DE102004021904A1 - Method and device for generating an analysis arrangement with discrete, separate measurement ranges for biological, biochemical or chemical analysis - Google Patents

Abstract

For producing an analysis arrangement (3) having a multiplicity of discrete, separate measurement areas (16), preferably in the form of a microarray (5) on carriers (4), it is proposed as a "spotting" method which is performed by a carrier (1 ) by the "spotting" to be transmitted samples (15) specifically with the laser beam of a laser light source (9) to irradiate the Prioben (15) with high precision and non - contact on the support (4) of the analysis device (3) for Generation of the desired measurement areas (16) to transfer, where they can be subjected to a subsequent biological, biochemical or chemical analysis. The "spotting" method according to the invention is also suitable for the contactless transfer of very small amounts of an analyte-specific reagent solution into or onto the measuring regions (16) of the analysis device (3).

Description

The The present invention relates to a method and an apparatus for generating an analysis array having a plurality of discrete ones and separate measuring areas, 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 in said measuring ranges previously immobilized biological, biochemical or synthetic recognition elements, in a biological, biochemical or chemical (and optionally also allow physical) detection method. In particular, it concerns the present invention provides such a method in the form of a "spotting" method, with the help of a "spotting" technology, the samples to be analyzed to the individual measuring ranges of the analysis arrangement or for generation the individual measuring ranges applied to the analysis arrangement be, as well as a corresponding device, which are also referred to as a "spotter" can.

in the According to the present invention, discrete, separate measuring ranges defined by two- or three-dimensional regions on a support become the immobilized binding partner for the detection of a or more analytes in one or more samples in a binding or affinity assay, taking. The discrete, separate measuring ranges can, but have to not identified prior to the attachment of the binding partners on said support be, for example in the form of a subdivision of the carrier in corresponding to the geometry of the measuring ranges discrete, separate Areas. Preference is given to the discrete, separate measuring ranges with the step of applying the binding partners to be immobilized on the carrier generated.

To each other complementary Bonding partners are characterized by being between them a certain affinity consists. Bonds between complementary binding partners (such as Analytes and their biological, biochemical or synthetic Recognition elements) reversible or irreversible.

different Such measuring ranges can for example, biological, biochemical or synthetic recognition elements 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 a or more to the respective analytes (which are specific recognized and bound by the corresponding recognition elements be contacted) to be examined samples. One- or two-dimensional arrays of a plurality of such arrays Measurement ranges of this first form are also referred to as "capture" arrays. The in the measuring ranges immobilized biological, biochemical or synthetic recognition elements are preferably in an enriched, purified Form, i. in one compared to their original occurrence in their production medium increased Concentration, before.

The Discrete, separate measuring ranges can also be achieved by applying the samples to be tested for their contents themselves, without or after execution one or more purification steps such as fractionation of the underlying sample material. In this Traps are analytes contained in the applied samples as immobilized View binding partner. The sample material may be whole, for example or disrupted cells, cell components, tissue sections, include other biological materials or chemical materials. In the case of lysates as applied samples become one or two-dimensional Denote arrangements of such measurement areas of this second form as "lysate" arrays, their contained analytes then typically after contacting with for it specific biological, biochemical or synthetic recognition elements in one step of an assay with so-called "inverted assay architecture".

to Generation of arrays of the first and second shapes are a variety of methods and devices known.

To the known application methods include the so-called "ink jet Spotting ", in which a similar to piezoelectric effect as exploited in the operation of an inkjet printer, around the samples on a support to produce the desired Apply measuring 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), which contains the liquids to be deposited on the carrier with on the carrier immobilized compounds and the surface of the support takes place Such mechanical "spotting" processes have several principal disadvantages. In the case of solid substrates with pressure or scratch sensitive surfaces, where a low surface roughness and freedom of the surface of mechanical damage of ent Of crucial importance for the quality and practicability of a detection method to be carried out therewith (such as in optical waveguides, glass or plastic plates or membranes as carriers), there is a high risk of quality degradation. Furthermore, there is the danger of a continuous change in the arrangements brought into contact with the carrier surface (eg capillary tips or pen tips), similar to a wear process, which can lead to a corresponding continuous change in the geometry of the measuring areas ("spots") and the quantities of liquid applied therein. This trend can tentatively be counteracted by continuous adaptation of 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 position on the solid support with such a force that the meniscus in the capillary channel is broken and a selected volume of the liquid located in the capillary channel (eg a reagent solution) on the solid Carrier is applied. However, this approach disadvantageously requires a very accurate adjustment of the distance between the solid support and the capillary.

