A1 suggests activating a photoactivatable group on a support surface by exposure to subsequently site-specifically bind biological or chemical materials or building blocks on the respective areas of the support, which may be in the form of a chip.

In the publication JP 2002-125654 A , Patent Abstracts of Japan (2002), the irradiation of a thin layer is proposed with a light beam to transfer by evaporation of this layer individual samples of a gene material disposed in a thin layer above genetic material on a micro-array substrate, which is opposite of a the two-layered transfer substrate is arranged.

In the US 5,143,854 A For example, exposure to activate selected regions of a microarray is disclosed to synthesize polypeptides of desired length and sequence.

From the pamphlets WO 01/71311 A2 . US 4,322,449 A1 and DE 201 21 386 U1 In each case a contactless transfer of reagent solutions to a substrate is known. According to the WO 01/71311 A2 the contactless transport of the reagent solution is carried out by applying an electrical voltage while in the US 4,322,449 A1 the contactless transport of the reagent solution by means of a piezoelectric oscillator takes place. In the DE 201 21 386 U1 For the transport of the reagent solution, a compressed gas is used.

The present invention is therefore based on the object of proposing a method and a device for producing or producing an analysis arrangement having a large number of discrete, separate measurement areas which are suitable for biological, biochemical or chemical analysis of samples to be applied in or on the measurement areas in which, as a basis for a later analysis, samples or reagent solutions can not only be applied in a simple manner to a carrier for the production of individual measuring ranges, but with which, in particular, extremely small quantities of liquid or sample (for example in the US Pat Magnitude of individual cells or cell components) can be applied to or in the measuring ranges.

This object is achieved by a method for generating an analysis device having a plurality of discrete, separate measuring ranges according to claim 1 and an apparatus for producing such an analysis device according to claim 24. The dependent claims each define preferred or advantageous embodiments of the present invention.

The present invention makes it possible to dissolve a specific amount of said reagent solution by targeted, directed irradiation of electromagnetic energy into a storage volume of a reagent solution and to transfer it without contact to a carrier in order to generate discrete measuring ranges with amounts of the transferred reagent solution therein. In this case, light is preferably used as irradiated energy, in particular light from a laser light source.

The invention makes it possible to obtain very high densities of discrete, separate measurement areas on a support since the size of a measurement area is not necessarily determined by the interactions of a liquid with the surface of the support (eg due to wetting of the surface by the liquid ). This applies in particular to the inventive transfer of undissolved or solid material from samples from a sample material. Especially in this case, according to the present invention, much smaller amounts can be transferred than with the conventional methods and devices. Therefore, it is possible with the aid of the present invention to generate measuring ranges on a carrier with more than 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ measuring ranges per square centimeter.

• a) Bereitstellen eines Trägers,
• b) Bereitstellen eines Vorrats einer Reagenzlösung mit darin enthaltenen, auf dem Träger zu immobilisierenden Verbindungen als biologischen, biochemischen oder synthetischen Erkennungselementen für die in besagten Proben nachzuweisenden Analyten, und
• c) Übertragung mindestens einer bestimmten Menge der Reagenzlösung auf den Träger zur Erzeugung mindestens eines der Vielzahl von diskreten, separaten Messbereichen oder auf einen vorher ausgewiesenen Bereich des Trägers für einen diskreten, separaten Messbereich durch Bestrahlen des Vorrats der Reagenzlösung mit einem elektromagnetischen Energiestrahl, insbesondere einem Laserstrahl.

In order to produce an array of discrete measurement areas with biological, biochemical or synthetic recognition elements to be immobilized in the measurement areas, the object is achieved by a method for generating an analysis arrangement having a multiplicity of discrete, separate measurement areas which are used to detect one or more analytes in a biological , biochemical or chemical detection method in samples to be brought into contact with the measurement areas, comprising the steps

• a) providing a carrier,
• b) providing a stock of a reagent solution having compounds immobilized thereon as biological, biochemical or synthetic recognition elements for the analytes to be detected in said samples, and
• c) transferring at least a certain amount of the reagent solution to the carrier for generating at least one of the plurality of discrete, separate measurement regions or a previously designated region of the carrier for a discrete, separate measurement region by irradiating the supply of the reagent solution with an electromagnetic energy beam, in particular one Laser beam.

