EP1153282A2

EP1153282A2 - Method and devices for detecting optical properties, especially luminescence reactions and refraction behaviour of molecules which are directly or indirectly bound on a support

Info

Publication number: EP1153282A2
Application number: EP99966859A
Authority: EP
Inventors: Annemarie Poustka; Frank Breitling; Karl-Heinz Gross; Stefan Dübel; Rainer Saffrich
Original assignee: Deutsches Krebsforschungszentrum DKFZ; Europaisches Laboratorium fuer Molekularbiologie EMBL
Current assignee: Deutsches Krebsforschungszentrum DKFZ; Europaisches Laboratorium fuer Molekularbiologie EMBL
Priority date: 1998-12-14
Filing date: 1999-12-14
Publication date: 2001-11-14
Also published as: WO2000036398A8; WO2000036398A2; WO2000036398A9; US20020008871A1; WO2000036398A3

Abstract

According to the method for detecting optical properties, especially luminescence reactions and refraction behaviour of molecules (14) which are directly or indirectly bound on a support (12), electromagnetic waves, especially laser light (26), are radiated onto the support. The light that is emitted or refracted by the molecules is then detected by a detector (28). The detector and the support are moved in relation to each other during and after the radiation of the electromagnetic waves. The support used is especially a transparent support with integrated position markings (20) which can be detected by a detector, especially an optoelectronic scanning system. At least one of the integrated position markings is selected while the support and the detector are being moved in relation to each other or thereafter so that the light that is detected by the detector can be associated with a location on the support. A device for carrying out the method is provided with a detector, especially an optoelectronic scanning system, for detecting the position markings that are integrated in the support.

Description

Methods and devices for the detection of optical properties, in particular of luminescence reactions and refractive behavior, of molecules directly or indirectly bound on a carrier

The invention relates to methods and devices for detecting optical properties, in particular luminescence reactions and refractive behavior, of molecules directly or indirectly bound on a carrier. Here, the term "properties" is understood in the broadest sense and is intended not only to include the properties which are characteristic of certain molecules, such as, for example, their mass spectrogram, but also, for example, the ability to perform a specific reaction at all - namely by the mere presence of them show, so that the invention also relates to methods and devices in which a certain optical reaction is initially only intended to infer the mere presence of a substance - but not its type (the type of substance then being determined, for example, from its position is determined on the carrier).

The examined molecules are part of biological structures bound on the carrier, such as in particular cells, molecules, cell parts, cell extensions or tissues, or they are bound to biological structures immobilized on the carrier. The molecules to be examined are all types of molecules which are particularly relevant in biology, pharmacy and medicine, for example peptides, D-peptides, L-peptides and mixtures thereof, naturally occurring oligonucleotides, their mirror images and mixtures thereof, artificially derivatized oligonucleotides, such as those used to construct aptamers, oligosaccharides and modifications of the molecules mentioned. In particular, modular oligomers that do not occur in nature can have particular pharmacological relevance. In this context, special mention should also be made of non-natural substances that can be produced using chemical combinatorics Ligands of biological molecules can be used, in particular organic compounds, steroid derivatives, etc. Specific binders for a naturally occurring molecule, which modify the activity of this molecule, can be isolated from a large number of such molecules. However, since these binders can then often not be broken down by naturally occurring digestive enzymes, they are particularly suitable for use as a therapeutic agent.

Such methods and devices are known in various forms, but they all have certain disadvantages. For example, the known methods require complex and expensive special devices for their implementation and are comparatively slow in reading out a luminescence signal. In particular, if - as will be explained below for different reasons - many different groups of molecules are arranged on a common carrier and have to be examined individually, a very complex mechanism must be used to approach the individual groups of molecules, which is not only expensive and is prone to failure, but which, even with the highest precision work, always has manufacturing tolerances that are several orders of magnitude higher than the minimum size of the molecular groups sufficient for an investigation. As a result, the number of the maximum number of molecules or groups of molecules to be accommodated on a carrier is limited in the known methods and devices and is approximately in the order of some 10 ⁵ molecule groups. In particular for certain blood serum or DNA analyzes, however, it would be desirable if approximately 10 ⁸ to 10 ⁹ molecules could be applied and examined on a carrier.

Lithographic methods are known for applying the molecules to the respective carriers, in particular on so-called “diagnostic chips”, but - as in the later investigation - the difficult exact assignment of the molecule and the repeatedly positionable carrier position to the number of the maximum molecules that can be applied limited, because it is not enough to arrange a large number of different molecules densely packed on a carrier, but without knowing repeatedly, which molecules are in which position on the carrier. In particular, it is a problem with the known methods and devices to read a very large number of luminescence reactions on a support in an appropriate time and at the same time very precisely. In the dyeing tests which can advantageously be carried out with the methods and devices concerned here, in which a substance to be examined is applied to the support on which different molecules have previously been anchored, conclusions are to be drawn about the substances present in the substance to be examined, such as, for example certain antibodies in a blood serum are drawn from, with which of the molecules anchored on the carrier the substance or its constituents are bound, so that one must know very precisely which molecule is located where on the carrier.

Finally, in the known devices and methods, once the molecules have been applied, they can generally not be detached at all or only with great effort. In particular, if there were a very large number of molecules (a "molecular library") on the support, which would have been associated with a substance to be investigated or a mixture of substances (a second "molecular library", for example a protein mixture) it is often desirable to be able to deliberately detach the binding partners from the second molecule library, that is to say the molecules which have formed bonds with molecules from the first molecule library, from the support and to be able to subject them to a further investigation. This then made possible the parallel identification of binding pairs from the two molecule libraries, the binding partner from the first molecule library being known in each case by its position on the support, while the binding partner from the second molecule library can be identified after the targeted detachment from the support. It would be particularly advantageous if first those carrier-bound molecules could be identified which have bound molecules from the second molecule library at all. A sample carrier for the microscopic examination of a large number of samples by means of fluorescence spectroscopy is known from German Offenlegungsschrift 197 52 085 A1, in which a plurality of separate wells forming recesses are made in one of the side surfaces of a disk-shaped substrate. However, the use of such a sample carrier requires the ordered arrangement of the samples, since the position information cannot be read out simultaneously with the fluorescence signal in the same high resolution. In particular, the known sample carrier does not allow a cell lawn or irregular structures to be detected in a tissue section living on the sample carrier.

From EP 0 198 513 A2 a device for detecting fluorescence or phosphorescence reactions is known, in which a sample holder after excitation of the samples on it to avoid incorrect measurements by detecting unwanted background fluorescence must be rotated in such a way that the examining samples in the detection of the luminescence reactions are in a different location than when the excitation. The simultaneous excitation and detection of luminescence reactions is not possible according to the teaching of the European patent application mentioned.

Proceeding from this, the object of the invention is to provide improved methods and devices for detecting optical properties, in particular luminescence reactions and refractive behavior of molecules bound on a carrier, in particular biological molecules, which are also inexpensive to manufacture or carry out. If possible, the methods and devices should also be able to be carried out particularly quickly or work particularly quickly.

The object is achieved by a method according to claim 1. Advantageous implementation forms are the subject of the dependent claims. The relative movement of carrier and detector is preferably generated by rotating the carrier. In an advantageous further development, the rotation of the carrier can even be used to make certain desired physical changes on the carrier in the manner of a centrifuge. For example, it is possible to allow the biological structures or fluorescent dyes provided on the support to migrate along a direction essentially radial to the axis of rotation and only then to read out the optical properties. Such a movement can also be brought about by applying an electromagnetic field or a hydrodynamic or osmotic pressure instead of a rapid rotation of the carrier in the manner of the substances present on the carrier.

The invention advantageously allows the use of a preferably transparent CD, DVD, MOD or FMD, which is essentially commercially available with regard to the position markings. This has the advantage that position detection systems can be used to detect the position of the carrier, which have already proven themselves in practice to be very reliable and which can also be produced inexpensively. It should be emphasized at this point that the term “integrated position markings” is understood to mean all types of markings which are firmly connected to the carrier and allow a clear determination of the position of the carrier relative to a detector. In the case of the CDs, MODs, DVDs and FMDs mentioned as examples of carriers, these markings are integrated in the respective carrier, some of them not sitting on the surface on which the molecules to be examined are located, but in an underlying layer.

The use of carriers whose position markings can be detected optoelectronically has the further advantage that the laser light which is generally used for reading out the markings can simultaneously be used for examining the molecules, in particular for their luminescence excitation. The method can advantageously be carried out in such a way that the optical properties are measured several times in succession at a specific location on the rotating carrier, in order, for example, to enable statistical evaluation at a low signal strength or to change the optical properties at a specific location over time to grasp on the carrier. Alternatively or additionally, it is also possible to bleach or otherwise deactivate the fluorescent dyes used to color the molecules on the carrier between two measurements of the optical properties at one and the same location on the carrier and then to re-color the molecules, but with a different one Fluorescent dye f.

It is advantageous if an exchange or modification of the molecules, the optical properties of which are to be recorded, can take place at any location on the support and, in particular, can be measured again after the exchange of the molecules or the modification of the molecules.

The process of exchanging or modifying molecules whose optical properties are to be recorded can be repeated as often as desired.