the previously described methods are advantageous in droplets Application methods in which droplets of the liquid to be applied to cover a certain free way through the air and no contact between the support surface and the liquid required before the deposition step containing container (or its ends) is. An example of this is described in U.S. Patent No. 5,763,170, wherein for training an array of different biological molecules proposed a carrier surface In a gas environment, a single droplet becomes a variety to form liquid containing different biological molecules, each liquid droplet on average has at least one biological molecule, and wherein the liquid droplets for Generation of the desired Measuring ranges in the form of an array on a solid support below sterile conditions are applied.

One crucial problem of all previously described application methods is that while these are appropriate, discrete measurement ranges are relative small diameter (for example, less than 100 microns, minimal currently approx. 80 μm) with the application of individual, relatively small amounts of liquid (for example in the order of magnitude from picoliters to nanoliters volume per measuring range), however, all the devices used for this purpose are relatively complex Arrangements of guide lines for the require applied liquids and the required inventory volumes of each to be applied liquids are relatively large (typically of the order of magnitude from microliters to milliliters). In the from the prior art known devices and methods may additionally disadvantageous a variety from losses to those to be transferred amounts of liquid substances to be subjected to analytical proof as well as systemic distortions the following analysis results occur: In the storage vessels and guide lines There may be a loss of substance due to adsorption on their surfaces. If different, contained in a sample substances in one different dimensions It can also be adsorbed, in addition to a falsification the absolute quantities to be determined are distorted the concentration ratios different substances come. In case of removal of the transferred Samples or reagent quantities from small storage vessels can it to drying losses of solvent liquid with the adverse result of incorrect determination of excessive concentrations come. additionally is typically, in the case of cells as sample material to be transferred Lysis of these cells is required, also with a potential Loss of cell material is connected. These disadvantages of conventional Arrange orders and procedures to that, typically the transmission efficiency on a carrier for generating an array with discrete, separate measurement ranges 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, separate measurement generated using conventional deposition techniques Typically, domains may therefore contain only averaged information over a relatively large initial set, such as across a plurality of cells, and provide no information about the composition of individual cells or of a few cells of existing regions 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.

Besides that is the amount of sample transferred by conventional "spotting" methods and "spotting" arrangements in general difficult to quantify, and by limiting to liquid samples connected drying process, after their transfer into discrete measuring ranges a carrier, can cause quality issues 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, i. Fluctuations in "spot morphology" or "spot diameter".

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

Of the The present invention is therefore based on the object, a method and an apparatus for producing an analysis device with a variety of discrete, separate measurement ranges, which for biological, biochemical or chemical analysis of in or suitable for application on the measuring ranges, which as a basis for a later to be performed Analysis samples or reagent solutions not just in a simple way to a carrier for Generation of individual measuring ranges can be applied, but which in particular also extremely low Liquid or Sample quantities (for example, in the order of individual cells or cell constituents) applied to or in the measurement areas can be.

These The object is achieved by a A method of generating a multiparameter analysis array discrete, separate measuring ranges according to claim 1 or 37 or a device for producing such an analysis device according to claim 41 or 42 solved. The dependent ones claims each define preferred or advantageous embodiments of the present invention.

The present invention enables it, by targeted, directed irradiation of energy, in particular electromagnetic energy, in a stock volume of a reagent solution or in a sample material, a certain amount of said reagent solution or detach a sample from said sample material and transfer it to a carrier without contact, there discrete measuring ranges with amounts of transmitted therein reagent or the transferred To produce samples.

Prefers In the process, light is emitted as radiated energy, in particular light a laser light source used.

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 der entsprechenden Menge der Reagenzlösung mit einem Energiestrahl, insbesondere einem Laserstrahl.

