DE4301005A1 - Identifying molecules, esp. biopolymers, by fluorescent correlation spectroscopy - Google Patents

Abstract

Identifying one or a few molecules, esp. at a dilution of 10nM or less, uses laser-induced fluorescence correlation spectroscopy (FCS) with measurement time not over 500 ms and with short diffusion paths for the molecules (I) being analysed. Measurements, in small unit vols. (pref. not over 10 femtol), are made of substance-specific parameters determined by luminescence measurements on (I).Also claimed is an appts. for the above process.

Description

The present invention relates to a method to identify one or a few molecules, especially in a dilution of 10 nM with Ver application of laser-excited fluorescence correlation spectroscopy, the use of the method for be agreed applications, as well as a device for through implementation of the method according to the invention.

The analysis of biologically active molecules has been described in the last few years in terms of specificity and sensitivity constantly improved and through fundamentally new techniques added. In this context, the cloning method the method or methods of enzymatic amplification genetic material referenced to single cells or To increase the number of molecules so that they are a con conventional analysis. But it is in in many cases more advantageous if the sensitivity of a Analytical method would be sufficient to single molecules or ensembles with a few molecules directly qualitatively and quantitatively. Electron microscopy is for example a procedure with which single moles cooler can be detected. So it is tried with the Tunnel electron microscopy to individual DNA molecules sequence. However, this is very complex.  

Beyond the pure analysis of individual molecules statements about the state parameters of the Molecules as important as their conformation and change effect with molecular structures.

Deal with modern methods of evolutionary biotechnology deal with highly complex collectives of molecules. It applies doing molecules with specific interaction properties to identify target structures, the means a certain fitness regarding a desired one Measure function. Such fitness can affect thermodynamic parameters such as binding constants or Have the speed constants reduced. The the The object underlying the invention is therefore under other in specifying a procedure that allows beyond the pure detection of individual molecules, Information about their specific interaction with to get other molecules.

It is not enough for a number of important problems crucial, the sensitivity of a method a limited availability of the to be analyzed Increase molecule, rather the fact that it a very large number of samples out there, more or less must be analyzed at the same time. If inside a period of hours e.g. B. 106 analyzes lead, it is obvious that only an analysis can come into question, the samples in one Measure the time interval from approx. 1 ms to a maximum of 1 s and be evaluated.

The method according to the invention is based on a Luminescence detection and uses a method called Fluorescence Correlation Spectroscopy (FCS) per se is known. Chromophore molecular structures with fluorescence  properties can be used to make statements about the molecular environment of a chromophoric ligand hold. Rotation diffusion and trans measure the diffusion of a luminophore, and ver different ways of transferring energy to interacting Molecules, chemical kinetics and lifespan excited states.

The method according to the invention offers on the basis familiar physical and chemical phenomena new Solutions to problems with single or a few molecules using spectroscopic measurement parameters statements to get about the nature of the molecules, as well as statements about their fitness for a particular change effect function or different about the degree of occupation whose molecularly defined states of a luminophore.

The method of correlation fluorescence spectroscopy, as described by the groups D. Magde (Elson, E.L. & Magde, D. (1974) Fluorescence correlation spectroscopy. Con ceptional basis and theory. Biopolymers 13, 1-27) and R. Rigler (Ehrenberg, M & Rigler, R. (1974) Rotational Brownian motion and fluorescence intensity fluctuations. Chem. Phys. 4, 390-401) for almost twenty years was not technically possible be implemented in a practicable analysis procedure. It was not possible to meet the above requirements regarding measurement times and the problem of light-induced Bleaching (photobleaching) of the dyes fairly become. Rigler et al. could rotation times of moles determine cool. Magde et al. could do some chemical Determine reaction constants via fluctuation times.

The principle of measurement of CFS is that fluorophores Molecules can be measured in extremely dilute solutions  (10 nM) by adding a relatively small volume element to the Solution of a strong excitation light from a laser is exposed. Only the molecules corresponding to An excitation spectrum, which is in this volume hold are stimulated by the light. The emitted Fluorescence from this volume element can then affect one Photomultiplier with high sensitivity. If the solutions are dilute, they result significant fluctuations in the concentration of the the respective volume element. At a very dilute solution gives a Poisson ver division of the number of simultaneously in the volume element existing molecules per time interval. A molecule that diffused into the volume element once the trans lation in an average, but for the be matching molecule characteristic time again from the Remove volume element and therefore no longer to be observed be careful. If now one and the same molecule during his average length of stay in the ent speaking observation element optically many times can be excited, many of this molecule Detect fluorescence signals. In other words, the Probability that once diffused Molecule before it leaves the volume element can be stimulated again is with dilute solution much larger than the signal from a new one emerging molecule can be sent. So you can a correlation of the change over time current emission signals with the relative diffusions create times of the molecular species involved. Will the Rotation of the plane of polarization of excitation light and emitted light measured as another parameter, so the rotational diffusion coefficient of the be Determine involved molecules, from which information about  Molecular weight, shape parameters or the surrounding matrix can be won.

