DE102007010860A1 - Particle from a polymer material, useful e.g. in the medicinal diagnostic kit, comprises monodisperse in the material embedded fluorophore with absorption and fluorescence maxima in the ultraviolet/visible/near infra red-spectral area - Google Patents

Abstract

Particle from a polymer material, comprises two or more monodisperse in the material embedded fluorophore with absorption- and fluorescence maxima in the ultraviolet/visible/near infra red-spectral area, where the fluorophore is selected in such a manner that the absorption maxima of the fluorophore are at least around 5 nm; the fluorescence maxima of the fluorophore are at least around 10 nm; and the absorption maxima and the fluorescence maxima of the fluorophore are at least around 5 nm. Independent claims are included for: (1) a test-kit containing two or more charges of the particles, where the charges contain the same fluorophore with different concentration ratio; (2) production of the particles comprising polymerizing a solution/molecular or colloidal dispersion of the fluorophore in a monomer/monomer mixture; and (3) identification of the particles in a sample, comprising (i) irradiating particles preferably present in monolayer with one or more excitation wavelength in the overlapping area of the absorption spectra of the fluorophore and/or in the areas of the absorption maxima of the fluorophore (+- 10% of the extinction), (ii) capturing the intensities of the fluorescence at the above mentioned measuring point, where each fluorophore contained in the particle is given with a measuring point, which lies in an area of at least 5% of the intensity of the fluorescence maximum of the respective fluorophores and where the measuring points are spaced around at least 5 nm from one another, (iii) determining an intensity ratios of the intensities captured at the measuring point and comparing the determined intensity ratio with a reference value for the identification of the particles.

Description

The The invention relates to particles of a polymeric material, the two or more monodisperse fluorophores embedded in the material with absorption and fluorescence maxima in the UV / VIS / NIR spectral range. The invention further relates to a process for the preparation of such Particles as well as a test kit containing two or more batches of particles contains within each batch the same fluorophores with but deviating concentration ratio are included. Finally, concerns the invention a method by which an identification of the particles in a sample becomes.

Technological background and state of the art

monodisperse Particles (i.v., microparticles) of polymeric materials in increased measure of medical diagnostics, as a carrier for immobilized enzymes, as Markers in environmental analysis as well as in numerous microbiological Investigation used in which, for example, target compounds the surface the microparticle bound and identified by spectroscopic means become. Polymer particles of uniform shape and size can by precipitation, Suspension or emulsion polymerization can be obtained.

It is state of the art to add such particles fluorophores, that is, fluorescent chromophores whose fluorescence (emitted Fluorescent light) can serve to identify the particles. In the rule contains the particle thereby only a single fluorophore, so that for the identification either the location of the fluorescence maximum or the absolute intensity of the fluorescence must be recorded. The latter identification was about the absolute intensity already realized with particles containing two fluorophores. The intensities The dyes are determined separately.

Lie However, there are basically several fluorophores in a particle Possibility, that photophysical processes, such as radiationless deactivation or bimolecular processes (sensitization, quenching), and optionally also photochemical primary processes under spin reversal affect the fluorescence. When aggregating the Fluorophores in the particle undergo intermolecular interactions on. It is with a concentration-dependent changed spectroscopic behavior to count. In addition, the measured fluorescence intensity of the absolute total amount of fluorophores in the particle and thus also of the respective size of the particles dependent. Lying in a sample to be examined a number n different Particles before, so must these be distinguishable by fluorescence spectroscopy; is appropriate in conventional Proceed with a number n of different and spectroscopically distinguishable fluorophores or for a given fluorophore are particles to produce with graded concentrations of the fluorophore. evidently The disadvantage is that the number of suitable for the purpose, in sufficient dimensions accessible, and limited in their cost economically viable fluorophores is. It is also to be considered that an exchange of a component (i.e. H. a fluorophore or a component of the polymer material) of the particle under circumstances requires a modification of the entire system, be it through a necessary adjustment of the manufacturing route or the replacement of others Components, otherwise the one spectroscopic examination disturbing Could cause interaction. Finally is also the identification method for samples with a variety different particles with different fluorophores consuming: They are suitable excitation wavelengths for the different fluorophores pretend and the detection of fluorescence is simultaneous for the multiplicity to optimize the different fluorophores. The assignment of particles, the same dye in different high concentrations contained, alone by means of capturing the absolute intensity can be out different reasons disadvantageous be. Even small variations in particle size or inhomogeneities in terms of dye distribution lead to ambiguous results or to a very limited Number of distinct populations.