In order to generate an array of discrete measurement areas of the former form (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 with a multiplicity of discrete, separate measurement areas, which are used to detect one or more a plurality of 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 to a previously designated region of the carrier for a discrete, separate measurement region by irradiating the appropriate amount of the reagent solution with an energy beam, in particular one Laser beam.

• a) Bereitstellen eines Trägers,
• b) Bereitstellen eines Probenmaterials und
• c) Übertragung mindestens einer Probe aus dem Probenmaterial 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 der mindestens einen Probe in dem Probenmaterial mit einem Energiestrahl, insbesondere einem Laserstrahl.

In order to produce an array of the second-mentioned form (with samples to be applied 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 is designed to detect one or more analytes in a biological, biochemical or chemical detection methods in samples to be applied in the measurement areas, comprising the steps

• a) providing a carrier,
• b) providing a sample material and
• c) transferring at least one sample of the sample material 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 at least one sample in the sample material with an energy beam; in particular a laser beam.

there it may be advantageous if one of the sample material to be transferred Sample, optionally as part of a pretreatment of the sample material, stained or if, for example, by phase contrast using a Microscope, which forms part of a subsequently described Apparatus for carrying out the method according to the invention This can be advantageous to recognize and / or the selection to be transferred Samples are facilitated.

Corresponding the said method according to the invention for generating analysis arrangements of the type mentioned are also corresponding devices for generating such analysis arrangements Subject of the present invention.

The subject of the present invention for generating arrays of the former type is therefore an apparatus for generating an analysis arrangement having a multiplicity of discrete, separate measurement areas which are in contact with the measurement areas for detecting one or more analytes in a biological, biochemical or chemical detection method comprising samples to be prepared
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 measurement 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 reservoir with an energy beam, in particular a laser beam, thereby to transfer the at least a certain amount of the reagent solution from the liquid reservoir to the carrier to produce at least one of the plurality of discrete ones To transfer measuring ranges or on a previously designated area of the carrier for a discrete, separate measuring range.

A inventive device This type is, in a more general form also suitable, smallest amounts of liquid from a liquid supply a reagent solution of any kind on a carrier with discrete, separate measuring ranges or on already on the carrier to generate generated discrete, separate measuring ranges.

The subject matter of the present invention, for the generation of arrays of the second-mentioned type, is correspondingly a device for generating an analysis arrangement having a multiplicity of discrete, separate measurement areas, which is designed to detect one or more analytes in a biological, biochemical or chemical detection method in samples, which are to be applied in the measuring areas, comprising
a first arrangement for receiving a sample material,
a second arrangement for receiving a carrier on which the plurality of discrete, separate measurement areas is to be generated, and
an energy source, in particular a laser light source, for targeted irradiation of at least one sample of the sample material with an energy beam, in particular a laser beam, thereby the at least one sample of the sample material on the carrier for generating at least one of the plurality of discrete measurement areas or on a previously designated area to transmit the carrier for a discrete, separate measuring range.

It is advantageous if the species according to the invention each additionally an arrangement for He facilitating the detection and / or selection of the specific amount of a reagent solution or the samples of a sample material, for example in the form of a microscope, and / or arrangements for suppressing electrostatic charging of the liquid quantities or samples and / or the carrier for the measuring ranges to be transferred.

The The present invention additionally includes other preferred and advantageous embodiments the inventive method and apparatus for generating analysis arrangements of said Species.

According to the invention, it is provided a sample or a specific amount of a reagent solution by means of Energy, especially laser energy, directly from a corresponding Stock, for example from a conventional glass slide, a microtiter plate, a petri dish, etc. to transfer to a carrier, in order to said carrier Several discrete, separate measurement ranges for a biological, biochemical or chemical analysis in the form of arrays of measuring ranges (also referred to as "micro-arrays").

Of the carrier 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. A membrane as a carrier can for example, be stretched in a frame or a holder or on an additional rigid Carrier substrate, such as a glass slide, applied, for example laminated. The one to be produced Measuring areas facing surface of the carrier can be liquid-free or with a liquid be wetted, especially if this the liability of the in the discrete, separate measuring ranges to be generated Substances conducive is.