A characteristic profile can thus be created for a specific location of the material applied to the carrier, in particular cells or histological sections.

Here, the optical properties of several different molecules are preferably measured simultaneously at a specific location.

Are the examined molecules according to claim 10 or claim 11 according to the

Detection of optical properties detached from the carrier can be advantageous for

Detachment of the molecules of laser light can be used on the same source that also supplies the laser light used for the investigation of the optical properties. at Using appropriate carriers, a single light source can therefore perform three different tasks: scanning the position markers, stimulating luminescence reactions and detaching molecules. Depending on the type of substances applied to the carrier, the light source can also be used to trigger light-dependent reactions in the chemicals, biological structures or the substances bound to the carrier.

Since the repeated detection of optical properties at precisely defined locations on the carrier is possible according to the invention, various investigations can be carried out in succession, between which surface modifications have taken place, for example by applying bioactive substances, minerals, binding molecules, antibodies, ligands, receptors, molecules of one extracellular matrix, cell adhesion molecules or other biological structures such as membranes, cells, cell parts, viruses or tissues.

The principle of the convocal laser scanning microscope can be used to record the optical properties. The fluorescence signals of individual pixels of an array of molecules are advantageously measured in different focusing levels.

To increase the precision and facilitate the evaluation, an optical imaging system can be introduced between the excitation system and the carrier as well as between the carrier and the detection system. Such an imaging system should include at least one lens system or an array of lens systems, an optical grating, an optical mirror or an array of optical mirrors, optical fibers or an array of optical fibers and / or a gradient index lens system or an array of gradient Index lens systems include.

If selected molecules are automatically detached from the carrier, it can advantageously be provided that the molecules do not, before the detection of further ones necessarily optical properties are reproduced, for example by polymerase chain reaction or biological replication.

The molecules can be detached from the support in various ways, for example via a photolabile linker, via ionization, by electromagnetic radiation, by applying an electrical voltage, by an enzymatic reaction, by changing the ion concentration, by changing the pH. Value or with the help of a catalyst.

The detached molecules can be a component of liposomes, virus particles, retroviruses, bacteriophages, litroids or cells, in particular D-lymphocytes, T-lymphocytes, leukocytes or bacteria, of complexes of nucleic acid and protein, in particular of ribosomal display complexes, of with DNA binding fusion proteins of loaded DNA, of complexes of nucleic acid and other molecules, in particular artificially produced complexes of DNA and a ligand.

In the further investigation, a mass spectrogram of the detached molecules can be generated in particular. A carrier can advantageously be used to which a preferably complete peptide library containing L-amino acids, in particular a 4-mer, 5-mer, 6-mer, 7-mer or 8-mer peptide library, has been applied. Alternatively, a peptide library containing D-amino acids can also be applied to the support. It is also possible to use supports from which a peptide library of L- and D-amino acids, an aptamer library, an oligosaccharide library or an oligonucleotide library is applied. Such libraries can preferably be applied by means of chemical combinatorics. The individual monomers in the oligonucleotide library can be mirror images of the naturally occurring monomers. The method advantageously allows a particularly rapid analysis of the luminescence behavior of the molecules under investigation. In addition, a carrier can be used in which the samples are arranged as desired, since the position information can be read out simultaneously with the luminescence signal in the same high resolution. This makes it possible, for example, to detect cell turf or irregular structures, such as those present in a tissue section, on the examined carrier.

The method can be used particularly advantageously as an alternative for fluorescence-activated cell sorters or line scanners (FACS) in fluorescence-activated cell sorters or line scanners (FACS), which allows working with adherent cells and e.g. can be used in the screening of molecular libraries of bioactive substances. It is also possible to quickly and easily examine epithelia and other exclusively adherently growing cells arranged on the carrier used, so that apoptosis triggers and new malaria agents can be searched for. Adherent cells are particularly important when looking for new drugs. The method according to the invention can also be used for neural

Quantify differentiation processes (the axon growth) that only take place on solid matrices and often require a special coating of the carrier.

If an impulse processor is used, differences in the cell geometry can also be measured. The method thus also allows the repeated detection of each individual cell, while in known FACS the examined cell is lost after the measurement. Thanks to the position information contained in another layer of the carrier, for the first time it is also possible to measure different properties of one and the same cell in succession. Individual cells in tissue sections attached to the carrier used can also be counted after specific staining (for example of immigrated T lymthocytes in biopsies of inflammation spots in autoimmune patients). It is also possible to analyze tumor biopsies, if necessary after repeated staining. With a correspondingly fast rotation of the carrier, centrifugation can be carried out directly for analytical ultracentrifugation, in particular if the carrier is in a sealed compartment during rotation.

In a method for detecting a luminescence reaction of molecules bound on a carrier, wherein electromagnetic waves, in particular laser light, are radiated onto the carrier, it is provided that essentially only the light possibly emitted by the molecules is detected by the detector. This has the advantage over the known methods, which generally work on the principle of the confocal laser scanning microscope, in which a part of the scattered excitation light also reaches the detector, that only the emitted light is actually detected and therefore an essential one improved signal-to-noise ratio is achieved.

It should be emphasized at this point that the term "light" is understood here to mean all types of electromagnetic waves, in particular also waves with wavelengths outside the sensitivity range of the human eye.

In order to ensure, without complex screens or the like, that only the emitted light actually arrives at the detector or is detected by the detector, one can proceed in such a way that the electromagnetic waves are only briefly, in particular pulsed, irradiated onto the carrier and that the detector does not detect any light emitted by the irradiated molecules during the irradiation, the term "detection" being understood here to mean the determination of a measurable quantity, including the generation of corresponding signals which can be used for the respective examination, so that the actual one Sensor of the respective detector can very well get light that does not result from a luminescence reaction, as long as only - for example, by switching off the detection devices downstream of the sensor, in particular thus the signal generation and evaluation during the irradiation of the electromagnetic excitation for luminescence. table waves - it is ensured that this light is not converted into disturbing signals.

It should be emphasized at this point that the short-term excitation and the time-delayed reading of the luminescence reaction in the dark phases of the excitation can improve the signal-to-noise ratio for all types of fluorescence measurements, in particular for fluorescence measurements based on the principle of the confocal laser Scanning microscope, in the detection of chromatographically separated fluorescence-labeled molecules, especially when sequencing DNA, and in the fluorescence-activated cell sorter or cell scanner (FACS). As an alternative or in addition to the pulsed irradiation of the waves, this can be achieved in that the detector and the carrier are moved relative to one another, in particular rotated relative to one another, after the irradiation or during the irradiation of the electromagnetic waves, so that the irradiated with the electromagnetic waves Move molecules in a range that can be detected by the detector.

Finally, it is also possible to use a plurality of light sources and a plurality of detectors, which are alternately switched on and off, so that it is advantageously possible to excite molecules in specific areas on the carrier by irradiation of electromagnetic waves to luminescence and, at the same time or with a time delay, any luminescence reaction from others, to detect previously excited areas of the carrier.

Another approach to improve the signal-to-noise ratio of luminescence signals is based on the topography of a mixed array of individually controllable silicon elevations with light-emitting diodes. The individually controllable light-emitting diodes are arranged in recesses, so that the light emitted by them only stimulates a luminescence reaction in the case of adjacent silicon elevations. Of course, the topography of the mixed array described can also be associated with brief excitation of the luminescence reaction and reading out of the signals in the dark phase become. The short-term excitation of fluorescence molecules also enables a particularly simple assignment of fluorescence signals to individual molecules in a molecule library when these are applied to an array of detectors. The current generated in the dark phases of the excitation due to the luminescence of the molecules can be read out in parallel in the individual photodetectors and thus assigned to the individual members of the molecule library.

Decoupling of excitation and detection is advantageously achieved both by the described pulsed irradiation / pulsed detection and by the movement of the carrier and detector relative to one another and by the alternate operation of several light sources and detectors, which leads to a considerably clearer luminescence signal and the known methods thus leads to an improved signal-to-noise ratio. The latter can be further improved if one or more wavelength filters are connected between the carrier and the detector or detectors, which also allows any stray light from the light used for excitation to be separated from the fluorescent light.

In an advantageous method for detecting optical properties of molecules bound on a rotatable carrier, in particular of molecules bound to biological material, it is provided that electromagnetic waves are radiated onto the carrier, the irradiated molecules are observed with a detector, and selected molecules are automatically detached from the carrier and a second detector for detecting further, not necessarily optical, properties are supplied. This method has the advantage that after a first analysis step (for example a dyeing reaction known per se), in which certain molecules have been bound to molecules already bound to the support and new molecular complexes have been formed, these new molecular complexes are specifically investigated further can be, wherein the second detector can, for example, generate a mass spectrogram of the detached molecular complexes. It can be done in such a way that the selected molecules are detached from the support by irradiation of electromagnetic waves, in particular by irradiation of laser light, which can be implemented relatively simply and inexpensively for a very large number of samples.

In a particularly advantageous manner, a mixed array of individually controllable silicon elevations and light-emitting diodes can be used for the detachment of selected molecules. The individually controllable light-emitting diodes serve to excite neighboring fluorescence-labeled molecules and, by identifying binding events, to preselect which molecules should be detached from the carrier and fed to a second detector. If these molecules are located on individually controllable silicon elevations, they can be detached from the carrier very easily, in particular by applying an electrical voltage.