In accordance with the abovementioned method according to the invention for producing analysis arrangements of the type mentioned, a corresponding apparatus for producing such analysis arrangements is also the subject matter of the present invention.

The subject of the present invention is therefore an apparatus for generating an analysis arrangement with a multiplicity of discrete, separate measuring ranges, which is designed to detect one or more analytes in a biological, biochemical or chemical detection method in samples to be brought into contact with the measuring ranges
a first arrangement for receiving a liquid reservoir with a reagent solution containing compounds to be immobilized on a carrier as biological, biochemical or synthetic recognition elements for analytes to be detected in samples to be investigated,
a second arrangement for receiving a carrier, on which the plurality of discrete, separate measuring areas is to be generated, and an energy source, in particular a laser light source, for selectively irradiating at least a specific amount of the reagent solution in the liquid supply with an electromagnetic energy beam, in particular a laser beam, thereby transferring the at least a certain amount of the reagent solution from the liquid supply to the carrier for generating at least one of the plurality of discrete measurement regions or to a previously designated region of the carrier for a discrete, separate measurement region.

A device according to the invention of this type is, in a more general form, also suitable for transferring the smallest quantities of liquid from a liquid reservoir of a reagent solution of any kind to a carrier having discrete, separate measurement areas to be generated thereon or to discrete, separate measurement areas already generated on the carrier.

It is advantageous if in addition an arrangement for facilitating the detection and / or selection of the specific amount of a reagent solution, for example in the form of a microscope and / or or provisions are provided for suppressing electrostatic charging of the quantities of liquid to be transferred and / or of the carrier for the measuring ranges.

The present invention additionally comprises further preferred and advantageous embodiments of the method according to the invention and of the device according to the invention for generating analysis arrangements.

According to the invention, it is provided to transfer a specific amount of a reagent solution by means of electromagnetic energy, in particular laser energy, directly from a corresponding supply, for example from a conventional glass slide, a microtiter plate, a Petri dish, etc. onto a support in order to apply a plurality of discrete, create separate measurement ranges for biological, biochemical or chemical analysis in the form of arrays of measurement ranges (also referred to as "micro-arrays").

The carrier of the measuring ranges to be generated can be elastic or rigid. It may be in the form of a membrane, such as a nitrocellulose membrane. For example, a support membrane may be stretched in a frame or fixture or may be applied to an additional rigid support substrate such as a glass plate, such as laminated. The surface of the carrier facing the measuring regions to be generated can be liquid-free or wetted with a liquid, in particular if it is conducive to the adhesion of the substances to be applied in the discrete, separate measuring regions to be produced.

The surface of the carrier facing the measuring areas to be generated may be smooth or porous. In the case of a porous surface, it is preferable that the pore size is within a limited distribution, for example, between 3 nm and 10 nm, 10 nm and 30 nm, 30 nm and 100 nm, 100 nm and 1 μm, 1 μm and 30 μm. Closer limitations on the pore size of a porous material as a carrier are included in this sample listing.

For a variety of applications, it is preferred that the surface of the support be substantially pore-free, i. H. is smooth. In this case, the carrier is preferably substantially planar.

For many applications, in particular for the detection of analytes bound in or to the discrete, separate measuring ranges by means of optical detection methods, it is advantageous if the material of the carrier as a whole or one or more layers of a carrier constructed of several layers contributes to the measuring ranges the wavelength of a excited in the detection step of a biological, biochemical or chemical detection method excitation or measurement light are substantially optically transparent. By "substantially optical transparency" at a particular irradiated wavelength is meant that the intensity of the incident light is attenuated by less than 50% when passing through the relevant material or the relevant layer.

It is preferred that the material of the carrier comprises a material from the group which form, spray or millable plastics, metals, metal oxides, silicates, such as. As glass, quartz or ceramics.