The the surface of the surface facing the measuring areas to be generated carrier can be smooth or porous be. In the case of a porous one surface It is preferred that the pore size is within a limited distribution, for example between 3nm and 10nm, 10nm and 30nm, 30nm and 100nm, 100nm and 1μm, 1μm and 30μm. Narrower limits 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 nonporous, d. H. is smooth. In this case, the carrier is preferably substantially planar.

For many Applications, especially for the Detection of bound in or to the discrete, separate measuring ranges Analytes by means of optical detection methods, it is advantageous if the material of the carrier whole or one or more, facing the measuring ranges Layers of a built up of several layers support at the wavelength one in the detection step of a biological, biochemical or chemical detection method irradiated excitation or measuring light are substantially optically transparent. Under "substantially optical transparency" for a given irradiated wavelength is understood that the intensity of the incident light when passing through the relevant material or the relevant Layer is attenuated by less than 50%.

It it is preferred that the material of the carrier is a material from the group includes which moldable, injectable or millable plastics, metals, Metal oxides, silicates, such as. As glass, quartz or ceramics.

In a particularly preferred embodiment is the carrier as an optical waveguide, more preferably as a planar waveguide optical thin-film waveguide with a first high refractive, waveguiding at least at a wavelength a radiated excitation or measuring light substantially optically transparent layer, on, directly or mediated via a Primer layer, the discrete and separate measuring ranges to be arranged on at least a second layer with lower Refractive index designed as 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 planar waveguide is known in the prior art Technology. embodiments planar waveguides which are suitable as carriers are, for example in International Patent Applications WO 95/33197, WO 95/33198 and WO 96/35940, so that in this regard to these documents Reference can be made.

The simplest form of immobilization of the samples or biological, biochemical or synthetic recognition elements to be applied in the discrete, separate measurement areas on the support consists in physical adsorption, for example due to hydrophobic interactions between the recognition elements and the support. However, these interactions may occur in a subsequent biological, biochemical or chemical detection method by the composition of a contacted with the measurement areas medium (reagent) 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 or samples 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.

When to be applied in the measuring ranges biological, biochemical or synthetic recognition elements, i. in particular for the production of so-called "capture" arrays, for example Components from the group are applied, which nucleic acids (DNA, RNA) or nucleic acid analogs (e.g., PNA), antibodies, Aptamers, membrane-bound and isolated receptors, their ligands, Antigens for Antibody, cavities formed by chemical synthesis for uptake of molecular Imprints, chemical binding molecules, recognition sequences for "protein tags", such as "His tags." It is may also be provided that as biological, biochemical or synthetic Detecting elements whole cells or cell fragments are applied.

The with the inventive method a carrier in discrete, separate measuring ranges to be transmitted samples or the appropriate sample material can for example, be selected from the group of healthy or morbid, treated or untreated Cells (e.g., human, animal, bacterial or plant Cells) or their extracts or components, human, animal or plant tissue or its extracts, as well as body fluids, such as blood, serum. Plasma, synovial fluid, tear fluid, Urine, saliva, tissue fluid, Urine, and its components or extracts. To be transferred Cell material may be more open-minded or not more open-minded ("whole") form. The to be transferred Samples or sample materials can also include other biological or chemical materials.

When biological, biochemical or chemical detection methods for in In particular, analytes to be detected for the samples to be tested are suitable biological, biochemical or chemical processes involving a mass spectrometric or optical detection. The proof one or more analytes in or on a discrete measuring range For example, using a mass spectrometer, especially one MALDI-TOF instrument ( "Matrix Assisted Laser Desorption Ionization Time Of Flight "), or by means of arrangements for measuring one or more luminescences or fluorescences, especially in the evanescent field of a waveguide, as well as measurements the effective refractive index (e.g., grating coupler, surface plasmon or interference measurement systems) or changes to this Metrics. such Arrangements are known in the art.

Of the Transfer of the samples or the specific amounts of a reagent solution takes place particularly according to the invention contactless and preferably requires only one laser shot, the transfer across phase boundaries away, i. out of the supply (sample material or reagent solution) Air on the carrier, he follows.