The Invention should be at least alternative to existing systems Offer method of particle identification and preferably one or more of the above-described disadvantages of conventional Overcome approaches. If possible In particular, a system of particles should be provided, in terms of the nature of the particles and the applied Identification method a particularly large number certainly distinguishable Allows particle populations. Safe in this context means that deviations of the particles in terms of their size or the total amount of dye present the identification as far as possible unaffected and thus a clear assignment of different particle populations of any mixture is.

Inventive solution

• (i) die Absorptionsmaxima der Fluorophore um jeweils mindestens 5 nm voneinander beabstandet sind;
• (ii) die Fluoreszenzmaxima der Fluorophore um jeweils mindestens 10 nm voneinander beabstandet sind; und
• (iii) die Absorptionsmaxima und Fluoreszenzmaxima der Fluorophore um jeweils mindestens 5 nm voneinander beabstandet sind.

A first aspect of the invention is to provide a particle of a polymeric material containing two or more monophosphorus embedded in the material fluorophores with absorption and fluorescence maxima in the UV / VIS / NIR spectral range. The fluorophores are selected such that

• (i) the absorption maxima of the fluorophores are at least 5 nm apart;
• (ii) the fluorescence maxima of the fluorophores are at least 10 nm apart; and
• (iii) the absorption maxima and fluorescence maxima of the fluorophores are each at least 5 nm apart.

Of the The invention is based on the finding that with the help of a particle system from two (possibly more) fluorophores a variety via fluorescence spectroscopy distinctive efficient Particles can be provided if the mentioned selection criteria are observed. As a result of that the wavelengths the fluorescence maxima of the fluorophores at least 10 nm from each other are spaced, the wavelengths the absorption maxima are at least 5 nm apart and fluorescence and absorption maxima of all fluorophores around may differ by at least 5 nm in terms of their location, a relationship the fluorescence intensities the individual fluorophores are detected. Out of this relationship can one - like closer below explained - identification of particles. A sample to be examined can therefore be different Types of particles included, each in proportion to the recorded fluorescence intensities and thus differentiate relative proportions of the fluorophores, otherwise however, have an identical structure. Of course, there may be particles as well be used with different fluorophores. The in the polymeric material distributed fluorophores are distributed monodisperse, are therefore ideally as a homogeneous solution of the fluorophores in the polymeric matrix before, or are, if it is a dispersion acts as a dye particles largely the same size in the embedded polymeric matrix. Preferably, the absorption spectra the fluorophores have an overlap area on, in this area for each fluorophore has an extinction greater than 0.001 and a quantum yield greater than zero is. This can - in even closer Illustrated Way - that Identification method initiated by excitation with only one wavelength become. Across from usual Procedure becomes a significantly reduced number of individual particle instances a population needed to identify this population.

Preferably are the absorption maxima of the fluorophores by 10 to 100 nm from each other spaced. Independently or in addition For this purpose, the fluorescence maxima of the fluorophores are around 20 to 100 nm apart.

The Absorption and fluorescence maxima are in the UV / VIS / NIR spectral range, that means in the wavelength range from 250 to 2500 nm. Preferably, the absorption maxima are in the Wavelength range of 350 to 850 nm. Independent or in addition For this purpose, the fluorescence maxima are preferably in the wavelength range from 400 to 900 nm.