For example, in the mass spectrometer, the initially unknown binding partner is determined from a second molecule library to a member from the first molecule library that is already known from the position on the support, and also for several members in parallel. The identity of the binding partner from the second molecule library is obtained e.g. by comparing the mass (or tryptic fragments of a bound protein) with the known sequences from an already sequenced genome. Another example is the detachment of a recombinant phage antibody that has bound a known antigen through its surface-expressed Fv antibody. An E. coli bacterium can be infected with this phage antibody and the recombinant antibody can then be produced.

Another example is the sequencing of complex DNA by means of solid-phase-coupled oligos, in which a sequencing reaction takes place "in situ", ie in parallel with millions of different oligos. The oligo originally coupled to the carrier is changed, it is extended using a polymerase and a template until a ddNTP incorporated by the polymerase leads to chain termination. Instead of the originally coupled oligos, a group of somewhat longer oligos is obtained, which are terminated, for example, by a ddC. This group of modified oligos has to be separated from the template (e.g. by heating) and then from the carrier (for example by installing an S = S bridge in the connector to the carrier, which can be split using reducing agendas such as mercaptoethanol). The mass spectrometer then determines the masses of the individual members of this group of ddC-terminated oligos and, by comparison with the expected masses, the sequence of the extension.

A carrier which is advantageously to be used according to the invention has integrated position markings which are as close as possible and can be detected by a detector, so that any inaccuracies in the respective travel mechanism no longer have to lead to incorrect test results, since the position markers integrated in the carrier control the actual position of the carrier relative to a detector allow. It is particularly advantageous to use an essentially commercially available compact disc (CD), a digital versatile disc (DVD), magneto-optical disc (MOD) or, in particular, a fluorescence multilayer disc (FMD), which already have close-meshed internal position markings, which allows a position determination to the micrometer, the methods for reading and checking the markings on these known carriers have already proven themselves. Due to the similarity of CD, DVD, MOD and FMD with regard to the properties required here, for the sake of simplicity, only the CD is mentioned below, which should always include DVD, MOD and FMD.

The CDs and readers that can be used in the method according to the invention are, moreover, significantly cheaper than the known diagnostic chips.

It is particularly advantageous to use a translucent support in the methods described above. This allows a laser light used in a manner known per se for scanning the position markings to be simultaneously used Use excitation of luminescence reactions. Alternatively, a CD with a wavelength filter integrated in the CD can be used, whereby a laser with known technology can be used for the exact, close-meshed position determination, the light of which does not reach the molecules applied or applied on the other side of the CD. Another laser can then be used for the synthesis or for reading out the luminescence reaction, the light of which can penetrate the wavelength filter mentioned. This second laser is fixed to the first laser, so that when the position of the first laser is determined, the positioning of the second laser beam on the CD is also automatically known.

The flat screens used in computers also have integrated position markings in such a way that the individual LED / LCD pixels are assigned very precisely to specific positions and can be controlled individually, so that a transparent support for biological materials attached in close proximity above the light-emitting diodes / liquid crystals at precisely defined points can be illuminated. This applies all the more if the liquid crystals used up to now are replaced by an array of miniaturized lasers or light-emitting diodes packed as densely as possible.

A particularly advantageous solution is based on the topography of a mixed array of individually controllable silicon elevations with the molecules with light-emitting diodes applied to them. The individually controllable light-emitting diodes are arranged in recesses, so that the light emitted by them only stimulates a luminescence reaction in the case of adjacent silicon elevations. A repeatable, exact assignment of excitation light and applied molecules is also possible with this.

The use of an array of microlasers or light-emitting diodes or the use of the said mixed arrays offers the possibility of carrying the biological materials over a whole cycle - starting from the preferably lithographic or voltage-mediated synthesis of the molecular library, via the Color the library with a second one Molecular library up to the reading of the binding events - to keep fixed over the array. This ensures a very simple, very fast and yet repeatedly extremely precise control of the individual pixels, the time-consuming control and focusing of the individual pixels being eliminated. The possibility of imaging the array of light-emitting diodes or microlasers using a simple imaging system on the carrier in a reduced form also allows a very high packing density of the molecular libraries applied by lithographic synthesis. In addition, the carrier and array can then be separated, after which the array can be used again.

As detectors e.g. Arrays of photomultipliers, of PIN diodes, of avalanche diodes or a so-called multi-channel plate can be used. The detectors can also be incorporated directly into the arrays of excitation light sources, resulting in mixed arrays of detectors and excitation light sources.

If the molecules to be examined are applied on one side and the position markings on the other side of the compact disc, this allows the number of molecules that can be arranged per unit area on a carrier in a precisely defined and, above all, retrievable position compared to the known ones To increase the process enormously - up to now about 10 ⁵ molecules could be arranged on a carrier of a size comparable to the size of a CD in such a way that their position could be exactly determined. Now 10 ⁸ to 10 ¹⁰ can be arranged on a CD, exactly found and targeted to be examined. The same applies to the use of a type of flat screen, in particular if the previously used liquid crystals are replaced by an array of microlasers or light emitting diodes. In this case, the exact position information results from the relative arrangement of the microlaser to one another, so that precisely defined points on a preferably transparent support of molecular libraries arranged parallel to the flat screen can be illuminated. This makes it possible for the first time to arrange very large molecule libraries on a single, very handy carrier. For example, can a preferably complete peptide library, in particular a 4-, 5-, 6- or 7-mer peptide library, can be applied to the carrier, the carrier being able to be brought into contact with a blood serum to be examined, for example, after the peptide library has been applied. It is also possible to apply a preferably complete oligonucleotide library, in particular a 15-mer oligonucleotide library, to the carrier after the application of the oligonucleotide library, for example, to be brought into contact with DNA to be examined, in particular fluorescence-labeled and amplified with the aid of random oligonucleotide primers. This makes it possible for the first time to test a blood serum or DNA sample for a large number of infectious, autoimmune or inherited diseases in a single staining reaction, the detection of any serum antibodies bound to molecules of the peptide library in a manner known per se by fluorescence-labeled Anti-human antibodies can be made.

The invention thus creates completely new diagnostic possibilities and in particular also increases the chances of finding diagnostic markers and therapeutic agents. If, for example, complete 6-mer peptide libraries are associated with a larger number of patient sera and, for example, a staining reaction is used to examine which peptides have contained constituents of the sera, correlations between the disease and the colored peptides will result. This is due to the fact that every person carries in their blood serum a very complex individual pattern of antibody reactivities, which in particular reflects the confrontation of their immune system with acute, chronic, hidden or already survived diseases. A large part of the antibody reactivities can be defined by the specific binding to penta- or hexapeptides, whereby the above-mentioned individual pattern of antibody reactivities can be determined in an unprecedented complexity when analyzing the binding reactivities to a complete penta- or hexapeptide library. Longer binding motifs, in particular those that also occur frequently and have a helical structure, can be determined by libraries in which not every amino acid is random, but only those at certain positions derived from the structure. This enables new diagnostic markers and previously unknown correlations between illness and specific antibody reactivities to be found, including e.g. markers for tumor diseases, for cardiovascular diseases such as heart attacks, for multiple sclerosis and Parkinson's, for all types of autoimmune diseases and allergies and for all types of infectious diseases.

The resulting pattern of markers can make it possible to make a diagnostic statement even by correlating them to certain clinical pictures. The newly found markers can also be applied separately to carriers and used in future investigations.

In an analogous manner, attempts can also be made to correlate diseases with binding patterns to other molecular libraries, such as D-peptide libraries or oligosaccharide libraries. This method is not limited to human diseases, but is suitable for the investigation of forensic and veterinary questions as well as for the analysis of other liquids, from plant extracts to extracts from microorganisms.

In another application, potentially therapeutically interesting molecules, such as D-peptides that cannot be broken down by human digestive enzymes are arranged on a support and then brought into contact with medically relevant molecules, in particular with pathogen-specific proteins or with mixtures of pathogen-specific proteins. This enables the targeted and quick search for binding partners to these medically relevant molecules. Analogously, the search for enzyme ligands, enzyme substrate analogs or enzyme inhibitors is also possible.

The binding to the medically relevant molecules can then be detected, for example by means of biotinylation or fluorescence labeling, so that the D-peptides or aptamers can be identified that bind at least parts of the pathogen. These D-peptides or aptamers can then be tested successively to determine whether they inhibit the pathogen. If, for example, an enzyme of the pathogen (for example protease of HIV, reverse transcriptase etc.) is present in a suitable amount, this enzyme can be fluorescence-labeled (either directly or by the recombinant expression of a small peptide tag, which is carried out using a monoclonal Antibody can be stained). It can be used to determine which D-peptides the enzyme has bound to. A further staining reaction is then carried out, which is caused by the enzyme activity. For example, the cleavage by the HIV protease precipitates a fluorescent peptide that can be detected. This gives D-peptides that not only bind the enzyme, but also inhibit it at the same time.