In a particularly preferred embodiment, the carrier as an optical waveguide, particularly preferably as a planar optical thin-film waveguide with a first high-refractive, waveguide at least at one wavelength of an excitation or measurement light to be irradiated substantially optically transparent layer, on, directly or mediated via a Adhesive layer to be discrete and separate measuring areas to be arranged on at least one second layer with a lower refractive index than the former layer.

It is preferred that the refractive index of the high refractive, waveguiding layer is greater than 1.8. In addition, it is preferred that the material of the high-refractive, waveguiding layer include substances from the group comprising TiO ₂ , ZnO, Nb ₂ O ₅ , Ta ₂ O ₅ , HfO ₂ and ZrO ₂ .

The technology of planar waveguides is known in the art. Embodiments of planar waveguides which are suitable as carriers are disclosed, for example, in the international patent applications WO 95/33197 A1 . WO 95/33198 A1 and WO 96/35940 A described, so that reference can be made in this regard to these publications.

The simplest form of immobilization of the biological, biochemical or synthetic recognition elements to be applied to the support in the discrete, separate measurement areas consists in physical adsorption, for example due to hydrophobic interactions between the recognition elements and the support. However, in a subsequent biological, biochemical or chemical detection process, these interactions may be due to the composition of a medium (reagent) contacted with the measurement regions and its physico-chemical properties, such as Polarity and ionic strength, are greatly changed in their extent. In particular, in the case of sequential addition of various reagents in a multi-step assay, the adhesion of the samples or recognition elements to the adsorptive immobilization on the surface is often insufficient. In a preferred embodiment, the adhesion is improved by applying an adhesion-promoting layer to the immobilization of biological, biochemical or synthetic recognition elements on the support. For the preparation of the adhesion promoting layer, a variety of materials are suitable. Without limitation, it is preferred that the primer layer comprise compounds selected from the group consisting of silanes, functionalized silanes, epoxies, functionalized, charged or polar polymers, "self-assembled monolayers or multilayers", thiols, alkyl phosphates and phosphonates, multifunctional block copolymers such as for example, poly (L) lysine / polyethylene glycols.

As applied in the measuring ranges biological, biochemical or synthetic recognition elements, d. H. In particular for the production of so-called "capture" arrays, it is possible, for example, to apply components from the group comprising nucleic acids (DNA, RNA) or nucleic acid analogs (eg PNA), antibodies, aptamers, membrane-bound and isolated receptors, their ligands, Antigens for antibodies, cavities generated by chemical synthesis for incorporation of molecular imprints, chemical binding molecules, recognition sequences for "protein tags", such as "His tags". It can also be provided that entire cells or cell fragments are applied as biological, biochemical or synthetic recognition elements.

Suitable biological, biochemical or chemical detection methods for analytes to be detected in the samples to be investigated are, in particular, biological, biochemical or chemical methods which comprise mass spectrometric or optical detection. The detection of one or more analytes in or on a discrete measuring range can be carried out, for example, by means of a mass spectrometer, in particular a MALDI-TOF device ("matrix-assisted laser desorption ionization time of flight"), or by means of arrangements for measuring one or more Luminescences or fluorescence, in particular in the evanescent field of a waveguide, as well as measurements of the effective refractive index (eg, grating coupler, Oberflächenplasmonen- or interference measurement systems) or changes in these parameters. Such arrangements are known in the art.

According to the invention, the transfer of the specific amounts of a reagent solution takes place without contact and preferably requires only one laser shot, the transfer across phase boundaries, ie. H. out of the supply (sample material or reagent solution) out through air onto the carrier.

The invention thus provides a highly sensitive technology for the transfer of even the smallest amounts, so that correspondingly also the analysis of individual cells or individual cell components on their ingredients, for example nucleic acids such as DNA or RNA, or proteins, is made possible.

The invention will be explained in more detail with reference to the accompanying drawings.

1 1 schematically shows an apparatus for producing an analysis device having a plurality of discrete, separate measurement ranges according to a preferred embodiment of the present invention.

2 shows an exemplary structure of an analysis arrangement, as shown in 1 can be used, and

3A and 3B show examples of transferring individual samples to the analysis assembly according to the present invention.