Of the polymeric material should be insoluble in water and optionally swellable so that the use of the particles in aqueous media or in vivo possible is. The polymeric material may contain a polymer that is one or more monomers selected from the group consisting from substituted or unsubstituted acrylates, vinyls and Allylene is made. In addition, in particular polysilicates and Melamine resins find use. The polymeric material is preferably selected from the group comprising polysilicates, polyadducts (in particular Polyurethanes, polyureas, polyisocyanates and polydiols), polycondensates (in particular polyester / polycarbonates, polyamides, polyimides, polysulfones, Polyethersulfones and melamine resins), polyacrylates (especially Polymethyl methacrylate, polymethyl acrylate, polyhydroxyethyl methacrylate, Polyethylene glycol monomethacrylates of various chain lengths, poly-N-isopropylacrylamide, Polydiethylacrylamide, polyhexylmethacrylate, poly-tert-butylmethacrylate and polyacrylonitrile), polyolefins (especially polystyrene, polypropylene, Polyethylene, alipatic polyvinyls, aliphatic polyallylene), whose Copolymers or misery. The materials mentioned allow the Production of particles of defined size and are therefore as polymeric Matrix for the fluorescence spectroscopic examination of the particles especially suitable because they have no or negligible interference with the chromophore system and the photophysical processes, especially on fluorescence the fluorophores show. The polymer is particularly preferably Material made of polymethyl methacrylate (PMMA) or polystyrene (PS). In particle production, preference is also given to suspension stabilizers added, such as the substances listed in the embodiment PVP K-30 and Aerosol OT.

A particle diameter is preferably in the range of 10 nm to 2000 μm, in particular 1 μm up to 50 μm.

When Fluorophores can organic or organometallic compounds, but also from one Semiconductor material or metal oxides or metal sulfides existing Quantum dots find use. Preferably, the particle contains one or more fluorophores selected from the group of polyenes, Azo Compounds, Carboximides / Nitro Compounds / Quinacridones, Chinoids, Oxazines, indigoids, diphenylmethanes / triphenylmethanes and their derivatives, Polymethines, porphyrins / phthalocyanines, metal complexes, in particular the Precious metals, rare earths or transition metals, conjugated betaines and multiple chromophores. Especially preferred contains the particle is a combination of coumarin and rhodamine derivatives as fluorophores.

One Testkit, which builds on the aforementioned particles, contains two or more batches of particles, each batch being the same Fluorophores but with different concentration ratio included. Naturally can also be different test kits with different fluorophores or combine fluorophore combinations. The particles of different Batches can accordingly have the same size and be composed of the same components; they differ differ from each other in the concentration ratio of the fluorophores. The Particles of the various batches can be determined with the aid of the identification method according to the invention in even closer Illustrated Different way. In other words, in one to be measured Sample can be a large number of particles of the same size, the all also contain the same fluorophores. By the special Selection and definition of concentration ratios independent of However, it is possible for the total amount of fluorophores to be individual particles attributable to the known batches. Another aspect of the invention is therefore aimed at a test kit containing two or more batches of Particles of the aforementioned embodiment contains.

One Another aspect of the invention is to provide a Process for producing such particles. The inventive method provides that the particles by polymerization of a solution or molecular or colloidally dispersed dispersion of the fluorophores in a monomer / monomer mixture he follows. In other words, the fluorophores are initially dissolved in the monomer or at Preparation of a copolymer - im Monomer mixture before. Unless they dissolve sufficiently, lie they are preferably molecularly disperse (particle size <1 nm) or at least colloidally disperse (Particle size 1 nm up to 1 μm) in front. This ensures that the fluorophores in the produced Particles are monodisperse distributed, so interactions that the Emission characteristics could be avoided.