It becomes apparent that it is theoretically possible Thereby single molecules in dilute solutions touch the same molecule several thousand times is excited and the corresponding fluorescent light in many individual measurements is accumulated.

The implementation of this measurement principle in practice many technical difficulties. The Despite the use, the observation element was more modern Laser technology so large that it is biologically interesting biomolecules with low translation diffusion coefficients on the order of about 50 ms in Observation element stopped. This time is significant too long, because it causes a strong fading of the dye ligands used in each case. Frequent light excitation increases the chemical reactivity of the chromo phoren structure with surrounding molecules, in particular with oxygen, whereby the fluorescence activity ver is changed or deleted. Of course, this leads from bleaching (photobleaching) directly to incorrect measurement data, there is a loss of fluorescence the exit of the molecule feigned from the measuring element and a distinction is hardly possible by standardizing the measurement method or only through disproportionate effort can be achieved.

Thus was the broad practical implementation of the measurement principles in a generally applicable process so far set narrow limits by the inventive However, procedures are overcome.  

The decisive breakthrough on the way to a routine procedure for the CFS tasks defined above is achieved according to the invention by introducing ultra-small measurement volumes (10 ^-14 -10 ^-17 l) in the case of total sample volumes in the µl range, the implementation of which determines the simultaneous use according to the invention of certain Elements of excitation optics, single photon registration and sample handling are required. The measurement volume of the device described in the experimental arrangements is 2 × 10 ^-16 l about 1000 times smaller than the measurement volumes described in the literature. The illuminated area is therefore approximately 0.1 µm ² . If one assumes that the CFS provides particularly correct data for a concentration of chromophoric molecules of 0.1-10 molecules per volume measuring element, the working concentration is approx. 10 ^-7 -10 ^-9 M. Measurements were made with maximum detection efficiency and background discrimination by using confocal optics with high aperture in combination with single photon detection.

Binding constants can then be translated diffusion can be determined if the response time is slow compared to the diffusion time, that is to say careful ligand while entering the mess compartment and its exit is not its molecular Structure changed. Otherwise there is only a correlation measured, which describes the mixed state. Here again the importance of the Ver driving with very small measuring volumes clearly, since the Residence time of a molecule is approx. 1000 times smaller than in the previous compartments and thus the usual ones Dimensions for equilibrium constants and speed constants of specific, biological recognition  reactions, e.g. B. ligand / receptor interaction, for opens up a measurement.

At lower concentrations, correspondingly more volume elements have to be subjected to a measurement. Since the average measurement time for a measurement is between 10 and 100 ms, depending on the required quality of the measurement data (signal / noise ratio), 10,000 to 100,000 volume elements can be checked in 1000 s. This means that extremely low concentrations of 10 ^-14 to 10 ^-15 M can be measured. This also means that specific biological interactions with binding constants K _ass 10 ⁶ mol / l up to K _ass = 10 ^-15 mol / l can be measured.

An example shows how binding constant or reaction rate constants with of the technique according to the invention. Which he measurement of a binding balance according to the invention between reactants is measured by at least one Preferred reactant with at least one dye molecule is chemically coupled, and the rotation diffusion speed and / or the translations diffusion rate of the reactants through the Complex binding changes. If there are equilibrium constants experimentally not directly with the condition high ver to bring thinner solutions into line that means if low binding constants higher con require concentrations of reactants, this can be done either z. B. achieve that the unmarked Reaction partner is offered in excess, or by the labeled compound an excess of unlabelled Reactants is added.

The measurement of a reaction kinetics via molecular fluctuation with CFS, as described by Magne, could not be solved satisfactorily because of the long diffusion paths in the measuring compartments realized so far. With the measurement technique according to the invention, the determination of the dissociation rate constants of a complex is possible in a range from 10 ^-6 s ^-1 to about 10 ³ s ^-1 , a range which is relevant for biochemical reactions. The measurement can e.g. B. be determined by the exchange of fluorescence-labeled tracer molecules.

The technique according to the invention also allows the measurement of Conformational changes of biological macromolecules that Determination of thermodynamic and kinetic constants. Fluctuations in the structure can e.g. B. about the Measurement of rotational diffusion or via signal changes by so-called energy transfer (forester transfer).

The method can also be used for DNA / RNA analysis. In genetic analysis, especially for determination of infectious agents is the sensitivity of the diagnostic procedure often crucial interpretation. This has just been in Zu in recent years in connection with the introduction of enzymatic methods for Amplification of genetic target sequences clearly. By the use of this method is to be expected for a number of diagnostic procedures on the need a previous enzymatic amplification ver can be waived, whereby z. B. Problems of contact bypassed by heavily amplified single sequences could become.