1 shows an apparatus for producing or generating an analysis assembly having a plurality of discrete, separate measurement areas in or on each of which samples are to be applied for detection of one or more analytes contained therein in a biological, biochemical or chemical detection method. The example shown is a holder 3 with a variety of straps 4 , each containing one or more micro-arrays 5 from the previously described discrete, separate measurement ranges 16 are arranged or to be arranged, in 1 only four such carriers are shown by way of example. This will be explained in more detail below 2 be explained.

As in 2 In the form of a plan view of the analysis arrangement, the analysis arrangement in this exemplary embodiment comprises a frame or holder 3 , wherein according to 2 six side by side arranged carrier or carrier arrangements 4 from the holder 3 being held. On every carrier 4 are six individually addressable micro-arrays 5 with a matrix-like arrangement of discrete and separate measurement areas 16 wherein, for example, up to 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ such measuring ranges 16 can be provided per micro-array, in or on each of which individual samples or biological, biochemical or synthetic recognition elements to be immobilized are to be applied for a subsequent biological, biochemical or chemical analysis. The number of measuring ranges that can be generated in an array, in particular per unit area, on the carrier is essentially determined by the diameter of the laser beam used for the transfer and by the positioning resolution and positioning accuracy of the adjusting devices 7 and 8th , At the carrier 4 it may preferably be a solid support, more preferably a solid support having a planar, smooth or porous surface. In a special, particularly preferred embodiment, it is a planar waveguide, in particular a planar thin-film waveguide with a first high-refractive, at least at one wavelength of an excitation or measurement light to be irradiated substantially optically transparent layer on which, directly or mediated via an adhesive layer to be discrete measurement areas on at least one second lower refractive index layer than the former layer. By means of the excitation and detection of luminescence of luminescent molecules or molecular components in the evanescent field of the light guided in a thin-film waveguide for the step of analyte detection, a sensitivity which is, for example, fifty to a hundredfold higher when reading out the signals from the discrete, separate measurement ranges of a microliter. Arrays can be achieved on a planar thin-film waveguide as with conventional carriers, such as glass or microscope slides, when reading the signals using conventional laser scanner.

The luminescent molecules or molecular components used for the purpose of analyte detection can be bound, for example, as labels to the analytes or analyte analogues (molecules of the same type) in a competitive assay or to one of the binding partners of the analytes in a multi-step assay. In the case of nucleic acid hybridization assays, for example, it may also be so-called intercalators, which are incorporated in double-stranded nucleic acid strands and there, for example, to an increased luminescence (compared to the radiation behavior in homogeneous solution) can be excited.

The application of the samples to be analyzed or the specific amounts of reagent solutions to a carrier 4 for the production of micro-arrays 5 of individual discrete and spaced measuring ranges 16 is called "spotting". In the 1 shown device is designed such that the "spotting" allows a transfer of the corresponding samples or amounts of reagent solutions on the support, to generate the desired measurement ranges, by means of laser energy, wherein the transfer without contact by irradiation of the respective liquid reservoir of reagent solution or the supply on sample material with a laser beam. By irradiating the individual objects or samples or specific areas in a reagent solution (generally referred to as regions) with the laser beam, an impulse transmission from the laser beam to the regions irradiated therewith takes place, the exact cause of this momentum transfer not yet being fully clarified , However, it is presumed that the momentum transfer and the resulting acceleration of the object irradiated with the laser beam (or region according to the preceding definition) is due to a plasma formation in the area of the surface of the respective object (or region). By irradiation of the selected regions, which may be individual cells or individual cell constituents or even the smallest droplets of a reagent solution, etc., there is accordingly an acceleration in the direction of the propagation direction of the laser beam, so that a non-contact transfer across phase boundaries to the analysis arrangement 3 is possible.

At the in 1 illustrated embodiment, the samples to be analyzed by a on a support 1 Sample material (hereinafter also referred to as "preparation") 2 to the individual measuring ranges of the analysis arrangement 3 be transferred. At the carrier 1 For example, it may be a conventional glass slide, a membrane support having a membrane clamped on a frame, a microtiter plate, a Petri dish, or the like. The type and design of the carrier 1 is of no significance to the "spotting" method described and suggested herein.