Preferably the polymerization is carried out under free-radical conditions, in particular takes place a thermally induced free radical polymerization at 20 to 90 ° C. The conditions mentioned on the one hand allow the use of a Variety of known fluorophores, which under the experimental conditions mentioned are stable and allow on the other hand the recourse to known manufacturing methods for monodisperse microparticles. Preference is given to the polymerization of a or more of the monomers selected from the group consisting of substituted or unsubstituted Acrylates, vinyls and allylene used. It is also conceivable one ionic polymerization.

Further is preferred when the solution / dispersion one or more surface-active Substances are added.

Finally will preferred when the solution / dispersion Polyvinylpyrrolidone (PVP) and / or a small amount of other polymers such as polyethylene glycol (PEG) is added.

• (i) Bestrahlen von vorzugsweise in Monoschicht vorliegenden Partikeln mit einer oder mehreren Anregungswellenlängen im Überlappungsbereich der Absorptionsspektren der Fluorophore bzw. im Bereich der Absorptionsmaxima der Fluorophore (+/– 10% der Extinktion);
• (ii) Erfassen der Intensitäten der Fluoreszenz an vorgegebenen Messpunkten, wobei für jeden im Partikel enthaltenen Fluorophor wenigstens ein Messpunkt vorgegeben wird, der in einem Bereich von mindestens 5% der Intensität des Fluoreszenzmaximums des jeweiligen Fluorophors liegt und wobei die Messpunkte um mindestens 5 nm voneinander beabstandet sind;
• (iii) Bestimmen eines Intensitätsverhältnisses der an den Messpunkten erfassten Intensitäten; und
• (iv) Vergleichen des bestimmten Intensitätsverhältnisses mit einem Referenzwert zur Identifikation des Partikels.

Another aspect of the invention is directed to an identification method of particles of the aforementioned embodiment in a sample. The identification method comprises the steps:

• (i) irradiating particles which are preferably present in monolayer and having one or more excitation wavelengths in the overlap region of the absorption spectra of the fluorophores or in the region of the absorption maxima of the fluorophores (+/- 10% of the extinction);
• (Ii) detecting the intensities of the fluorescence at predetermined measuring points, wherein for each fluorophore contained in the particle at least one measuring point is set, which lies in a range of at least 5% of the intensity of the fluorescence maximum of the respective fluorophore and wherein the measuring points by at least 5 nm from each other are spaced apart;
• (iii) determining an intensity ratio of the intensities detected at the measurement points; and
• (iv) comparing the determined intensity ratio with a reference value for identification of the particle.

To the identification method according to the invention are therefore first in step (i) ideally in a monolayer present particles irradiated, in a wavelength range in which the fluorophores be stimulated to issue. The particles can only be identified if they are each directly accessible with the excitation light. Not directly accessible particles, the identification u. U. disturb and represent in any case an unused material use. It is therefore desirable to arrange the particles such that a preferably size Number of particles present in a sample directly irradiated can be. About that In addition, it is desirable that many particles in the shortest possible time, d. H. if possible several particles simultaneously, directly excited and identified become. These criteria lead to that the optimal distribution of particles in a monolayer exists and not, as in conventional Method (eg, flow cytometer), in the separation of the particles.

to Creating a monolayer will be in a solution Particles distributed in a microtiter plate. The bottom of the cavities of the Microtiter plate is prepared in such a way that the particles stick to it. Non-sticky Particles are taken off again and can be used at another time be used for evaluation.

One sufficiently accurate identification result is also achievable if a second particle layer over the interstices of the Monolayer lies. In the case, both the lower and the the overhead particles identifiable. Last it is too possible, the particles in solution, so random distributed, assign. However, it is disadvantageous in that the detection of moving particles is more complex and time-consuming and on top of each other lying particles u. U. can not be clearly identified and therefore not available for evaluation.

Provided the absorption spectra of the fluorophores have an overlap area, in the for each fluorophore has an extinction greater than 0.001 and a quantum yield greater than zero is preferably irradiation in step (i) is carried out using a single excitation wavelength, in the overlap area lies.