The method can also be used instead of the known diag nostic procedures of the RIA, ELISA or other ver driving can be used. A particular advantage of the inventive method can be seen in that the  System is self-calibrating. On provisions of Eich curves or internal standardization can be omitted become. Every experiment draws its inner potash bration on the total number determination of the considered Molecules. For example, fluctuations in the lasers intensity has no influence on the measuring accuracy. A Drift problem with successive ones Measurements do not appear. Measurements are without recalibration at different times same result repeatable. A device calibration not applicable.

Charged molecules (cations and anions) can be specifically within the measuring compartment of the inventor inventive method by using an "electrical Analyze trap ". This can be done, for example, by a molecular flow induced by the measuring compartment is, with or without overlay by a mechanically induced flow an electric field for a concentration of certain ionic molecules in the Observation compartment, or a single one Molecule directed into the compartment is transported. This can be done by a rectified field z. B. between the outlet ends of two capillaries with one Field strength of 1 µm can be achieved. It succeeds according to the invention, one or more captured molecules in the observation compartment in an electrical Alternating field to oscillate as soon as it enters this Volume element has occurred.

The inventive method is technically with a high quality microscope optics regarding the Image quality of the focus realizable. Especially that Lens system before the excitation light emerges be chromatically and spherically corrected. Prefers  the Neofluar system from Zeiss is used, Oberkochen, Germany, with high aperture 1.2 N.A for use with and without cover glass or release film. Of the Working distance is 0.17-0.9 mm. The lens is for Water corrects and offers at maximum working distance a maximum aperture. Oil immersion lenses are badly suited. The amount of light is according to the invention through a confocal pinhole in the object plane limited to the microscope objective.

As a laser light source for wavelengths from <200-1000 nm continuous lasers are preferred, esp special argon, krypton, helium-neon lasers, Helium-Cadmium, and modulatable diode lasers (red to Infrared range) with the possibility of each Frequency doubling. The use is also patented high-frequency pulsed laser of 10 MHz possible.

The laser intensity is already as strong as 0.5 mW a few percent of the dye molecules in the observation volume are excited. With a laser intensity of 5 mW is the percentage of excited molecules already 50%. Another increase in laser power thus appears for the increase in light output no longer makes sense.

As a couplable luminophore or fluorescent color fabrics come in a wide range of possible dye bases structures and oligomers of these dyes, such as them for a long time for fluorescence spectroscopic Detection methods are used. Preferred color substances are those that are either not too specific fishing and interfering interactions with target contribute molecules, or as in P 42 34 086.1 (Henco et al.) specifically using specific binding properties  like the ability to double-strand nucleic acid Intercalation.

All dyes used preferably have an Ab sorption coefficient between about 30,000 and 100,000 with a quantum yield of 0.1-1.

Have proven themselves. B. dyes from the series Coumarins or rhodamine B derivatives with a high content hydrophilic residues to prevent hydrophobic changes effects or dyes based on thiazole basic orange structures with the ability of inter calibration in double strands.

The resistance is for the measuring method according to the invention of dyes against light-induced bleaching (Bleaching stability) an important property. she is however, as mentioned above, no longer outstanding Significance for the execution of the measurement, since at the use of very small measurement volumes according to the invention compared to the measurement times shortened by orders of magnitude approx. 40 ms in measuring compartments approx. 1000 times larger.

The measuring compartments must be 1000 µm to the output lens of the laser-focusing Optics can be introduced. This is done in the simplest way Trap by a drop hanging on the lens itself, which contains the molecules to be analyzed. Such Measuring arrangement can only be used for a few analyzes, because here is the risk of contamination between different Samples is great. Techniques can be compared those of conventional microscopy with cover slip and Oil immersion. The method according to the invention is used Water immersion and very thin glass or art to remove the aqueous sample from the optics  separate. The foils can be closed at the same time the underlying compartments in the form of flat Serve carriers or in the form of capillaries.

For the application for screening large numbers of samples, such as those obtained in experiments for the evolutionary optimization of biopolymers, membranes are used that are chemically modified facing the sample side. Preferred modifications are surface structures with specific binding properties such as. B. Ion exchange property for fixing nucleic acids and / or specific binding properties against proteins such as antibody coating or coating with chelating agents, in particular NTA (nitrilotriacetic acid) or IDA (iminodiacetic acid) for the targeted fixing of recombinant proteins or peptides with binding oligopeptides such as (His) ₆ that can be bound to surfaces with high binding constants (K _ass 10 ¹⁰ ) via metal chelates in order to analyze them for their respective interaction properties with target structures.