In order to demonstrate to which of the molecules bound to the carrier have attached molecules, the blood serum or the DNA can, in particular after or before the carrier has been brought into contact with the blood serum or the DNA with one that reacts with the blood serum or the DNA Bindings incoming material are brought into contact. The substance reacting with the blood serum or the DNA is expediently colored with a substance which can be excited to luminescence, in particular a dye which can be excited to fluorescence by irradiation of laser light, before being brought into contact with the serum or the DNA. Such dyes are e.g. commercially available under the names "Cy3", "Cy5", "FITC" or "TRITC", whereby a whole range of conjugates of these fluorescent dyes is advantageously already available (e.g. goat-anti-human-antibody-conjugated Cy5).

If the blood serum to be examined is brought into contact with a detection reagent specific for type E immunoglobulins, it may be possible to determine existing allergies in the patient, since type E immunoglobulins are responsible for allergic reactions such as asthma or hay fever. Non-allergy sufferers have practically no IgEs in their blood serum, allergy sufferers have different ones Quantities that reveal different allergens. Finally, the invention enables the search for specific binding partners for a target molecule from a library of 10 ⁸ to 10 ¹⁰ different molecules and thus - by the simultaneous identification of many (differently binding) binding partners - according to the structural parameters responsible for the binding of the ligand to the target molecule . This greatly simplifies the path to lead structures. For example, signal patterns obtained with the abovementioned methods can be automatically correlated with structural parameters or structural models of the identified ligands from the library used.

In order to arrange a peptide library on a support, in particular on a support that can be used in one of the processes just described, a surface of the support can first be coated with a plastic layer that contains free amino groups for the solid phase synthesis of a peptide or oligonucleotide library (eg the derivatized polystyrene " CovaLink "from Nunc), are coated (for the introduction of primary amino groups into polystyrene, see FIG. 15). After inserting a suitable spacer, the free amino groups are then blocked with a protective group that can be removed by light. An example of many for an activated, i.e. protective group reactive with amino groups which can be removed from light is nitroveratryloxycarbonyl (NVOC).

Protective groups in specific areas of the carrier are then created using a

Lasers cleaved, whereupon an activated amino acid, whose own amino group is blocked by a protective group which can be removed by light, is applied to the support and distributed there, so that the amino acid can couple to the free amino groups. Thereafter, the process steps "cleaving protective groups in certain areas of the support" and "supplying an activated amino acid whose own amino group is blocked by the light-cleavable protective group" are repeated for different, preferably every 20 amino acids, and finally the protective groups are removed from all cleaved synthesized peptides. Alternatively, another lithographic method for applying a peptide library can be used. For example, the sophisticated conventional standard syntheses (eg use of fMoc protective groups in peptide synthesis) can be combined with the inclusion of the activated monomers in larger, photo- or electrolabile particles. The radiated electromagnetic waves or an applied voltage then do not split off the photo- or electrolabile protective group on the growing oligomer, but only release the normal activated monomers as used for the standard syntheses. The activated monomers are preferably dissolved at a higher temperature in a solvent, the melting point or transition of which to a gel-like state is close to 20 ° C., whereupon a laser-absorbing dye is added, the mixture is cooled and ground to small solid particles at low temperature which are atomized over the support for the lithographically synthesized molecular libraries. The activated monomers are only released from these particles locally where a laser heats the particles due to the absorption of the included dye. As a result, the particles liquefy or gel and the activated monomers can couple to the free amino groups in solution (or to free hydroxyl groups as in oligonucleotide synthesis). The liquid solidifies right next to the heated place.

The heating of the solid particles takes place particularly advantageously with the aid of a repeatedly briefly illuminating light source, in particular a laser or a light-emitting diode, as a result of which particularly sharply delimited transitions between solid particles and substances released locally from the particles occur.

As an alternative to melting particles, it is also possible to use a cage inclusion, in particular in fullerenes, or another method of release which can be triggered, for example, by electromagnetic waves or an electrical voltage. Furthermore, as an alternative, selected areas can also be heated by the repeated application of a voltage in rapid succession, in particular if a mixed array of individually controllable silicon elevations with light-emitting diodes is used. Instead of activated monomers, combinations of monomers can also be coupled, for example all 20 × 20 possible activated dimers of L-amino acids.

In the next step, the uncoupled monomers, the undigested particles and / or the solidified particles are either simply blown away, or dissolved and washed away, again solid particles, which contain activated monomers, are applied to the support in selected areas, and this step is preferably carried out all available different activated monomers are carried out one after the other, then the standard protective groups newly introduced due to the couplings are split off using known techniques and the next cycle begins until, for example a complete peptide library was synthesized. Additional modifications of a different kind by means of other chemical reactions are also possible, in particular biologically relevant modifications such as glycosylations, phosphorylations, or alkylations.

Another option when using a mixed array of individually controllable silicon elevations with light-emitting diodes is to control the combinatorial molecular synthesis by applying a voltage in selected areas. Appropriately charged activated monomers for molecular synthesis in selected areas are thereby either repelled or attracted.

The procedure for applying an oligonucleotide library to one of the abovementioned supports can be carried out analogously to the synthesis of the peptide library, with the difference that instead of the 20 different activated amino acids, only 4 different activated nucleotides have to be used. Four different 3'-O-phosphoramidite-activated deoxynucleosides are suitably used for this purpose, which carry a photolabile protective group at the 5 'end or at the 3' end or which, as described for the synthesis of a peptide library, are atomized in particles over the carrier and then made mobile by the radiation of electromagnetic waves. The free hydroxyl groups usually used for oligonucleotide synthesis can be introduced simultaneously with the insertion of a suitable spacer.

Analogous methods can also be used for the production of aptamer libraries, i.e. for oligomers based on ribonucleotides and their derivatives. In addition, reactive molecules of other types, such as those e.g. are used in combinatorial chemistry, are included in the activatable particles and are released site-specifically. Thus, in addition to nucleotides or amino acids, a variety of other groups can also be used as monomeric combinable building blocks for oligomer synthesis.

Alternatively, a molecular library can also be applied to the support by one of numerous printing or spot methods, such as by means of a nozzle, similar to that used in an inkjet printer. Subsequently, selected areas of the support can be irradiated with laser light or with the aid of light-emitting diodes in such a way that the applied molecules are anchored in these areas on the support. This is particularly advantageous because it later excites and reads out a luminescence signal in exactly the same area in which the applied molecules were previously anchored due to the activity of the excitation laser.

To achieve this, e.g. B. prior to the application of the molecules to be anchored, a solid substance at the respective ambient temperatures can be placed on the support, which is then melted to anchor the molecules. The procedure can be such that the surface of a support is activated (for example by uniform chemical coupling of streptavidin or of biotin on the entire support) is then at 40 ° C a solid at 10 ° C (possibly - if hydrophilic molecules are to be coupled - containing hydrophobic) dye molecules solvent is applied to the support and allowed to solidify there, whereupon those to be coupled to the support Molecules are applied, selected areas of the carrier are heated by means of lasers, light-emitting diodes or voltage, as a result of which the solvent liquefies or locally volatilizes and the attached molecule to be coupled can bind to the carrier. The binding takes place preferably via biotin streptavidin. Then unbound molecules are washed away and the cycle can begin with a new spot or print process until a tightly packed and complex library of molecules is finally applied to the support.

However, the application or synthesis of a molecular library on a support is not restricted to the described methods; Examples of other methods are:

- the spotting of micro-quantities of molecules with the aid of a principle comparable to that of a fountain pen, in particular gene sequences reproduced by PCR products;

the spotting of micro-amounts with the help of a kind of screen printing process, in particular activated monomers for oligonucleotide synthesis, the synthesis of peptides or the synthesis of PNAs;

the spotting of micro-amounts with the help of a kind of ink jet printer, in particular activated monomers for oligonucleotide synthesis, the synthesis of peptides or the synthesis of PNAs; the synthesis of PNAs, whereby at any point in the polymerization cycles or after the molecules have been printed on, other compounds can also be added or the already coupled molecules can be modified.

A device suitable for solving the above-mentioned object is given in claim 10. A carrier used to carry out the methods according to the invention is the subject of claims 12 to 15.

A particularly advantageous embodiment is based on the topography of a mixed array of individually controllable silicon elevations with the molecules with light-emitting diodes applied to them. The individually controllable light-emitting diodes are arranged in recesses, so that the light emitted by them only stimulates a luminescence reaction in the case of adjacent silicon elevations. An exact, in particular short-term excitation of selected areas is thus repeatedly detectable. This embodiment even offers the possibility of directly assigning a voltage change resulting from the luminescence signal to each individual silicon elevation, so that in this case the carrier of the molecules is identical to the detector.

Due to the short-term excitation of fluorescence molecules, the targeted luminescence excitation of selected areas can also be dispensed with if the different molecules of a molecule library are applied to an array of detectors. In this case, the excitation can preferably be carried out by a pulsed laser, the light of which detects a larger number of detectors. The current generated in the dark phases of the excitation due to the luminescence of the molecules can then be read out in parallel in the individual photodetectors and thus assigned to the individual members of the molecule library.

In the case of excitation, the spatial decoupling of excitation and detection can also be carried out very simply by means of an array of microlasers or light-emitting diodes a relatively coarse grid of detectors takes place, which detect the luminescence of the molecules excited by the excitation laser scattered in all spatial directions. To do this, only the one detector, which detects the light radiated by the excitation laser, has to be masked out. Of course, the temporal decoupling of excitation and detection can also be combined with the spatial decoupling.