In the subsequent one Step (ii) becomes the intensities the fluorescence detected at predetermined measuring points. These measuring points are at the fluorescence maximum or in a wavelength range at least 5%, preferably at least 50%, more preferably at least 90% of the intensity of the Fluorescence maximums are present for every single fluorophore. In other words, the sample contains two Fluorophores, so at least two measuring points are to be specified; one each for the first and one for the second fluorophore. Naturally can To increase the accuracy of measurement also several measuring points in the above Wavelength range be assigned to the respective fluorophore, but this is in usually not necessary. If the above conditions for the Position of the absorption and fluorescence maxima are observed, can the intensities clearly detected at the measuring points.

in the Step (iii) becomes the intensities previously detected at the measurement points put into proportion. The thus determined intensity ratio becomes in step (iv) compared with a stored reference value and allows an identification of the particle. That is, for the individual Batches of particles with fluorophores of different concentration ratios be prepared in advance by reference values by the intensities of the Measure measuring points and set in relation to each other. The inventive method is characterized by the fact that the intensity ratios already a clear identification of the particles different Batches possible is. The apparatus for carrying out the spectroscopic investigation as well as the identification procedure even only once has to go to a very specific spectral system set / optimized to a variety of different Identify particles. This is a great advantage of the method according to the invention.

When Further identification parameters can be the fluorescence lifetime be used. In other words, in step (ii), for selected fluorophores additionally a fluorescence lifetime are detected. This is special then makes sense, if an additional Fluorophore (eg n-th dye) to increase the number of distinguishable Particles is used and this fluorophore is not sufficient of the other fluorophores have different properties. absorption and fluorescence behavior.

It is also conceivable that the particles to be measured consist of batches of different diameters and in step (ii) additionally an absolute intensity of the fluorescence is detected. In step (iv), a particle diameter is then determined on the basis of the detected absolute intensity of the fluorescence by comparison with a reference value. Taking into account this parameter, it is therefore possible to use particles of the same fluorophores and the same concentration ratios of the fluorophores which, however, are present differ in diameter. The difference in the particle diameter causes a different absolute total amount of the fluorophores, which in turn leads to differences in the absolute intensity of the fluorescence (at the measuring points or in a predetermined wavelength range). The latter difference then serves as the distinguishing criterion for the particles of different sizes.

simultaneously also calls for a different absolute total amount of fluorophores with the same particle size one each different intensity which were used in the described way for particle identification can be. In other words, the microparticles to be measured can same diameter but different absolute amounts of fluorophore In addition, an absolute intensity is detected in step (ii) and assigned to the respective batch.

Lie in a sample particles of the same dye coding and the same absolute total chromophore levels but different Particle diameter, in addition to detecting the intensities of the Fluorescence carried out an optical object identification. With others Words, if the particles to be measured from batches of different Diameter and same absolute amounts of fluorophores exist may additionally to detect the absolute intensity of the fluorescence, an optical object identification for determining the particle surface or the particle diameter carried out become.

The recorded absolute intensity the fluorescence as area integral is a function of particle size and the contained total amount of fluorophores. An identical detected intensity So the fluorescence of two particles can be both from the actual Equality of the objects as well as differences in the particle size at the same absolute amount of dye result and thus leaves no clear conclusions the particle diameter too.

Over a Evaluation of the projection of the emitted light on a picture surface can the particle surface or the diameter can be determined in such cases. The capture the absolute intensity together with the determination of the particle diameter then makes a clear distinction possible.

A alternative detection of particle size is in the detection of the backscattered Excitation light. The detection of the particle size can both, as described, for distinguishing different populations as well as for correction purposes be used. In other words, the particles to be measured can consist of batches of different diameters and in the step (ii) will then be additional an intensity of the backscattered Excited light detected and in step (iv) based on the detected intensity of the backscattered Excitation light a particle diameter by comparison with a Reference value determined.

in the Step (ii) preferably additionally becomes a surface potential (eg ζ potential) certainly.