Another possibility for the described recording of individual molecules in small volume elements consists of the use of a molecular trap according to the invention Using an electric field, which later be should be written. However, this analysis can only applied to ionic molecules.

Registration is preferably done via the optics a fluorescence microscope with single photon counting, an avalanche diode detector is preferably used is coming. Has proven itself. B. the use of the SPC-100 and SPC-200 from the manufacturer EG & G. The signal analysis takes place with a digital correlator or multichannel counter MCS.  

The correlation method provides direct information about three characteristic molecule sizes, the number (N), the translation diffusion coefficient D _t and the rotational diffusion coefficient D _r . Both are a function of the molecular size (ie radius, shape and volume) and provide information about changes in the molecule due to enzymatic cleavage or complexation with other ligands. Since the measurement of the diffusion times correlated to diffusion can be carried out quickly and sensitively according to the invention, the method for analyzing the molecular sizes and their distribution in a population in solution can be used without the need for a chromatographic separation.

As Rigler has shown, the analysis of the corrections shows lationsfunktion for interacting molecules that by measuring the number of molecules N and the weight factors the changes in the characteristic diffusion times effect can be determined. In the event that Re action rate constants slower than that Diffusion times are whatever is specific Reactions that will be the case is the correlation function by the sum of the weighted diffusion times given;

G (t) = 1 + 1 / N (x (1 + t / τ _x ) ^-1 _{+ y} (1 + t / τ _y ) ^-1 )

where x, y and τ _x , τ _{y are} the fraction and diffusion _times of molecule X and Y. τ = ω ² / D. ω is the radius of the sample volume and D is the diffusion constant.

In the event that reaction rate constants are faster than the diffusion times Correlation function

G (t) = l + l / N (l + 4 <D <t / ω ² ) ^-1

where <D <= x D _x + D _y .

If D _x and D _{y are} different, the binding of a (small) ligand to a large molecule (protein, nucleic acid, antibody) can be monitored in a simple manner without molecular separation methods, as are common in so-called inhomogenous assays. Corresponding relationships can also be found for the rotational diffusion coefficients. Derive rotational diffusion times. The high measuring sensitivity exceeds radioisotope methods, as are known from the techniques of radioimmunoassays (RIA). These are only equivalent if they are marked with high radioactivity. Molecule separation processes and complex calibration are omitted in the implementation according to the invention. The correlation method thus represents an alternative to the radioimmunoassay used today. According to the technique described above, instead of the radioactively labeled antigen reagent, a fluorescence-labeled antigen is used, the binding of which to an antibody in a homogeneous phase or inhomogeneous phase (solid phase-coupled) in competition with the antigen to be determined is analyzed. The advantage of the method resides in the absence of unwanted radioactivity with comparable sensitivity.

Where enzymatic catalysis changes the Molecular structure and molecular weight leads, can the emergence of reaction products via the Ver Track changes in the number of molecules and diffusion times. Typical applications are replication and splitting of Nucleic acids, cleavage of proteins and peptides, however also the selection of catalytic antibodies.  

The ability to measure rotational diffusion of large molecules in a viscous environment using the FCS method is of particular importance for the analysis of the dynamics of specific receptors on the cell surface but also inside the cell. At the same time, the binding of labeled ligands can be measured by measuring rotational diffusion and translational diffusion on cell structures, such as receptors and others. Examples of this are neuro transmitters, tissue factors such as growth hormones, but also cationic ligands such as Ca ²⁺ .

The ability to determine diffusion times in fractions of a second allows the kinetic interaction of two molecules with high association constants to be analyzed and recombination and dissociation rates to be determined. This is of particular interest for the characterization of the interactions of high biological specificity, such as antigen-antibody, ligand / receptor interaction, etc. The analysis of particularly slow processes with constants up to 10 ^-6 s ^-1 is readily possible because of the initially mentioned mentioned intrisical method characteristics of self-calibration.

The high sensitivity allows the detection of individual molecules as they move through at least one detec ting laser beam. By using so-called "molecular funnels" in the form of special glass pipettes with openings of 1 µm, individual molecules can be introduced into a laser beam with a diameter between 1-5 µm by flow. By using electric fields, the Brownian molecular motion is so narrowed that each molecule is pulled through the intensity maximum of the laser beam. The optical setup with opposing detectors and immersion optics guarantees very good photon flow and detection accuracy and detection efficiency of the luminescence emitted in all spatial directions ( Fig. 1). The arrangement is suitable for example for the analysis of sequences of individual DNA or RNA molecules with the aid of exonucleolytic degradation (JH Jett et al., US 4,962,037), but also for the detection of individual molecules provided with markings and charges.