In addition, in 1 indicated that the carrier 1 via an adjusting device 7 with a computer system 11 coupled, which takes over the function of an automatic control for the execution of the "spotting" method and as output means, for example, a commercially available monitor 12 and as input means a graphical user interface in conjunction with a keyboard 13 and a computer mouse 14 includes. For example, the graphical user interface allows a user to select individual desired cells of a cell assemblage (such as a piece of tissue) or cell constituents of individual cells of the preparation 2 for the individual "spotting" operations, for which purpose the area of the carrier 1 with the preparation on it 2 from a microscope with integrated CCD camera 10 is taken, so that the lens (not shown) of the microscope correspondingly enlarged image of the preparation 2 on the monitor 12 is representable. About the adjustment 7 can the carrier 1 with the preparation 2 both two-dimensionally in x- and y-direction as well as vertically in z-direction procedure, so that the entire range of the preparation 2 moved over the lens of the microscope and thus the entire area of the specimen 2 can be traversed, for example, to find the cells or cell components of interest and to select.

The samples or amounts of reagent solution (collectively referred to hereinafter as "objects") selected for each "spotting" operation may then be graphically displayed on the monitor by the user via appropriate user input 12 displayed magnified image and thus selected. It can also be determined in this way by the user, which previously selected samples or specific amounts of a reagent solution on which micro-array 5 the analysis arrangement 3 and in particular for generating which measuring range 16 of the desired micro-array 5 to be transferred. For this purpose, an imaging matrix can be created by the user, which clearly assigns the selected samples or specific amounts of a reagent solution to the individual measurement areas 16 the individual micro-arrays 5 the analysis arrangement 3 defines what follows in more detail on the basis of the representations of 3A and 3B should be clarified.

In the presentation of 3A It is assumed that by the user four objects of the preparation 2 have been selected and selected for the subsequent "spotting" process, in which case the selected objects 15 essentially while retaining the original topography in the preparation 2 on the carrier 4 for generating correspondingly arranged measuring ranges 16 any micro-array 5 the analysis arrangement 3 to be transferred.

At the in 3B On the other hand, it is assumed that during the transfer of the selected objects 15 the original topography in the preparation 2 is no longer maintained, with the objects 15 on the carrier 4 for generating measuring ranges 16 in a common column of a micro-array 5 be transmitted. For that on the basis of on the measuring ranges 16 However, in the case of biological, biochemical or chemical analysis methods to be subsequently carried out on transferred samples, it is necessary to know which sample or object 15 in or on which measuring range 16 of the micro-array 5 located. This information may be retained by the above-described mapping matrix, wherein in the mapping matrix in the 1 computer system is clearly stored, which sample or which object of the preparation 2 in or on which measuring range 16 the individual micro-arrays 5 the analysis arrangement 3 has been transferred. In principle, creating and saving a simple two-dimensional table is sufficient.

A significant advantage of the present invention, however, is that not only a two-dimensional "spotting" is possible, but that individual cells or cell components of the preparation 2 Also, as described above, can be spotted three-dimensional, so that superimposed areas or entire layers of the preparation sequentially removed by laser irradiation and in or on the individual measuring ranges 16 the different micro-arrays 5 the analysis arrangement 3 transmitted, that is spotted, can be. In this case, the user must correspondingly assign the measuring ranges 16 to the selected objects or samples 15 a three-dimensional mapping matrix can be managed.

Of course, it is contrary to the representations of 3A and 3B not necessary for the preparation 2 All of the selected measuring ranges are shared by a common micro-array 5 assigned. Rather, the samples selected for the "spotting" method may be arbitrary in or on the measurement ranges 16 all micro-arrays 5 the analysis arrangement 3 be distributed. In addition, it is also possible that different biological objects 15 of the preparation 2 on one and the same measuring range 16 be transmitted.