Prefers is further that the two or more fluorophores of a particle an overlapping one Have absorption and irradiation in step (i) below Using a (in the overlap area) or more excitation wavelengths he follows. With a single excitation source can be simultaneously in the case of the above criteria, both fluorophores fluoresce stimulate, so that the identification process can be accelerated can. In any case, however, a very high identification accuracy (low Deviation). Preferably, the excitation wavelength is in the case of two present fluorophores at the intersection of the absorption spectra and in the area around the point of intersection with a difference of up to 10% of the value of the extinction on Intersection. In the case of three or more fluorophores, all must Fluorophores have a common excitation wavelength range, wherein in this area for everyone Fluorophore has an extinction of at least 0.001, preferably 0.1 to 1, and a quantum yield of at least 0.01, preferably 0.5 to 1, and the irradiation in step (i) using a single excitation wavelength takes place in the overlap area lies.

The Excitation can therefore be with a single or with different Light sources or different wavelengths take place.

When Light sources can Lamps (gas discharge lamps possibly in combination with appropriate Filters), lasers, LEDs be used.

Brief description of the figures

The invention will be described below with reference to two embodiments and the associated Fi explained in more detail. Show it:

1 to 3 Absorption and fluorescence spectra of the Coumarin 314 / Rhodamine 6G system at two different concentration ratios, showing possible excitation with only one wavelength (abscissa: wavelength [nm]; ordinate: intensity); and

4 Fluorescence behavior in PMMA at 11 different mixing ratios of a system of fluorophores Coumarin 334 / rhodamine 6G, which was excited at two wavelengths (abscissa: concentration ratio of the fluorescent dyes, ordinate: intensity ratio of the detected intensities at 550 nm and 490 nm).

5 Particles of the dye system coumarin 334 / rhodamine 6G of different diameters and at different concentration and thus determined intensity ratios (abscissa: particle diameter [μm]; ordinate: intensity ratio of the determined intensities at 550 nm and 490 nm).

Detailed description of embodiment - production the particle

In general, all methods which produce adjustable particles by reaction in size can be used; specifically for precipitation polymerization, emulsion polymerization and suspension polymerization. The production of particles will be explained in more detail on the basis of the precipitation polymerization. Reagents used for the polymeric matrix: methanol 70 ml Methyl methacrylate (MMA) 10 ml Polyvinylpyrrolidone (PVP K-30) 5 g Sodium bis (2-ethylhexyl) sulfosuccinate (Aerosol OT) 320 mg Azobisisobutyronitrile (AIBN) 40 mg

PVP and aerosol OT were presented dissolved in methanol. The solution became a solution of the radical initiator AIBN added in the monomer MMA. Subsequently was at 65 ° C Bath temperature for Stirred for 24 h at about 100 rpm. The still warm suspension was poured into 300 ml of water, and the rainfall over sucked a frit and washed with water.

Fluorophors used:
Rhodamine 6G and coumarin 314 and 334, respectively

The fluorophores were added in the amounts specified in Tab. 1 of the solution of AIBN in MMA. Particle number. Rhodamine 6G [mg / g plastic] Coumarin 334 [mg / g plastic] determined intensity ratio 1 0,475 0,015 2748 2 0,375 0,075 15 3 0,250 0,150 4 4 0,125 0.225 1 5 0.063 0,270 0.5 6 0.031 0,356 0.18 7 0.028 0.422 0.17 8th 0.016 0,366 0.12 9 0,006 0.422 0.04 10 0,004 0.422 0.02 11 0,002 0.422 0.01 Tab. 1 - proportions of fluorophores

A total of 11 different concentration ratios of Coumarin 334 and Rhodamine 6G were prepared in said PMMA matrix (cf. 4 ).