In the case of ionic structures, individual molecules can also be retained in the observation volume element by forced directional translation in the electric field. Alternatively, the one-off or repeated translation can be enforced by the volume element. This is preferably done in an experimental arrangement, which is shown in Fig. 1. A molecular flow runs through a larger sample volume in the center of which is the observed volume. The migration of charged molecules through the observation element can be forced by an electric field, rectified or as an alternating field. This molecular focus is important if there are only one or a few molecules in the total volume element that need to be quantitatively determined.

Technically, the task is solved by preferring that Sample volume as microdroplets between the openings two microcapillaries as fixed by B. Sackmann and E. Neher have been described. The capillaries are vapor-coated with a conductive metal layer before pulls gold on chrome primer, which at the openings of the Capillaries in contact with the aqueous buffer system stand. The measuring element is inside the Sample droplets, the starting lens of the  Microscope is in direct contact with the drop or is separated from the lens by a film.

Is a single molecule or a molecular complex after the exit from the capillary end once in the measuring element kinetic data can also be obtained. It is not technically complex, in the small volume element to realize a field or jump in temperature. When reaction complexes show a Wien effect or one have sufficient enthalpy of reaction can be use this relaxation method, for example To determine reaction rate constants.

The method is part of evolutionary screening assays to determine the binding of ligands to selek ionizing molecules (proteins, nucleic acids, antibodies) suitable. The extreme sensitivity allows in particular especially the analysis of highly specific interactions genes that are of particular biological interest. The Interaction is through binding of the labeled ligand or by competition with the unlabeled ligand measured a labeled inhibitor. So you can Fitness regulations on variant spectra especially at large sample groups.

The following fitness parameters apply:

• - affinity parameters
• - kinetic parameters
• - enzymatic parameters

under certain milieu conditions of the test.

In the analysis and evaluation according to the invention, large Sample collectives of phenotypic variants are from ent  of crucial importance that subsequent access to the corresponding genotype, e.g. B. the coding Plasmid or the coding mRNA can be made to the To continue the evolutionary process. This problem is by no means trivial. The process is all the more successful the more targeted the access, that is, the more precise the corresponding genotype can be prepared without contaminating him with sequences that are of no value Encode phenotypes.

If the number of samples is medium (<10,000), they can be used together Volume elements arranged in a fixed spatial separation elements to be worked, which are referred to below as cuvettes be designated. This can be both parts of a slide systems, comparable to those used for PCT / EP 89/01320, PCT / EP 89/01387, PCT / DE 91/0082, PCT / DE 91/00081, PCT / DE 91/00083, PCT / DE 91/00704 have been, with individual volume elements samples in sealed foil elements included. From the ver closed elements can be identified once Phenotypes together with their coding genes or mRNA transcripts via direct mechanical access isolate.

The method of optical measurement according to the invention and Variant evaluation in ultra-small sample volumes allowed also very small total sample volumes in the form of a Micro-compartmentalization, which, however, is not trivial are available. This applies to both the individual Be filling as well as for their targeted emptying paration of a genotype identified as positive.

Microcompartments can consist of regular and irregular porous supports should be constructed as from parallel arranged capillary elements, as described in the patent message P 42 37 383.1 are described, or flat  Carrier made of porous materials such as glass with control gated pores or capillaries, their volume elements one-dimensionally within the capillary are separated, but in pairs with each other in direct Are in contact.

A particularly preferred form of microcompartmentation is shown in Fig. 2. The volume elements present in a sample to be analyzed are applied in the form of recombinant or natural cells or artificial vesicular elements with their respective phenotypes and genotypes under an optically transparent flat carrier. Alternatively, volume elements are only generated when the order is placed or after the order is placed on the carrier. Gel or vesicle-forming polymers are particularly preferred, in particular polymers based on thermoreversible structures such as caprolactam derivative polymers.

The application can initially be made from a homogeneous solution subsequent separation of the polymers with formation separate aqueous volume elements, or by Application in the form of microdisperse drops using a piezo-controlled microdispenser.

In the described way, the moleku Analyze laren contents of cells. For example cells can be described in the vesicular Include structures and later at higher tempera lyse doors. At least there is one partial mixing of vesicularly enclosed Solution with its reactive molecules or moles cooling complexes with the content of the lysed cell. Right Actionable molecules can e.g. B. nucleic acid probes, Enzymes or proteins with cellular components  enter into specific interactions or reactions, that can be detected with the CFS methodology according to the invention and are quantifiable. This technique is analogous to in situ hybridizations or cell-specific protein see colorings.

Also carriers for the simultaneous fixation of nucleus acids and the phenotypic moles to be analyzed cooling structures, as described in the application K. Henco et al., DE 42 37 381.6 are preferred Reaction carriers suitable for genotype / phenotype coupling to reach.