In another embodiment of a device for detecting optical properties of molecules bound on a carrier, in particular biological or biologically relevant molecules, with means for irradiating electromagnetic waves onto the carrier and a detector for observing the irradiated molecules, means for detaching selected molecules are provided, which makes it possible, in particular, to detach molecules from the carrier in a targeted manner if they have been noticed in a first analysis step. The detached molecules can then be subjected to a further investigation, in particular a detector of the second type, e.g. a spectrometer, in particular a mass spectrometer, with which certain properties of the detached molecules can be detected.

In an advantageous development of the device mentioned, the means for detaching can comprise a laser. However, the use of an electrically controllable and chargeable carrier is particularly simple and advantageous for this purpose, the molecules bound to the carrier being able to be detached in selected areas very simply by applying a voltage.

As supports for molecules, in particular biological molecules, in particular for use in one of the methods mentioned, supports according to the invention can be used which have a close-knit network of integrated position markings, so that it is possible to determine the position of a detector, for example by means of conventional mechanics to inspect the area to be examined that has been approached by the wearer. The position markings are preferably designed such that they can be detected by an optoelectronic scanning system. If an essentially commercially available compact disc is used as the carrier, this has, in addition to cost advantages over the known diagnostic chips, the advantage that for reading the carrier for essentially commercially available devices, in particular the reading and reading devices provided in CD players and CD burners Writing laser can be used. If the compact disc is then made translucent, the light from the laser can not only be used for optoelectronic position detection, but also for stimulating luminescence reactions of molecules bound on the CD. Alternatively, CDs or analog media can be used with a wavelength filter integrated in the CD, whereby a laser whose light cannot penetrate the wavelength filter is used for the exact positioning of the CD, while a second laser whose light can penetrate the wavelength filter due to its distance from the first laser can be used for the repeated location-specific synthesis or excitation of a luminescence reaction.

Similar advantages are achieved by using an array of microlasers or light-emitting diodes to excite the luminescence reaction. In this case, the exact position information results from the relative arrangement of the microlaser to one another, so that precisely defined points can be illuminated almost any number of times on a preferably transparent support of molecular libraries arranged in parallel above the array. The carrier can consist of almost any materials, in particular glass derivatized for the synthesis of a molecular library.

In particular in the lithographic synthesis of a molecular library, the carrier of the molecular library can be fixed particularly advantageously above the array when using an array of microlasers or light-emitting diodes. In addition, an array of detectors can also be attached to it, which in particular enables the individual signals obtained to be calibrated with a uniformly fluorescence-labeled carrier. The same applies to the mixed array of individually controllable silicon elevations (with the molecules applied to them) with light-emitting diodes described above. Such mixed arrays can also be fixed to a detector or an array of detectors, if not even the voltage change caused by a luminescence signal is directly assigned to each individual silicon elevation, so that in this case the carrier of the molecules is identical to the detector. This ensures a very simple, yet extremely precise, repeatable control of the individual image points during the lithographic synthesis or the synthesis of the molecular library, which is controlled by applying a voltage, and the subsequent dyeing and readout steps. Furthermore, the time-consuming activation and focusing of the individual is eliminated Pixels. Due to the absence of any moving parts, such an embodiment is particularly robust and easy to use.

Highly complex molecular libraries, e.g. a peptide library, in particular a preferably complete 4-, 5-, 6- or 7-mer peptide library, or an oligonucleotide library, in particular a preferably complete 12-, 13-, 14- or 15-mer oligonucleotide library or aptamer library, are applied such that the position of the individual Molecules or groups of molecules made up of several molecules of one type of molecule can be repeatedly approached precisely and thus can be specifically examined.

For the application of molecules to an essentially flat surface of a carrier, the invention proposes, on the one hand, a device which has means for rotatably holding the carrier about an axis of rotation essentially perpendicular to the surface of the carrier, means for applying various liquids to the surface of the carrier in the region of the axis of rotation and at least one laser that can be moved relative to the carrier for irradiating selected regions of the carrier with laser light. When using an array of microlasers or light-emitting diodes, a suitable, preferably transparent support is placed in the light beam of the light sources and fixed there. In this case, the exact position information results from the relative arrangement of the microlaser or light emitting diodes with respect to one another, so that precisely defined points on the carrier can be illuminated repeatedly. This can be used in particular for the lithographic synthesis and the subsequent reading out of molecular libraries.

If the molecular libraries are applied to the support independently of the activity of the excitation laser (s), then regularly arranged, appropriately detectable "guide dots" in relation to the applied molecular library serve as a reference point for determining the position of the applied different molecules.

Alternatively, a device can also be used in which nozzle-like means for applying the smallest quantities of molecules to be anchored on the carrier, means for moving the means for applying the molecules and the carrier relative to one another, and at least one laser for irradiating selected regions of the carrier with laser light are.

Alternatively, molecules or molecule libraries can also be applied to the support used using various printing processes which are already known, for example using a modified screen printing process. For example, PCR fragments are currently printed on the principle of a fountain pen. Although the latter allows the synthesis of highly complex molecular libraries, the individual spots must be adapted to the readout mechanism in printing processes, which is very time-consuming since each spot has to be approached and generally focused through until the setting that gives the maximum light output is finally selected , which is not necessary when using a CD or an array of microlasers or light-emitting diodes, since the individual pixels are particularly important in the case of an array of microlasers be irradiated with almost parallel light. The same applies if an array of detectors is used as the carrier of the molecules.

Even with a CD or analogue carriers, the problem mentioned is very much alleviated due to the extremely close-knit network of position information, since there is always a pit in the immediate vicinity, relative to which the molecules can be anchored and read out on the carrier.

In particular, if an array of individually controllable silicon elevations or a mixed array with light-emitting diodes is used as the carrier of the molecules, printed molecules can also be applied to the carrier due to a voltage applied in selected areas if the molecules to be applied carry a corresponding charge or with With the help of selectively controllable light sources in selected areas.

The invention thus advantageously allows the person skilled in the art to choose the device which is optimally suitable for applying the respective molecules to the respective supports, in which case he also combines both devices in individual cases and first a part of the molecules with one and then another part of the molecules with the other device can apply to the carrier.

Further details and advantages of the invention result from the purely exemplary and non-restrictive description of some exemplary embodiments in connection with the drawing.

1 illustrates the functional principle of a device for detecting a luminescence reaction of biological molecules 14 bound on the top 10 of a carrier 12, the carrier 12 rotating about an axis 16 perpendicular to the top 10, as indicated by the arrow 18 . The carrier 12 is an essentially commercially available CD, but is designed to be translucent and which is provided on its side opposite the top 10 with depressions, so-called "pits", which are indicated by the lines 20 and which are located under a transparent protective layer.

The pits form internal position markings which can be read by the optoelectronic detection system of a conventional CD player or CD burner, which is only indicated here. As is known, the detection system essentially consists of a laser (not shown here) and - as indicated by the movement arrow 22 - an adjustable or movable focusing coil or lens 24, which allows the beams 26 generated by the laser to be advantageously used both for scanning the pits 20 and thus for position determination as well as - as at the point in time shown in the figure - through the CD for irradiation and eventual excitation of the molecules 14.

The location of the irradiation is followed by a light-sensitive detector 28 in the direction of movement of the CD, to which a lens 30 is assigned, which bundles light beams 32 emitted by possibly excited molecules and directs them onto the detector 28. This arrangement ensures that the light from the laser does not interfere with the detection process through superimposition. In addition, luminescence reactions of excited molecules can be detected while new molecules are already being irradiated, so that the analysis of the molecules arranged on the CD can advantageously be carried out at very high speed.

2 shows the use of an array 40 of microlasers 42, 44 for the excitation of molecules 50, 52 bound on a flat side 46 of a carrier 48. Since the microlasers can be controlled individually and are arranged in a fixed position relative to one another, precisely defined points can be illuminated on the support 48, which is attached parallel to the array 40 in the example shown, and, if necessary, molecules located there can be excited to luminescence. At the time shown, the microlaser 42 is inactive, while the microlaser 44 is active and the light is on the molecular group 50 irradiates. It should be emphasized at this point that the carrier 48 is transparent in this exemplary embodiment, but that the carrier does not necessarily have to be transparent, since the exciting light source and detector can also be arranged on one and the same side of the carrier, namely on the side on which the molecules to be examined are located.

In this exemplary embodiment, an array 54 comprising a number of detectors 56, 58 is provided to detect any luminescence reactions. This allows a particularly simple separation of excitation and detection by "switching off" the detector (s) 58 located in the direct radiation range of an active laser 44, ie, an excitation of the molecules, at least during the emitting of the laser, e.g. in such a way that any radiation incident on the detector or detectors is no longer detected or signals from the respective detectors are not passed on or evaluated. The other detectors 56 can also be switched off, preferably with pulsed radiation, for the duration of the excitation phase, but this is not absolutely necessary. In particular, it is possible to arrange a wavelength filter 60 between the array 54 of detectors and the carrier 48, which filters out radiation 62 with the wavelength of the excitation laser or at least weakly attenuates it (as indicated by the rays 62 '), but allows luminescence radiation 64 to pass through , so that it can be detected by the active detectors 56 and converted into corresponding signals, which are then sent to a signal evaluation device known per se and therefore not described here, for example a computer that can be forwarded.