The microparticles obtained by the process have a middle Diameter of approx. 9 μm.

identification method

To above-described process dye mixtures were gem. Tab. 1 introduced into the material. The resulting particles were on Glasslides attached and analyzed in the fluorescence spectrophotometer. The device used (Shimadzu RF-5301 PC) comes with a standard Xenon bulb which covers the entire UV / VIS spectrum.

Each specimen was exposed to light of wavelengths 470 nm and 525 nm and the values of the intensity of the emitted fluorescence radiation were determined at 490 nm and 550 nm, respectively. These intensity values were then set in relation to each other, the value obtained gives the identification parameter (cf. 4 ).

The 1 and 2 Exemplary excitation peaks and superimposed fluorescence spectra of the system coumarin 314 / rhodamine 6G can be seen. There are fluorescence spectra at two different concentration ratios, namely 1:15 ( 1 ) and 3: 1 ( 2 ), each shot at three different wavelengths.

The peak at 449 nm results from the irradiation at this wavelength and corresponds to the absorption maximum of Coumarin 314, 525 nm corresponds to the absorption maximum of rhodamine 6G. In the case of different concentration ratios present in a mixture, therefore, the total fluorescence intensity (integral) of one or the other fluorophore and also the intensity at selected measurement points dominates, as additionally in 3 illustrated.

Of the Peak at 470 nm results from the irradiation at the same wavelength, the the approximate Represents maximum of the common excitation range. Will the Mixtures of the fluorophores irradiated only at this one wavelength, sufficiently different fluorescence spectra arise, the an identification based on the ratio formation of intensities to two allow selected measuring points.

For the fluorescence Intensity ratios of (i) 2.43 (1:15) resulted in the measurement points and (ii) 0.30 (3: 1).

In the 4 are the intensity ratios at the different mixing ratios of the particle no. 1-11. The linear relationship of the two variables over a very wide range of concentrations can be seen. The ratio is therefore suitable as an identification parameter, possibly even without the deposit of concrete reference values, if the function is known.

5 shows particle populations that differ both in terms of set concentration and thus determined intensity ratio as well as in terms of their particle diameter.

Claims (29)