Of course, carriers can also be used, such as it by S. Fodor et al. within the so-called AFFYMAX technology used in the genotype is defined by the x / y position on the carrier.

A preferred way of addressing and marking variants selected according to the invention in their respective volume elements is the photo-optical marking of the position by using the analytical optics described ( Fig. 3). This can be done by a reflection of light of a wavelength with which a photoactivatable coating can be excited on the surface of the carrier in order to produce an easily recognizable reaction product, for. B. by targeted discoloration. The light signal is activated when the correlation analysis indicates a certain predefined value of the volume element analyzed in each case.

A common photoreactive coating can be used be set. An additional light source can be used Light source are used, preferably also a laser light source, or it can be used for analysis  set lasers can be used. Discrimination between measuring and marking the position Photoactivation can e.g. B. by using a frequency doubler for the labeling reaction can be achieved.

After marking the positions on a support leaves the desired volume element Nucleic acid either by mechanical access win.

It proves to be particularly suitable according to the invention place the marking of the selected volume element according to (1) the marking of the corresponding genotype self.

According to the invention, several alternatives are preferred:

• (1) the photoactivated attachment of the nucleic acids Surface structures of the volume element,
• (2) the photochemical activation of nucleic acid speci fischer ligands,
• (3) photochemical inactivation of nucleic acids in all volume elements except the positive selected.

For the purposes of (1), psoralen derivatives can be used, for example, which combine nucleic acid counterstrands as double-strand intercalating reagents under 360 nm light irradiation. Such ligands can either be chemically linked to the carrier surface, for example ( Fig. 4).

In the screening, a ge sufficient number of plasmid-coding plots encoding the positively selected surface segments stapled become. Subsequently, all unfixed nuclei acids are washed off. Then either selected nucleic acids directly on the surface be subjected to enzymatic amplification reaction.

Alternatively, the nucleic acids can be recovered be made by the reversibility of the psoralen linkage is exploited. This is done, for example, by Light of the wavelength of 260 nm. The nucleic acid binding ligands can also be mixed with other moles according to (2) specific elements are linked, which is a subsequent Cleaning and separation of unwanted nucleus enable acid structures in a simple manner. As with Games may be mentioned: the coupling of DNA or RNA knowing ligands, especially photoactivatable Psoralen derivatives or intercalating dyes, ge couples to affinity ligands, especially biotin, Avidin, streptavidin, immunoglobulin, or oligopeptides Oligonucleotides.

According to Fig. 4, after light-induced chemical linking of these compounds with the DNA or RNA from the positively selected volume elements, it is possible to purify the DNA and / or RNA of all volume elements together and at the same time to separate them from the nucleic acids that do not meet the selection criteria have fulfilled. The separation can preferably be carried out via hydrophobic chromatography, affinity chromatography or by using magnetic particles, the surfaces having corresponding binding properties to the ligand of the adapter molecule. Preferred examples of such a specific coupling are: oligo-dT / oligo-dA, avidin-streptavidin / biotin, NTA-IDA / (His) ₆ and similar known complexing agents.

The purified nucleic acids pooled in this way can subsequent step either directly to an enzymatic Subjected to amplification reaction or cDNA synthesis be, or previously by reversion of the photochemically induced linkage are decoupled from the ligand.

In the sense of the invention, it is also conceivable selectively inactivate all of the nucleic acids that the Selection criteria of the analysis are not sufficient (3). This can also with cross-linking substances like Psoralens happen, cross-linking in one Way happens that the so networked structures at no longer amplify subsequent enzymatic reactions are capable. However, the process has the disadvantage that the inactivation must take place very completely because otherwise the enrichment factor for the positively selected th volume units is adversely affected. Both Method (1, 2) is not the yield of the enrichment of primary importance, since with the help of enzymatic Amplification process also very low copy numbers are sufficient. However, for most people it is turns advantageous if the largest possible proportion of nucleic acids from a positively selected volume element can be displayed preparatively. This succeeds preferably with an optic that has a larger volume element ment as the volume element considered in the analysis illuminates.

As already explained above, the presented one is suitable Pay particular attention to procedures for analysis and evaluation moderately complex collectives of molecules previously in an evolutionary process has been generated. The  functional analysis of complex systems of molecular However, diversity is also of considerable importance. Not just because of evolutionary systems, in the sense of replica mechanisms, diversity arises, just as little as Compartmentalization of subpopulations only in cellular or vesicular structures.

In synthetic chemistry, they often arise unintentionally very complex systems of different types of molecules, where the complexity can also be generated in a targeted manner. Micro organisms or plants synthesize a large number Secondary metabolites, of which a large number pharmacologically active structures was derived. With chromatographic methods such as HPLC, FPLC, gas Chromatography etc. can be such connections fractionate efficiently.