The assignment of the selected samples to the desired measuring ranges 16 the individual micro-arrays 5 For example, this can be done by moving from the computer system to the monitor 12 also a digital image of each micro-array 5 with the corresponding measuring ranges 16 the analysis arrangement 3 is displayed, so that the selected samples from the user 15 of the preparation 2 simply the desired measuring ranges 16 the individual micro-arrays 5 can be assigned graphically. Of course, other possibilities of assignment are conceivable.

Once the user has determined which objects or samples are to be applied to the carrier to generate which measuring ranges 16 the individual micro-arrays 5 be spotted by the user, a "spotting" process automatically controlled by the computer system can be started by the computer system automatically the respectively required relative movement between the carrier 1 with the preparation on it 2 and the analysis arrangement 3 with the carriers on it 4 or micro-arrays 5 is caused.

For this purpose - as in 1 is shown - the analysis arrangement 3 from a bracket 6 held, which in turn via a further adjustment 8th computer-assisted in x- and y-direction and preferably in the z-direction can be moved. Of course, may also be on the bracket 6 be waived provided the scope of the analysis 3 itself is designed such that it directly with the adjustment 8th can be coupled.

During the "spotting" process becomes the carrier 1 with the preparation on it 2 over the adjusting device 7 adjusted by the computer system such that the respectively desired object or the respective desired sample 15 opposite the laser beam of a laser light source 9 , For example, a UV nitrogen laser is aligned. The arrangement and orientation of the laser light source 9 opposite the carrier 1 and the carrier 4 is in 1 merely to be understood as an example. Of course, it is with appropriate arrangement and orientation of the laser light source 9 also possible to perform a transfer from bottom to top or in a horizontal or diagonal direction, etc. From the computer system is the adjustment 8th controlled so that the on the carrier 4 intended area for generating the desired measuring range 16 of the desired micro-array 5 also opposite the laser beam of the laser light source 9 and consequently aligned with the sample to be transferred. Subsequently, the computer system automatically becomes the laser light source 9 activated and set a laser shot to the desired sample, whereupon due to the carried out momentum transfer the thus irradiated sample 15 from the preparation 2 solved, on the carrier 4 is transferred and there the desired measuring range 16 generated. Due to the acceleration of the sample caused by the laser irradiation 15 is a high-precision transfer possible. In addition, the resolution of the "spotting" process is limited in principle only by the diameter of the laser beam, wherein the laser beam diameter in the range of a few microns and can be reduced with appropriate focusing of the laser beam to a few 100 nm, and by the positioning resolution and positioning accuracy the adjusting elements 7 and 8th so that high-precision, for example, individual cells or cell components or extremely small cell areas can be spotted.

The procedure described above is for each previously selected sample 15 of the preparation 2 and for each correspondingly assigned measuring range 16 the individual micro-arrays 5 the analysis arrangement 3 repeated, so that finally all selected samples 15 one after the other on the carrier 4 the analysis arrangement 3 Fully automatic and computer-aided by appropriate control of the adjustment 7 . 8th and the laser light source 9 be spotted and there the desired measuring ranges 16 produce.

It is possible to digest the individual samples or cells to be scoffed before transfer (for example in a lysis buffer). However, it is also possible to directly, without prior digestion, to select one or more cells or components thereof in a cell assembly (for example, a piece of tissue) or in a cell culture or individual regions in individual cells, with the described method from their environment to solve and on the carrier 4 transferred to.

The carriers 4 or the measuring ranges formed thereon 16 the micro-arrays 5 are prepared in such a way that immediately a biological, biochemical or chemical analysis of the samples applied thereon 15 is possible. In principle, any analysis method can be used. These may in particular be qualitative and / or quantitative analysis methods for examining the individual samples with respect to their constituents, such as, for example, nucleic acids, such as DNA or RNA, or proteins, the samples being, for example, both living and non-living Cells / cell components can act. In general, the analysis methods can be designed such that the presence or the absolute or relative amount (in comparison of different samples) and / or the identity of one or more particular analytes, such as biopolymers, in particular polynucleotides or polypeptides, determined in each spotted sample can be. Such bioanalytical methods or devices are generally state of the art, so that it is not necessary to discuss them further here.