Particles made of a polymeric material, the two or more monodisperse fluorophores embedded in the material with absorption and fluorescence maxima in the UV / VIS / NIR spectral range contains where the fluorophores are selected are that (i) the absorption maxima of the fluorophores around each at least 5 nm apart; (ii) the fluorescence maxima the fluorophores spaced at least 10 nm apart are; and (iii) the absorption maxima and fluorescence maxima the fluorophores spaced at least 5 nm apart are. Particles according to claim 1, wherein the absorption maxima the fluorophores are spaced from each other by 10 to 100 nm. Particles according to claim 1, wherein the fluorescence maxima the fluorophores are spaced from each other by 20 to 100 nm. Particles according to one of the preceding claims, in the fluorescence maxima in the wavelength range from 400 to 900 nm lie. Particles according to one of the preceding claims, in the absorption maxima in the wavelength range from 350 to 850 nm lie. Particles according to one of the preceding claims, in the absorption spectra of the fluorophores overlap , wherein in this area for each fluorophore an extinction greater than 0.001 and a quantum yield greater than zero is. Particle according to claim 1, wherein the polymeric material insoluble in water is. Particles according to claim 7, wherein the polymeric material selected is selected from the group comprising polysilicates, polyadducts, polyacrylates, Polyolefins, their copolymers or blends. Particles according to claim 8, the polymeric material additionally contains a suspension stabilizer. Particle according to claim 1, wherein the particle has a Diameter in the range of 10 nm-2000 microns has. Particle according to claim 10, wherein the particle has a diameter in the range of 1-50μm. Particles according to claim 1, wherein the fluorophores organic or organometallic compounds or of a semiconductor material or metal oxide or metal sulfide are existing quantum dots. Particles according to claim 12, wherein the particle one or more fluorophores selected from the group of polyenes, Azo Compounds, Carboximides / Nitro Compounds / Quinacridones, Chinoids, Oxazines, indigoids, diphenylmethanes / triphenylmethanes and their derivatives, Polymethines, porphyrins / phthalocyanines, metal complexes, in particular of precious metals, rare earths or transition metals, conjugated Contains betaines and multiple chromophores. Particles according to claim 13, wherein the particle a combination of coumarin and rhodamine derivatives as fluorophores contains. Test kit containing two or more batches of particles according to one of the claims 1 to 14, wherein the batches each with the same fluorophores with however, contain a different concentration ratio. Process for the production of particles from a polymeric material that is two or more monodisperse in the material embedded fluorophores with absorption and fluorescence maxima in the UV / VIS / NIR spectral range, the fluorophores being so selected are that (i) the absorption maxima of the fluorophores around each at least 5 nm apart; (ii) the fluorescence maxima the fluorophores spaced at least 10 nm apart are; and (iii) the absorption maxima and fluorescence maxima the fluorophores spaced at least 5 nm apart are, and wherein the particle by polymerization of a solution / molecular or colloidally dispersed dispersion of the fluorophores in a monomer / monomer mixture he follows. The method of claim 16, wherein the polymerization under radical conditions. The method of claim 17, wherein a thermal induced free radical polymerization is carried out at 20 ° C-90 ° C. A method according to claim 16, wherein for the polymerization one or more of the monomers selected from the group consisting from substituted or unsubstituted acrylates, vinyls and Allyl used. The method of claim 16, wherein the solution / dispersion one or more surfactants be added. The method of claim 16, wherein the solution / dispersion Polyvinylpyrrolidone (PVP) and / or a small amount of other polymers such as polyethylene glycol (PEG) is added. Method for identifying a particle according to one of the claims 1 to 14 in a sample, comprising the steps: (i) Irradiate preferably present in monolayer particles with a or more excitation wavelengths in the overlap area the absorption spectra of the fluorophores or in the range of the absorption maxima the fluorophore (+/- 10% the extinction); (ii) detecting the intensities of the fluorescence at given Measuring points, where for each fluorophore contained in the particle at least one measuring point which is in a range of at least 5% of the intensity of the fluorescence maximum of the respective fluorophore and wherein the measuring points by at least 5 nm apart; (iii) determining an intensity ratio the intensities detected at the measuring points; and (iv) Compare the determined intensity ratio with a reference value for identification of the particle. An identification method according to claim 22, wherein in step (ii) for selected Fluorophores in addition a fluorescence lifetime is detected. An identification method according to claim 22, wherein the absorption spectra of the fluorophores have a common excitation wave range , wherein in this area for each fluorophore an extinction of at least 0.001 and a quantum yield of at least 0.01 and the irradiation in step (i) using a single Excitation wavelength takes place in the overlap area lies. An identification method according to claim 22, wherein the particles to be measured from batches of different diameters and in step (ii) additionally an absolute intensity the fluorescence is detected and in step (iv) based on the detected absolute intensity the fluorescence of a particle diameter by comparison with a Reference value is determined. An identification method according to claim 22, wherein the particles to be measured the same diameter but different have absolute amounts of fluorophore and in step (ii) additionally an absolute intensity recorded and assigned to the respective batch. An identification method according to claim 22, wherein the particles to be measured from batches of different diameters and in step (ii) additionally an intensity of the backscattered Excited light is detected and in step (iv) on the basis of the detected intensity of the backscattered Excitation light a particle diameter by comparison with a Reference value is determined. An identification method according to claim 22, wherein the particles to be measured from batches of different diameters exist and have the same absolute amount of fluorophores and in addition to record the absolute intensity of fluorescence an optical Object identification for determining the particle surface or of the particle diameter is performed. An identification method according to claim 22, wherein in step (ii) additionally a surface potential (e.g. B. ζ potential) is determined.