A method of this type thus reveals itself not only the parallel analysis of mutants / - Variants from so-called replicative systems such as Nucleic acids or proteins, but also chemical or metabolic complexity. This is traditionally the case up to now proceed that complex mixtures first in single fractions were preparatively cleaned, chemically after their structure were analyzed and if possible in form the pure substances are fed individually to biological assays were.

With the inventive method it is now possible to fractions initially obtained in analytical quantities preparative z. B. for pharmacological assays. In a preferred process, the falling fractions directly using a CFS assay ties. There are no individual substances Deliver before not positive in CFS assays speak.  

The Fig. 1 describes the preferred arrangement of the optical detection unit with respect to the sample volume and the measuring volume. One or two detectors (detector 1/2 ) register the emitted fluorescence signals from the measuring volume element, which are also confocally imaged via one or two optical units, as described in the text. The aqueous sample is either in direct contact with the surface of the exit lens or, as shown in FIG. 2, is separated from the objective by a thin film.

The sample is placed between at least two capillaries a clear width of the capillary end of approx. 1 µm held. The capillaries are in the case of the function as Molecular trap for ionic molecules with a conductive Coated surface, preferably gold on chrome Primer to which a rectified field or a Alternating field can be created. The control of the Field is preferably done by a computer that is linked to the optical detection unit and the Fields when a molecule of interest enters can regulate in a certain way.

The FIG. 2 describes a preferred embodiment of the arrangement according to the invention to pay for screening large mutants for specific fitness parameters is shown. Using an optical detection unit according to FIG. 1, the samples are in the form of droplets under a film-like surface which in turn is in contact with the objective in aqueous immersion. The film can carry certain coatings, which it allows to specifically bind molecules from the respective samples to the surface. The samples can be applied regularly at certain positions, e.g. B. by using a microdispensing system or in statistical distribution. To prevent the samples from evaporating, the drops can be surrounded by a protective matrix.

Fig. 3 shows schematically the CFS labeling of the selected genotypes. If certain samples correspond to the preselected fitness parameters in accordance with FIG. 2, access to the respective volume segments can be facilitated by providing the surface with a photo-activatable coating which, for. B. optically marked the position and then allows access to the sample.

Access to a selected volume segment or to molecules contained therein, such as coding nucleic acids, as shown schematically in FIG. 4, can also be achieved by having soluble reactants in the volume element photoactivated in order, for. B. to react with a nucleic acid. Nucleic acids labeled in this way can then be isolated relatively easily in order to subject them to further reactions, for example a PCR reaction.

Claims (24)