The Invention thus provides a highly sensitive technology for transfer Even the smallest amounts or samples, such as individual cells or cell constituents, available, so that accordingly also the analysis of individual cells or individual cell components on their ingredients, for example nucleic acids such as DNA or RNA, or Proteins, allows becomes.

Furthermore allows the invention transmission whole pattern of samples in different measuring ranges on the Carrier, where a 1: 1 transmission in accordance with the original one Topography just as possible is like a transmission using an imaging matrix in which the original Topography is not or not completely maintained.

There the invention even the selective transfer of individual cell components allows can disturbing Components that you are not interested in analyzing, or Impurities during transfer are minimized. Furthermore The invention also allows the unlocking of individual cells, cell aggregates or Tissue associations.

The invention also makes it possible to subject the sample material to a pretreatment before the transfer and subsequently to a pretreated one To transmit sample on a support for generating a corresponding measuring range by irradiation with the laser beam. This pretreatment may, for example, be an assay which is carried out with the sample material so that the compounds to be selected are then transferred by irradiation with the laser beam onto the carrier with the samples to be selected in order to generate corresponding measuring ranges. For example, such an assay may be the staining of a cell culture. The pretreatment may also include the addition of biological, biochemical or synthetic recognition elements to the sample material. In addition, the pretreatment may include adding a luminescent, preferably fluoresceable, material to the sample material. An array of measurement areas with samples pretreated in this way can be analyzed after transfer of these samples to a support by reading the signals from the discrete, separate measurement areas.

Also allowed the invention also a "three-dimensional Spotting ", ie. the transfer of samples of portions of the sample material which two or three-dimensionally spaced from each other, ie in one Plane (x / y direction) or with each other or one above the other (z-direction) arranged are on the carrier in different discrete and separate measuring ranges by irradiation with a laser beam. In this case, preferably an imaging matrix is used used to make a clear association between the place of origin the samples in the sample material and the arrangement of the measuring ranges on the carrier produce and avoid mixing information. These embodiment the method according to the invention is particularly suitable for the transfer of cells or cell components as a sample material.

The Invention allows on the one hand an "imaging" of the occurrence of Cell components on a cell surface, such as the distribution of surface proteins on a cell surface, through targeted selection of the corresponding regions on a cell and transmission the selected one Regions (parts of cells on the surface) in discrete, separate Measuring ranges on the carrier. On the other hand, the invention allows, by means of said embodiment of the "three-dimensional Spotting "a three-dimensional resolution Analysis of cell constituents, by selecting different areas within a cell or within a cell group (such as a tissue) and their transmission in discrete, separate measuring ranges on the carrier.

The Invention will become more apparent below explained with reference to the attached 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, especially before to act around a solid support with 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. With the aid 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, an example fifty to one hundred times higher sensitivity when reading the signals from the discrete, separate measurement ranges of a micro 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 technology of planar waveguides is known in the art and described, for example, in International Applications WO 95/33197, WO 95/33198 and WO 96/35940, so that it is not necessary to discuss this further here.

there can the luminescent molecules used for the purpose of analyte detection or molecular components, for example as a label to the analytes or analyte-analogous (analyte-like molecules) in one competitive assay or to one of the binding partners of the analytes be bound in a multi-step assay. In the case of nucleic acid hybridization assays they may also be so-called intercalators, for example, which are in double-stranded Store nucleic acid strands and there, for example, to increased luminescence (in comparison 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 about the Lens of the microscope moves 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 automatically controlled by the computer system "spotting" process can be started, automatically by the computer system, 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 has another 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 containing therein act biological, biochemical or synthetic recognition elements. 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" process remain the same, so that reference can be made to the preceding explanations regarding the transfer of biological samples, for example pamphlet EP 0 804 731 B1 disclosed type act.