Preferably, the in 1 to a device for bioanalysis, in which the in 1 illustrated arrangement for computer-aided laser-induced spotting is integrated in the form of a single device.

The laser-induced spotting described above can in principle not only be applied to the spotting of biological samples such as cells as described in detail above, but it is also particularly suitable, for example, from a liquid reservoir, for example a supply of a reagent solution, smallest amounts this reagent solution in or on discrete, separate measuring ranges 16 on a carrier 4 or on previously designated areas of the vehicle 4 for discrete, separate measuring ranges or on previously on the carrier 4 generated discrete measuring ranges by irradiating the appropriate amount of the reagent solution with a laser beam to transfer. In particular, said reagent solution may be an analyte-specific reagent solution with biological, biochemical or synthetic recognition elements contained therein. The method according to the invention therefore makes it possible, from a liquid supply with an analyte-specific reagent solution, ie contained therein biological, biochemical or synthetic recognition elements, smallest amounts of this reagent solution by energy irradiation, in particular by laser irradiation, in or on the measuring ranges 16 the analysis arrangement 3 to transfer micro-arrays with analyte-specific measuring ranges (so-called "capture" arrays) on the analysis device 3 manufacture. For this purpose, instead of the in 1 Sample material shown 2 the corresponding liquid supply with the laser beam of the laser light source 9 irradiated, wherein the respectively irradiated with the laser beam liquid droplets of the reagent solution are transferred. However, the general mode of operation and the general procedure of the "spotting" method remain the same, so that reference can be made to the preceding explanations regarding the transmission of, for example, biological samples. The reagent solutions may in particular be analyte-specific reagent solutions of the type described in the introduction EP 0 804 731 B1 disclosed type act.

Claims (26)