A carrier shown in Fig. 2 can, for example, if a very precise resolution of the individual pixels on the carrier is required for the synthesis and reading out of a highly complex molecule library, even before (e.g. ithographically synthesizing) a molecular library with the (but not insoluble) Array of microlasers are connected and this during the synthesis and staining with a second substance (for example the blood serum of patients) until the readout Luminescence response also remain. The carrier can then be disposed of and a new carrier firmly connected to the array of microlasers. In addition, an array of detectors can also be permanently connected to the array of microlasers, as shown in FIG. 10.

Alternatively, so-called "guide dots" can be inserted on the carrier at regular intervals, each of which gives a defined luminescence signal and thus takes over the function of the pits on the CD, i.e. submit an internal grid that serves for position information.

The decoupling of excitation and detection is even easier in these examples than in the example using a CD as a carrier, since one microlaser can be activated after the other. For example, a coarser grid of photodiodes can be built up over the array, the diode being switched off at the maximum intensity of the currently active excitation laser.

If the power of the laser (s) can be controlled, the laser (s) can also be used to detach selected molecules from the carrier, the detached molecules then being suitably sent to a further examination device, e.g. a mass spectrometer can be supplied.

8 once again shows the basic principle of the presented examination method by way of example: selected regions 7 of a carrier 12 with molecules or aggregates 4 coupled to these molecules or with molecules 8 that with the coupled molecules 2 or with the aggregates to these molecules 4 interact, are irradiated with electromagnetic waves 6.

FIGS. 9 and 10 schematically show how the physical environment, in particular the matrix layer 3 of substances 5 in locally narrowly limited areas 7 by the radiation of electromagnetic waves 13 (FIG. 9) or by the Applying a voltage 13 '(FIG. 10) can be changed so that the previously immobilized substances 9 are made movable (movable substances are indicated by 11) and can get close to the carrier 12. There they can couple to molecules 2 on the carrier (form bonds), form an aggregate or become part of another chemical reaction. 10 also shows that the carrier 12 can be embedded in a mixing array 17 comprising detectors 56 and sources of electromagnetic radiation, for example light-emitting diodes 19, which can be controlled in a targeted manner. Selected areas 7 of the carrier 12 are expediently separated from one another by a non-conductive insulator 21, which is also impermeable to the incident electromagnetic waves.

As illustrated in FIG. 11, selected areas of a carrier fixed above the array can be repeatedly irradiated with pinpoint accuracy using an array of individually controllable microlasers. As a result, molecules from a molecular library can be applied to selected areas of the support using lithographic methods. After the molecular library has been colored with a fluorescent dye, the fluorescent molecules are stimulated in successively selected areas with a brief pulse of light. The radiation generated between the excitation pulses by the fluorescence of the molecules is collected by the photodetectors and can be assigned to individual members of the molecule library, so that it can be determined exactly which molecules have caused the fluorescence reaction.

In the case of a suitably designed mixing array 17 consisting of individually controllable light sources 19 and detectors 56, the detectors 56 themselves can - as shown in FIG. 12 - be carriers 12 of different molecules 2, 4 and 8. Such a mixing array 17 can e.g. to be used in selected areas

7 trigger a combinatorial molecule synthesis, briefly excite selected areas 7 with electromagnetic waves 6 and directly measure the luminescent reactions that may arise with such an excitation. As detectors 56 In particular, silicon carriers, light-emitting diodes or an independent array of detectors can be used. A non-conductive insulator 21, which is impermeable to the incident electromagnetic waves, is provided to separate the regions 7 from one another.

13 and 14 schematically show how an array 23 of individually controllable detectors 27 (FIG. 13), which can be carriers 12 of different molecules 2, 4 and 8, or an array of individually controllable light-emitting diodes 29 (Fig. 14), which can also be used as photodetectors, can be used to trigger combinatorial molecule synthesis in selected areas 7, to briefly excite larger areas 25 or selected areas 7 with electromagnetic waves 6 and those which may arise with such an excitation Measure luminescence reactions directly in the dark phases of the excitation. Silicon carriers in particular can be used as detectors 27 (FIG. 13). A non-conductive insulator 21, which is impermeable to the incident electromagnetic waves, is again provided to separate the regions 7 from one another.

Some examples of the implementation of methods according to the invention or the use of devices according to the invention are described below:

a) Synthesis of a complete 6mer peptide library on a CD under anhydrous conditions

The surface of a CD is covered with a plastic layer that contains free amino groups for solid-phase synthesis. After coupling a suitable spacer of 2-3 amino acids in length using standard fMoc peptide synthesis, free amino acids are then blocked with a protective group that can be removed by light. The protective group to be coupled is fed through a hose to the inside of the rotating CD. It can be a slightly modified Synthesis program of a known peptide synthesizer can be used. A program that controls the activity of a laser burns off the protective groups in the areas where the amino acid alanine is to be coupled in the first step. The protective group is split off by means of two-photon activation with the aid of the combustion laser and a second laser which irradiates from above and is somewhat less focused. After the protective group has been split off selectively, the activated amino acid alanine is fed to the CD through the tube mentioned above. The activated amino acid couples to the free amino groups, the amino group's own amino group having previously been blocked by the same protective group as mentioned above. This process is repeated for the other 19 amino acids and that

Repeated a total of six times. In the last step, the protective groups are split off from all synthesized peptides.

b) Synthesis of a complete 5-mer peptide library using an array of microlasers

A suitable flat, clear support that contains free amino groups is firmly fixed over the array of microlasers. Particularly suitable are thin glass panes, which with conc. NaOH purified, then washed with water and derivatized for 2 hours at room temperature with 10% (vol / vol) bis (2-hydroxyethyl) aminopropyltriethoxysilane. Alternatively, a suitable flat, transparent support can be coated with a plastic layer which contains free amino groups for the solid phase synthesis.

A suitable spacer of 2-3 amino acids in length is first synthesized on the free amino groups using standard fMoc peptide synthesis familiar to the person skilled in the art under anhydrous conditions. The carrier is then divided parallel to the X axis into 20 separate areas, which are wetted by the 20 different activated fMoc derivatives of the amino acids, each of which is dissolved in DMF.

The activated amino acids are then coupled at room temperature for 30-60 minutes. After washing three times with dimethylformamide (DMF), the fMoc protective group is cleaved off using 20% piperidine in DMF and washed again with DMF.

The beam is again divided into 20 separate areas, this time parallel to the Y axis. These areas are in turn wetted by the 20 different activated fMoc derivatives of the amino acids, each of which is dissolved in DMF, followed by the standard fMoc peptide synthesis described above and familiar to the person skilled in the art.

After the cleavage of the N-terminal protective group as described above, the carrier described above is divided into 400 regions separated from one another at this stage, each with one of 400 possible C-terminally coupled dipeptides to the carrier by a spacer whose N-terminus is free, i.e. is present without a protective group.

For the next coupling steps, the activated amino acids described above are dissolved individually instead of in DMF in a suitable solvent which is liquid at 50 ° C. and solid at 4 ° C., a suitable additive which absorbs the laser light and is inert with respect to the activated amino acids, in particular a dye or Graphite particles, added, the solution frozen and ground into small particles.

These particles are dusted at 4 - 10 ° C on the carrier, which is firmly mounted over an array of microlasers, a cover plate is placed on top and selected areas are irradiated with the microlasers for 20-30 minutes. The absorption of the laser light by the added dye locally heats the solvent frozen at the selected temperatures in the irradiated areas and thus enables the Coupling of the activated amino acids to the free amino groups only in these areas. The cover plate is then removed and the uncoupled amino acids are washed away with DMF, this washing process being repeated twice with heated DMF.

This process is repeated a total of 20 times with all activated amino acids, so that the 400 delimited regions described above are divided into 20 x 400 defined regions, followed by the cleavage of the fMoc protective groups with 20% piperidine in DMF described above.

The peptides are then extended by a 4th and 5th amino acid analogously to the synthesis of the 3rd amino acid, with each area being divided into 20 areas in each synthesis step as described above.