1. Method for the identification of one or a few molecules, in particular in a dilution of 10 nM, by using the laser-excited FCS with measuring times 500 ms with short diffusion paths of the molecules to be analyzed, the measurement being carried out in small volume units of preferably 10 ^-14 l is, by determining substance-specific parameters, which are determined by luminescence measurements on molecules to be examined. 2. The method according to claim 1, wherein translations diffusion coefficients, rotational diffusions coefficients, the excitation, emission waves length, the lifespan of the respective excited Zu level of a luminescent substituent or Combinations of these measurands as substance-specific Parameters can be determined. 3. The method according to any one of claims 1 and / or 2, characterized in that the luminescence of a Substituents with the molecule to be determined in there is direct interaction, the substituent a luminophore ligand or a ligand complex whose spectroscopic parameters are related to Art or function of the molecule to be determined correctly are. 4. The method according to at least one of claims 1 to 3, being about the measurement of translation diffusion and / or rotational diffusion with evaluation of the corrections  the functional evaluation in particular the determination of the absolute number of moles present cooler and / or their temporal variation and / or by determining the specific conc structurally different ligands and / or the ligand complexes and the derived therefrom conductive thermodynamic binding constants spec interactions and / or the speed constant of specific recognition reactions or enzymatic processes with participation Ligand-coupled molecules is performed. 5. The method according to at least one of claims 1 to 4, the measured molecules or molecule comm are complex ionic or non-ionic in nature. 6. The method according to at least one of claims 1 to 5, characterized in that the measurement in one superimposed constant or time-varying electric or magnetic field takes place. 7. The method according to claim 6, wherein the ionic Molecules or molecular complexes of a sample volume with the help of a rectified electrical Field or an alternating electrical field for Passing through the measuring element and / or briefly timely remain in the measuring element. 8. The method according to at least one of claims 1 to 7, characterized in that the luminophore Ligand and / or the luminophore ligand complex an extinction coefficient of 30,000 for a Quantum yield 0.1.   9. The method according to at least one of claims 1 to 8, characterized in that the measuring compartments at a working distance of 1000 µm from the outside gear lens are arranged, the lens either directly in contact with the sample volume stands or where the sample volume by a thin Film is separated from the exit lens. 10. The method according to at least one of claims 1 to 9, characterized in that defined molecules and / or equilibrium mixtures of molecules and / or kinetic course of reactions in at least a sample volume element can be analyzed, whereby in the case of several sample volume elements a two-dimensional flat support order, in particular a membrane or film and / or a wafer surface, or in linear Way, preferably in a capillary system are ordered, the Sample volumes in or on natural or in vitro modified cells and / or in artificially herge vesicular structures exist, esp special vesicles based on liposomes or Based on soluble polymers with vesicle-forming properties create. 11. The method according to at least one of claims 1 to 10, characterized in that the individual samples volumes by using a microdispenser systems are generated. 12. The method according to at least one of claims 1 to 11, characterized in that access to pheno typically selected genotypes at the DNA or RNA level is made possible by photochemical labeling  speaking measuring positions by either the local Known position of the associated volume element is drawn, preferably by using the Phenotype analysis used laser system under Use of photochemically activatable substances for optical identification or by using photo chemically activatable reagents in soluble or surface-bound form with the content of the selected volume element are in contact and with structural elements of the selected genotype in a stable chemical interaction can occur, in particular through psoraline derivatives, and via a specific binding reaction to a coupled Structural element a targeted separation of the selek enabled genotype, in particular activated bare substances with oligonucleotides or Biotin or avidin or streptavidin or oligo peptides or metal complexing agents or combinations of which are linked. 13. Use of the method according to at least one of the Claims 1 to 12 for determining the reaction efficiency of a specific substance conversion or a binding reaction of a ligand-bearing Molecule by adding volume elements to be evaluated reaction conditions simultaneously or sequentially be set and after a defined reaction time carried out an analysis of the reaction products becomes. 14. Use of the method according to at least one of the Claims 1 to 13, for qualitative and / or quanti tative detection of specific molecules and / or Molecular complexes and / or the molecular environment of the molecules and / or molecular complexes, in particular  measurement and / or evaluation more physiologically active Receptors, in particular surface receptors or the assessment of receptor binding ligands or Li gandic complexes. 15. Use of the method according to at least one of the Claims 1 to 14 as an alternative to radioimmuno assays or enzyme immunoassays by the competitive Binding of a desired molecule with a Lumino phor-carrying ligands to a receptor molecule will measure. 16. Use of the method according to at least one of the Claims 1 to 15 for the analysis of complex molecules collective and replicative molecules in particular Nucleic acids and proteins derived therefrom or Peptides, complex chemical reaction products, com complex systems of synthesis products from chemical Reactions or complex mixtures of secondary meta bolites as cellular synthesis products. 17. Use of the method according to at least one of the Claims 1 to 13, wherein the analysis is more complex Mixtures of substances on line with an analytical Fractionation takes place. 18. Device for performing the method according to at least one of claims 1 to 17 with one well-known microscope optics for laser focus tion for fluorescence excitation in a small measurement compartment of a very dilute solution and Figure of the emitted light in the follow the measurement by confocal imaging, whereby min mostly an optic with a high numerical aperture  1.2 N.A. is used, the amount of light through  a confocal pinhole arranged in the object plane is limited according to the microscope objective and the measuring compartment at a distance between 0 and Positio 1000 µm from the observation lens is nated. 19. The apparatus according to claim 18, characterized in net that two lenses are used, one Form angles <90 ° to each other. 20. The device according to claim 18 and / or 19, characterized characterized in that as a light source continuous Laser with emitting wavelengths <200 nm turned on be used, especially argon, krypton, Helium-neon, helium-cadmium laser or high-frequency pulsed laser 20 MHz with a power of 0.5 mW. 21. The device according to at least one of claims 18 to 20, characterized in that facilities for single photon counting, like avalanche diodes detectors, in the beam path of the emitted light are arranged to register the emitted Light, and where the signal analysis by a digital correlator or multichannel counter. 22. The device according to at least one of claims 1 to 12 and 18 to 21, characterized in that the measuring compartment in a sample volume between two capillaries is fixed, the capillaries on the outside with a chemically inert conductive coating are provided, in particular a metal vapor deposition, especially a goldbe vaporization on a chrome background and the conduct capable coatings with a computer-controlled rectified field or an electrical  Alternating field are connected and via the measuring compartment ment are connected to each other. 23. The device according to at least one of claims 18 to 22, characterized in that two each other opposing microscope optics the measuring compartment include ment. 24. The device according to at least one of claims 18 to 23, characterized in that the film for Uptake of the samples by a molecular derivative specific binding properties for moles cooler, especially in the form of ions exchanger ligands or affinity ligands, in particular special oligopeptides, polypeptides, proteins, anti body or chelating agent, especially Iminodi acetic acid or nitrilotriacetic acid ligands, ins special foils that differed by location molecular structures of different bonds have specificity as ligands.