Method for generating an analysis arrangement ( 3 ) with a multiplicity of discrete, separate measuring ranges ( 16 ), which are used to detect at least one analyte in a biological, biochemical or chemical detection method with the measuring ranges ( 16 ) in contact with samples, comprising the steps of a) providing at least one carrier ( 4 b) providing a supply of a reagent solution, and c) transferring at least a certain amount of the reagent solution to the carrier for generating at least one of the plurality of discrete, separate measurement areas (US Pat. 16 ) or to a previously designated area of the carrier ( 4 ) for the at least one discrete, separate measuring range ( 16 ) or on a previously on the support ( 4 ) generated discrete measuring range ( 16 ) by irradiating the stock of the reagent solution with an electromagnetic energy beam. A method according to claim 1, characterized in that the electromagnetic energy beam is a light beam. A method according to claim 1 or claim 2, characterized in that the electromagnetic energy beam is a laser beam. A method according to claim 3, characterized in that for transferring the amount of the reagent solution to the carrier ( 4 ) for generating the at least one measuring range ( 16 ) a laser shot is placed on the sample. Method according to one of the preceding claims, characterized in that a plurality of regions of the carrier ( 4 ) for generating discrete measuring ranges ( 16 ) are selected, and then several quantities of the reagent solution successively by means of the electromagnetic energy beam to the carrier ( 4 ) for generating the corresponding measuring ranges ( 16 ) be transmitted. Method according to claim 5, characterized in that the selection of the areas on the support ( 4 ) for the measuring ranges ( 16 ) and the subsequent transfer of the amounts of reagent solution to the carrier ( 4 ) for generating the corresponding measuring ranges ( 16 ) computer-aided. A method according to claim 5 or claim 6, characterized in that the transfer of the amounts of the reagent solution to the carrier ( 4 ) for generating the corresponding measuring ranges ( 16 ) by causing corresponding relative movements between the reagent solution and the electromagnetic energy beam on the one hand and between the reagent solution and the carrier ( 4 ) on the other hand. Method according to one of the preceding claims, characterized in that the measuring ranges ( 16 ) in the form of at least one micro-array ( 5 ) on the at least one support ( 4 ) be formed. Method according to one of the preceding claims, characterized in that the at least one carrier ( 4 ) is rigid. Method according to one of claims 1-8, characterized in that the at least one carrier ( 4 ) is elastic. Method according to claim 10, characterized in that the at least one carrier ( 4 ) is in the form of a membrane. Method according to one of the preceding claims, characterized in that one of the measuring ranges ( 16 ) facing surface of the at least one carrier ( 4 ) is smooth or porous. Method according to one of the preceding claims, characterized in that the at least one carrier ( 4 ) is substantially planar. Method according to one of the preceding claims, characterized in that the material of the at least one carrier ( 4 ) is substantially optically transparent at the wavelength of an excitation or measurement light irradiated in the detection step of the biological, biochemical or chemical detection method. Method according to one of the preceding claims, characterized in that the material of the at least one carrier ( 4 ) comprises a material from the group comprising molding, sprayable or millable plastics, metals, metal oxides and silicates. Method according to one of the preceding claims, characterized in that the carrier ( 4 ) is configured as a planar optical thin-film waveguide with a first at least at one wavelength of an excitation or measurement light to be irradiated substantially optically transparent layer on at least one second layer having a lower refractive index than the first layer. Method according to one of the preceding claims, characterized in that on the support ( 4 ) an adhesive layer is applied. A method according to claim 17, characterized in that the primer layer comprises at least one compound selected from a group comprising silanes, functionalized silanes, epoxides, functionalized charged or polar polymers, self-assembled functionalized monolayers or multilayers, thiols, alkyl phosphates, alkyl phosphonates and multifunctional ones block copolymers. Method according to one of the preceding claims, characterized in that successively several quantities of the reagent solution by irradiation with the electromagnetic energy beam to the carrier ( 4 ) for generating different measuring ranges ( 16 ), wherein an imaging matrix is created, which records the quantities of the reagent solution on the carrier ( 4 ) for generating which measuring ranges ( 16 ) are to be transferred. A method according to claim 19, characterized in that the imaging matrix is stored for subsequent use. Method according to one of the preceding claims, characterized in that the method is performed automatically computer-assisted. Method according to one of the preceding claims, characterized in that the reagent solution provided in step b) is analyte-specific and on the carrier ( 4 ) contains compounds to be immobilized as biological, biochemical or synthetic recognition elements for the at least one analyte to be detected in the samples. A method according to claim 22, characterized in that the biological, biochemical or synthetic recognition elements are selected from a group comprising nucleic acids, nucleic acid analogues, antibodies, aptamers, membrane-bound and isolated receptors, their ligands, antigens for antibodies, cavities produced by chemical synthesis Recording of molecular imprints, chemical binding molecules and recognition sequences for "protein tags". Device for generating an analysis device ( 3 ) with a multiplicity of discrete, separate measuring ranges ( 16 ), which for detecting at least one analyte in a biological, biochemical or. chemical detection method in with the measuring ranges ( 16 ) to be contacted ( 15 ), comprising a first arrangement ( 1 ) for receiving a liquid reservoir with a reagent solution, a second arrangement ( 6 ) for receiving at least one carrier ( 4 ) with the multiplicity of discrete, separate measuring ranges ( 16 ), an energy source ( 9 for selectively irradiating the reagent solution in the liquid reservoir with an electromagnetic energy beam, and control means ( 11 ) for automatically controlling the irradiation of the reagent solution with the electromagnetic energy beam of the energy source ( 9 ), thereby at least a certain amount of the reagent solution from the liquid reservoir contactlessly on at least one of the plurality of discrete, separate measurement areas ( 16 ) of the carrier ( 4 ) or to a previously designated area of the carrier ( 4 ) for the at least one discrete, separate measuring range ( 16 ) transferred to. Apparatus according to claim 24, characterized in that adjusting means ( 7 . 8th ) for effecting a relative movement between the first arrangement ( 1 ) and the energy source ( 9 ) on the one hand and the first arrangement ( 1 ) and the second arrangement ( 6 ) are provided on the other hand. Device according to claim 24 or claim 25, characterized in that the energy source ( 1 ) is a laser light source for generating a laser beam as an electromagnetic energy beam.

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