Finally, all protective groups are cleaved with 10% silane in concentrated trifluoroacetic acid, the support is washed with DMF and methanol and dried, so that the end result is a support with 20 ⁵ = 3,200,000 different areas, each one of all possible C-terminal represent coupled pentapeptides.

c) Examination of blood serum using a compact disc with a peptide library fixed on it

The CD is stained with a patient's blood serum by first blocking non-specific bindings with a suitable aqueous solution, such as 2% milk powder in PBS, and diluting the blood serum in the same buffer, after which the surface of the CD is shaken gently for 60 Minutes with the serum. The CD is washed three times. The goat anti-human antibody coupled to the dye Cy5 is diluted in 2% milk powder in PBS; then the surface of the CD is shaken gently for 60 minutes wetted and then washed three times. The compact disc stained with the second antibody is read out in a modified CD player.

d) Reading a colored compact disc using a converted CD burner

A burning laser set to weak wattage scans the CD in the first pass. Any fluorescence signals are detected using the optics additionally built into the CD burner and assigned to the individual CD areas. This additional optic consists of a focusing lens that images one or, if desired, several pits on a photomultiplier or a CCD camera. The lens forms a point in the running direction of the CD outside the maximum of the incident laser. In addition, the laser scattered light is separated from the fluorescence signal by a suitable edge filter. The areas of the CD that gave a signal in the first run are targeted in the subsequent runs and scanned several times. The signals read are added up and assigned to the positive pits.

e) Examination of blood serum using a carrier with a peptide library fixed on it

As described in Example c), the support, which in this example is firmly mounted on an array of microlasers, is blocked with a lithographically synthesized complete pentapeptide library described in Example b) with a suitable aqueous solution, preferably 2% milk powder in PBS, with the diluted one Blood serum from patients was incubated and then stained with a goat anti-human antibody coupled with Cy5. Subsequently, one microlaser after the other is switched on sequentially and the light emitted by any bound fluorescence molecules is read out using a coarser grid of photodiodes over the array of microlasers, the diode being switched off at the maximum intensity of the currently active excitation laser (see FIG. 2 ). The scattered light caused by the excitation laser is additionally separated from the fluorescent light by a suitable wavelength filter.

In order to further improve the signal / noise ratio, the incident microlaser can be pulsed, the fluorescence signal being detected in the dark phases of the incident laser.

The fluorescence signals are divided into a total of 10 different brightness levels, which are assigned to the individual microlasers (i.e. in this case the different pentapeptides).

The microlaser that gave the highest fluorescence signals in a first run can then be used several times in succession for excitation, after which the fluorescence signals obtained are averaged.

f) Synthesis of a complete 12mer oligonucleotide library on a support with the help of an array of microlasers

As described in Example b) for the synthesis of a complete 5-mer peptide library, a suitable flat, transparent support with free amino groups is used.

If not already available through the first step, the free amino groups are added using standard synthesis familiar to the person skilled in the art under anhydrous conditions a suitable linker was synthesized, which in turn anchored free amino groups on the support, but this time around 22 atoms from the surface.

The carrier is then divided into 4 separate areas parallel to the X axis, which are wetted by 4 different activated nucleoside anhydrates with protective groups. The nucleosides then couple to the free amino groups (FIG. 3). Instead of the linker which can be split off in the base, a linker which is stable under these conditions can also be used.

The coupling of the activated nucleosides (with protective groups) to the solid support, the removal of the protective groups and the washing steps take place under the standard conditions known to the person skilled in the art for oligonucleotide synthesis. Examples of the protecting groups used are:

DMTr for the 5 'end of the nucleosides (FIG. 3) benzoyl in the case of the bases adenine and cytosine (FIG. 4) isobutyryl in the case of the base guanine (FIG. 4) - methoxy - or betacyanoethyl - in the case of the phosphate groups (FIG 4)

After the DMTr protective group has been cleaved from the 5 'end of the nucleosides, in the next step the support is again divided into 4 separate areas, this time running parallel to the Y axis. This time, these areas are wetted by the 4 different phosphoramidite derivatives activated with the help of a weak acid such as tetrazole. By coupling the activated phosphoramidites to the free 5'-OH end, a chain extension around a base takes place (FIG. 5).

In the next step, any remaining 5'-OH ends are provided with a "cap" so that they can no longer participate in later reactions (FIG. 6).

A final step in which trivalent phosphate groups are oxidized concludes the synthesis cycle (Fig. 7). The synthesis described above corresponds to the standard of oligonucleotide synthesis familiar to the person skilled in the art. In contrast to the conventional standard synthesis, however, the oligonucleotides are anchored on the solid support in such a way that after the protective groups have been completely cleaved off, they are not cleaved from the support but remain coupled to the support.

The DMTr protective group is then in turn cleaved from the 5'-OH end using TCA, so that there are 16 subdivided regions on the carrier described above at this stage, each with one of 16 possible regions via the 3 'end through a spacer the carrier-coupled dinucleotides whose 5 'end carries a free OH group.

For the next coupling steps, the activated phosphoramidite derivatives described above are dissolved individually instead of in acetonitrile in a suitable solvent which is liquid at 50 ° C. and solid at 4 ° C., a suitable dye which absorbs the laser light and is inert with respect to the activated nucleosides, in particular graphite particles , added, the solution is frozen and ground into small particles.

These particles are dusted at 4 - 10 ° C on the carrier, which is firmly mounted over an array of microlasers, a cover plate is placed over them and selected areas are irradiated with the microlasers for 20-30 minutes. The absorption of the laser light by the added dye locally heats the solvent frozen at the selected temperatures in the irradiated areas and thus enables the coupling of the activated phosphoramidite derivatives to the free 5'-OH ends only in these areas. The cover plate is then removed and the uncoupled phosphoramidite derivatives are washed away with cold acetonitrile. This washing process is then repeated twice with heated acetonitrile. This process is repeated a total of 4x with all activated phosphoramidite derivatives, so that the 16 delimited areas described above are divided into 4 x 16 defined areas, followed by the above-described "capping" of remaining free 5'-OH ends, the oxidation of the trivalent phosphate groups and renewed cleavage of the DMTr protective groups with TCA.

The oligonucleotides are then extended by a further 9 bases analogously to the synthesis of the 3rd base, with each region being subdivided into 4 defined regions in each synthesis step as described above.

Finally, all protective groups are cleaved off with dichloromethane and trichloroacetic acid, the support is washed with acetonitrile and dried, so that the end result is a support with 4 ¹² = 16,777,216 different regions, each of which represents one of all possible 12-mer oligonucleotides coupled via the 3 'end .

g) Examination of patient DNA using an array of microlasers with a 12mer oligonucleotide library fixed on a support

The support described in Example f) with a complete oligonucleotide library fixed thereon is stained with patient DNA. Non-specific bindings are e.g. saturated with DNA from herring sperm.

A tumor tissue sample and at the same time a sample of healthy tissue are taken from the patient and the genomic DNA contained therein is taken using one or more pairs of tumor gene-specific primers (specific, for example, for the genes of p53, pl6, ras, c-myc, n- myc) reproduced in a polymerase chain reaction. FITC-labeled dNTPs are built into the tumor sample, or the normal sample is labeled with biotinylated dNTPs, the samples are mixed and placed on the Hybridized carrier. The hybridized sample is then re-stained with a chemically coupled protein from streptavidin and phycoerythrin, to which the fluorescent dye Cy5 was additionally coupled. As a result, 2 different fluorescences can be measured with one excitation wavelength.

As described in Example e), one microlaser after the other is then switched on sequentially and the light emitted by any bound fluorescence molecules is read out using a coarser grid of photodiodes above the array of microlasers, the diode being switched off at the maximum intensity of the currently active excitation laser is. The scattered light caused by the excitation laser is additionally separated from the fluorescent light by a suitable wavelength filter. The wavelength filter is matched to the fluorescent dye FITC and the tandem dye phycorythrin-Cy5 (PE-Cy5).

The fluorescence signals are then divided into a total of 10 different brightness levels, which are assigned to the individual microlasers (i.e. in this case the different oligonucleotides).

The microlasers, which in a first run gave a strikingly different ratio of the FITC staining to the PE-Cy5 staining from the other pixels, are then used several times in succession for excitation, after which the fluorescence signals obtained are added up and the ratio of the FITC- Staining for PE-Cy5 staining is determined.

In this way, point mutations in genes that are important for the prognosis of tumor diseases can be diagnosed. In contrast to the systems on the market, many genes can be analyzed simultaneously with a complete 12-mer oligonucleotide library. In an alternative approach, the DNA taken from the patient serves as a template for the duplication with so-called Alu primers, which hybridize to the edges of repetitive Alu sequences which occur very frequently in the genome and which reproduce the non-repetitive DNA lying between 2 Aluse sequences. Again, FITC-labeled dNTPs are built into the tumor sample or biotinylated dNTPs into the normal sample, and the samples are hybridized to the carrier in a mixed manner.

The fluorescence signals are then read out as described above. In this way, the entire genome is scanned for differences between normal and tumor tissue, whereby new diagnostic markers can be discovered that provide important information for tumor progression.

h) Combination of the fluorescence detector according to Example d) with a mass spectrometer

The fluorescence detector according to example d) or only the CD burner part without additional fluorescence optics is encased in an evacuable vessel. In the place of the normal sample holder of a mass spectrometer is the focal point of the laser. The laser can drive to any point on the CD and blow away the molecules on it, which can then be examined using the mass spectrometer.