Claims (45)

Method for generating an analysis arrangement ( 3 ) with a multiplicity of discrete, separate measuring ranges ( 16 ), which is designed to detect at least one analyte in a biological, biochemical or chemical detection method in samples which are in the measuring ranges ( 16 ), comprising the steps of a) providing at least one carrier ( 4 ), b) providing a sample material ( 2 ) and c) transfer of at least one sample ( 15 ) from the sample material ( 2 ) on the carrier ( 4 ) for generating at least one of the plurality of discrete, separate measurement areas ( 16 ) or on a previously designated area of the carrier ( 4 ) for the at least one discrete, separate measuring range ( 16 ) by irradiating the at least one sample ( 15 ) in the sample material ( 2 ) with an energy beam. Method according to claim 1, characterized in that the at least one sample ( 15 ) by the irradiation with the energy beam without contact on the carrier ( 4 ) for generating the at least one measuring range ( 16 ) is transmitted. A method according to claim 1 or claim 2, characterized characterized in that the energy beam is a light beam. Method according to one of the preceding claims, characterized characterized in that the energy beam is a laser beam. Method according to claim 4, characterized in that for transmitting the at least one sample ( 15 ) on 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 samples of the sample material ( 2 ) as well as corresponding areas of the carrier ( 4 ) for generating discrete measuring ranges ( 16 ) and then the selected samples ( 15 ) successively by means of the energy beam on the support ( 4 ) for generating the corresponding measuring ranges ( 16 ) be transmitted. Method according to claim 6, characterized in that the selection of the samples ( 15 ) and the areas on the support ( 4 ) for the measuring ranges ( 16 ) and the subsequent transfer of the selected samples ( 15 ) on the carrier ( 4 ) for generating the corresponding measuring ranges ( 16 ) computer-aided. Method according to claim 6 or claim 7, characterized in that the transfer of the selected samples ( 15 ) on the carrier ( 4 ) for generating the corresponding measuring ranges ( 16 ) by causing corresponding relative movements between the sample material ( 2 ) and the energy beam on the one hand and between the sample material ( 2 ) 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 ) are formed. Method according to one of the preceding claims, characterized in that the measuring ranges ( 16 ) on the at least one support designed for the biological, biochemical or chemical detection method ( 4 ) are 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 1-10, characterized in that the at least one carrier ( 4 ) is elastic. Method according to claim 12, 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 20. A method according to claim 19, characterized in that the adhesion-promoting 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 block copolymers. Method according to one of the preceding claims, characterized in that the sample material ( 2 ) from which the at least one sample ( 15 ) on the at least one carrier ( 4 ) for generating the at least one measuring range ( 16 ) is carried on a support ( 1 ) is arranged. Method according to one of the preceding claims, characterized in that the at least one sample ( 15 ) is selected from a group comprising whole, disrupted, healthy, diseased, treated and untreated cells and their extracts and components, human, animal and plant tissues and their extracts and body fluids and tissue fluids, and their components and extracts and other biological or chemical materials. Method according to one of the preceding claims, characterized in that as the at least one sample ( 15 ) a single cell or a cell constituent or a cell aggregate or tissue composite of the sample material ( 2 ) on the carrier ( 4 ) for generating the corresponding measuring range ( 16 ) is transmitted by irradiation with the energy beam. Method according to one of the preceding claims, characterized characterized in that the sample material before carrying out the Step c) is unlocked. Method according to one of the preceding claims, characterized in that the sample material is subjected to a pretreatment before the implementation of step c), so that in step c) the at least one pretreated sample ( 15 ) on the carrier ( 4 ) for generating a corresponding measuring range ( 16 ) is transmitted by irradiation with the energy beam. A method according to claim 25, characterized in that the pretreatment comprises an assay which is compatible with the sample material ( 2 ) is carried out. A method according to claim 25 or claim 26, characterized in that the pretreatment comprises an addition of biological, biochemical synthetic recognition elements to the sample material ( 2 ). Method according to one of claims 25 to 27, characterized in that the pretreatment, the addition of a luminescent material to the sample material ( 2 ). Method according to one of claims 25 to 28, characterized in that the pretreatment, the addition of a fluorescent material to the sample material ( 2 ). Method according to one of the preceding claims, characterized in that successively several samples ( 15 ) by irradiation with the energy beam on the carrier ( 4 ) for generating different measuring ranges ( 16 ), wherein an imaging matrix is created, which records which biological samples ( 15 ) on the carrier ( 4 ) for generating which measuring ranges ( 16 ) are to be transferred. Method according to claim 30, characterized in that that the mapping matrix for a subsequent use is saved. Method according to one of the preceding claims, characterized in that a plurality of samples ( 15 ) of the sample material ( 2 ) by irradiation with the energy beam on the carrier ( 4 ) for generating different measuring ranges ( 16 ), whereby the samples ( 15 ) of sections of the sample material ( 2 ), which are spaced from each other two or three-dimensionally, on the support ( 4 ) into different measuring ranges ( 16 ) are transmitted by irradiation with the energy beam. Method according to one of the preceding claims, characterized in that the at least one sample ( 15 ) irradiated by irradiation of the energy beam onto a support ( 4 ) into the corresponding measuring range ( 16 ), followed by qualitative and / or quantitative biological, biochemical or chemical analysis. A method according to claim 33, characterized in that the biological, biochemical or chemical analysis comprises an analysis with respect to nucleic acids and / or protein constituents in the at least one sample ( 15 ). A method according to claim 33 or claim 34, characterized in that the biological, biochemical or chemical analysis for determining the identity and / or the absolute or relative amount of the at least one analyte in the at least one sample ( 15 ) is carried out. Method according to one of the preceding claims, characterized characterized in that the method is performed automatically computer-assisted. 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 regions ( 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 appropriate amount of the reagent solution with an energy beam. A method according to claim 37, characterized in that in step b) at least one analyte-specific reagent solution containing therein, on the carrier ( 4 ) is provided as immobilized compounds as biological, biochemical or synthetic recognition elements for the at least one analyte to be detected in the samples in order to obtain an analyte-specific measuring range (c) by the step c). 16 ) on the support ( 4 ) train. Method according to claim 38, characterized in that that the biological, biochemical or synthetic recognition elements are selected from a group comprising nucleic acids, nucleic acid analogues, Antibody, Aptamers, membrane-bound and isolated receptors, their ligands, Antigens for Antibody, cavities formed by chemical synthesis for uptake of molecular Imprints, chemical binding molecules and recognition sequences for "protein tags". Method according to one of claims 37 to 39, characterized in that the method according to one of claims 1 to 36 is configured, wherein instead of the defined in claims 1 to 36 transmission of at least one sample ( 15 ) or in addition thereto, the at least a certain amount of the reagent solution according to claims 1 to 36 is transmitted by the irradiation with the energy beam. Device for generating an analysis device ( 3 ) with a multiplicity of discrete, separate measuring ranges ( 16 ) which is designed to detect at least one analyte in a biological, biochemical or chemical detection method in samples ( 15 ), which in the measuring ranges ( 16 ), comprising a first arrangement ( 1 ) for receiving a sample material ( 2 ), a second arrangement ( 6 ) for receiving at least one carrier ( 4 ), on which the plurality of discrete, separate measurement areas ( 16 ) and an energy source ( 9 ) for the targeted irradiation of at least one sample ( 15 ) of the sample material ( 2 ) with an energy beam, to thereby obtain the at least one sample ( 15 ) from the sample material ( 2 ) on the carrier ( 4 ) for generating at least one of the plurality of discrete measurement areas ( 16 ) or to a previously designated area of the carrier ( 4 ) for the at least one discrete, separate measuring range ( 16 ) transferred to. Device for generating an analysis device ( 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 ) 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 ), and an energy source ( 9 ) for selectively irradiating at least a certain amount of the reagent solution in the liquid reservoir with an energy beam to thereby deliver the at least a certain amount of the reagent solution from the liquid reservoir to at least one of the plurality of discrete, separate measurement regions (US Pat. 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 41 or claim 42, characterized in that control means ( 11 ) for automatically controlling the irradiation with the energy beam of the energy source ( 9 ) are provided. Device according to one of claims 41 to 43, 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 one of claims 41 to 44, characterized in that the energy source ( 1 ) is a laser light source for generating a laser beam as an energy beam. Device according to one of claims 41 to 45, characterized that the device is carrying the method according to any one of claims 1 to 40 is configured.