A device combined in this way allows the binding partners that form when two highly complex molecular libraries to be combined to be analyzed, whereas it is only possible with the techniques known to date to combine and analyze two molecular libraries that are several orders of magnitude less complex . Instead of a mass spectrometer, a movable device can also be attached, with which phages or DNA can be recovered from individual pits on the CD.

i) Sequencing complex DNA using solid-phase-coupled oligos

The complete 12-mer oligonucleotide library described in Example f) is used. In contrast to example f), however, the synthesis direction must be "reversed", i.e. the protective group which can be removed analogously to DMTr must be on the 3'-OH end for this, so that at the end there is a solid-phase-coupled oligonucleotide library with free 3'-ends. The hybridization conditions are chosen so that there are very few, if any, mismatches between the oligonucleotides and the templates hybridized to them. The complexity of the solid-phase-coupled oligonucleotides should exceed that of the DNA to be sequenced. Non-hybridized cDNA is washed away. A sequence reaction according to Sanger with a comparatively large number of ddNTPs in the reaction solution is carried out, so that on average the oligoprimer is extended by an average of 20 nucleotides. Then the template is removed by heat. The sequence information is then read out in the combined CD burner mass spectrometer described in Example h), the combustion laser evaporating selected oligonucleotides which have been lengthened by the sequence reaction, so that the sequence information can then be detected by means of the mass spectrometer.

j) Repeated staining of a large number of defined lymphocytes

Lymphocytes are obtained from a person's blood serum using standard methods and fixed using standard methods (0.37% paraformaldehyde in PBS). Approximately 10 ⁸ lymphocytes fixed in this way are distributed on the surface of a CD or a fluorescence multilayer disc (FMD) and fixed there on the surface. The FMD or CD is then placed in a tightly fitting rubber sleeve and non-specific bindings are blocked with 5% milk powder in PBS.

About 150 different monoclonal antibodies are individually conjugated with fluorescent labels, including e.g. B. Phycoerythrin, EGFP, EBFP, EYFP, Cy3, Cy5, Cy5.5, Cy7. The corresponding Fab fragments can therefore optionally be prepared beforehand. The hallmark of the conjugated monoclonal antibodies is that they recognize different surface antigens of human lymphocytes, including CD antigens, receptors for growth hormones, apoptosis-associated proteins and homing receptors.

The lymphocytes fixed on the FMD or CD are then incubated with the fluorescence-labeled monoclonal antibodies (60 minutes at 37 ° C. in 5% milk powder in PBS and 0.5% Tween 20). For a staining ration of the fixed lymphocytes, preferably 2 to 5 antibodies conjugated with different fluorescences are used in each case. After unbound antibodies have been removed by washing three times with PBS, the CD or FMD is inserted into an essentially commercially available CD or FMD drive and played. Due to the integrated position markings (repeatable and controllable), the playback time is divided into> 10 ⁸ different time units. During each of these time units, the laser beam used for scanning detects any fluorescent molecules on the surface (ie antibodies that bind the lymphocytes). The fluorescent light is broken down into different color fractions and preferably recorded according to the principle of the confocal laser microscope. A fluorescence intensity is thus assigned (possibly repeatable) to each unit of time and stored, preferably several different fluorescences at the same time.

The FMD or CD is then removed, the fluorescence on it is bleached by irradiation with very intense light and, as described above, stained again with further fluorescence-labeled antibodies. How again the fluorescence intensities are assigned to the (unchanged) time units. Repeating this process several times assigns each time unit up to 150 different signal intensities corresponding to the monoclonal antibodies mentioned.

The signal intensities assigned to a unit of time are assigned to different cell categories and the number of the respective cell categories is determined (e.g. CD3 is characteristic of a T lymphocyte, CD4 defines T helper cells, CD8 cytotoxic T cells, CD 19 is characteristic of B lymphocytes a defined stage of differentiation, etc.).

k) diagnosis of lymphocytes

The staining of lymphocytes described in Example j) is carried out with the blood cells of clinically unremarkable patients. The signal patterns obtained in this way are compared with the signal patterns which are obtained when the blood cells of patients with diagnosed Crohn's disease, heart attack, Parkinson's disease, multiple sclerosis, lymphones, in particular preclinical lymphones or systemic lupus erythematosus, are stained.

Reference list

1 unused

2 molecules coupled to the carrier

3 matrix layer 4 aggregates that bind to molecules coupled to the carrier

5 substances

6 electromagnetic waves

7 locally limited, selected area

8 other molecules that interact with molecules (2) coupled to the carrier, or that interact with aggregates on these molecules (4)

9 immobilized substances

10 top

11 mobilized substances

12 carriers 13 electromagnetic waves or applied voltage

14 molecules

15 different substances

16 axis

17 Mixing array 18 Movement arrow

19 sources of electromagnetic radiation

20 pit

21 isolator

22 movement arrow 23 array of individually controllable detectors

24 focusing lens

25 larger area, which comprises several selected areas (7)

26 laser beams Detector detector Light-emitting diode that can also be used as a detector Lens-emitted light beams Array of microlasers Microlaser Microlaser Top side of a carrier Carrier Molecules / group Molecules / group Array of detectors Detector Detector Wavelength filter Excitation radiation (radiation from a Mirokaser) Luminescence radiation

Claims

Claims:

1. A method for detecting optical properties, in particular luminescence reactions and refractive behavior, of molecules bound directly or indirectly on a carrier, wherein electromagnetic waves, in particular laser light, are radiated onto the carrier and light emitted or refracted by the molecules is detected by a detector and wherein the detector and the carrier are moved relative to one another during or after the irradiation of the electromagnetic waves, characterized in that a carrier, in particular a transparent carrier with integrated position markings which can be detected by a detector, in particular an optoelectronic scanning system, is used, and in that at least one of the integrated position markings is read out during or after the relative movement of the carrier and detector, so that an assignment of the light detected by the detector to a location on the carrier is possible.

2. The method according to claim 1, characterized in that the relative movement of the carrier and the detector is generated by rotating the carrier.

3. The method according to claim 1 or 2, characterized in that as a carrier a correspondingly modified, in particular transparent, but with regard to the position markings designed commercially compact disc (CD), digital versatile disc (DVD), magneto optical disc (MOD) or

Fluorescence Multilayer Disc (FMD) is used.

4. The method according to any one of claims 1 to 3, characterized in that a carrier is used, on which at least one element of the group consisting of molecular library, cells, cell fragments, adherently grown cells or cell fragments, tissues, tissue sections, by Embedding in a gel-like matrix, in particular agar, agarose, polyacrylamide or gelatin, which is attached to the carrier, attached cells or pieces of tissue, is applied.

5. The method according to any one of claims 1 to 4, characterized in that the optical properties are measured repeatedly at a specific location on the carrier.

6. The method according to any one of claims 1 to 5, characterized in that an exchange or modification of the molecules, the optical properties of which are to be detected, can take place at any location on the carrier and in particular after the exchange of the molecules or the modification of the

Molecules can be measured again.

7. The method according to claim 6, characterized in that the process of exchange or modification of molecules whose optical properties are to be detected can be repeated as often as desired.

8. The method according to any one of claims 6 or 7, characterized in that a characteristic profile for a specific location of the material applied to the carrier, in particular cells or histological sections, can be created.

9. The method according to any one of claims 1 to 5, characterized in that the optical properties of several different molecules can be measured simultaneously at a specific location.

10. The method according to any one of claims 1 to 5, characterized in that the selected molecule or the biological structures carrying the selected molecules, in particular liposomes, virus particles, retroviruses, bacterial vague, viroids, cell fragments, in particular B-lymphocytes, T-lymphocytes, leukocytes, parasites, unicellular organisms or bacteria, detached from the carrier, in particular by irradiation with electromagnetic waves, in particular laser light, and fed to a method for detecting further, not necessarily optical properties.

11. The method according to claim 10, characterized in that the detachment of the selected molecules or the biological structures carrying the selected molecules by interaction of the electromagnetic waves with a provided between the carrier and the molecules or the biological structures, preferably meltable or photosensitive

Substances.

12. The method according to any one of claims 1 to 11, wherein optical properties of the molecules by applying a dye to the molecules or the molecules optionally carrying biological structures, characterized in that a further dye is applied to the carrier and that a change in optical properties of the molecules by fluorescence energy transfer from one dye to another.

13. The method according to any one of claims 1 to 12, wherein the carrier is rotated, characterized in that the optical properties of biological structures, in particular of molecules, cell fragments or cells, or bound dyes are determined after a migration of the biological structures or Fluorescent dyes have taken place along a direction essentially radial to the axis of rotation of the support.

14. Device for detecting optical properties, in particular of luminescence reactions and refractive behavior, directly on a carrier or Indirectly bound molecules, with means for irradiating electromagnetic waves, in particular laser light onto the carrier and a detector for detecting light emitted or refracted by the molecules, and means for generating a relative movement of the detector and carrier during or after the irradiation of electromagnetic waves, characterized that a detector, in particular an optoelectronic scanning system, is provided for detecting position markers integrated in the carrier.

15. The apparatus according to claim 14, characterized in that the optoelectronic scanning system is a position detection system of a CD, MOD, DVD or FMD drive.

16. A carrier for use in a method according to any one of claims 1 to 13.

17. Carrier according to claim 16, characterized in that the carrier is a preferably transparent, but with respect to the position markings essentially commercially available CD, DVD, MOD or FMD.

18. A carrier according to claim 16 or 17, characterized in that a molecular library, preferably adherently grown cells or cell fragments, tissue or tissue sections, is applied to it or by embedding in a gel-like matrix attached to the carrier, in particular agar, agarose,

Polyacrylamide or gelatin are embedded.

20. A carrier according to any one of claims 16 to 18, characterized in that a coating with meltable and / or photosensitive substances is provided between the molecules to be examined and the actual carrier body.