DE202004002064U1 - Microarray carrier containing colored glass fluorescent standard, useful for preparing DNA or protein arrays for e.g. diagnosis, enables instrument calibration and comparability of measurements between laboratories - Google Patents

Abstract

Microarray carrier (1) comprises a substrate (2), preferably essentially non-fluorescent, and at least one standard (A) for fluorescent measurements, made of colored glass. An independent claim is also included for a biochip that comprises (1) and, on (1), at least one microarray of probe molecules.

Description

The present invention relates to a microarray carrier, which is a substrate and one or more standards for fluorescence measurements comprises, wherein the standard or the standards contains / contain a colored glass. Furthermore, the invention relates to a biochip which comprises a microarray carrier according to the invention.

The fluorescence spectroscopy has in the last few years especially in the diagnostic area and quality control greatly increased in importance. An area of application of particularly great importance is for example the biochip technology, binding tests using microarrays (e.g. DNA or protein microarrays) can be read out by means of fluorescence measurements.

The fluorescence is generally in arbitrary and relative units measured. Such measurements allow fluorescence intensities compare with each other, measured with one and the same device were. A comparison of measurements with different devices is difficult or impossible. This is particularly disadvantageous because comparisons of Measured values that are obtained in different laboratories are hardly possible. To the solution This problem therefore requires defined standards that can serve as a reference for individual measurement.

Described in the prior art Fluorescence standards usually consist of an organic material. These materials include following, usually together Disadvantages: photo-induced degradation, low chemical resistance and fluorescence fluctuations from standard to standard.

This also applies to a fluorescence calibration standard for the Microscopy, which is described in WO 01/059503. Doing so thin fluorescent Layer on a carrier plate used. A polyimide serves as the broadband fluorophore in the layer. WO 02/077620 also describes a fluorescence standard which one or more polymer layers on a substantially non-fluorescent solid support includes.

Furthermore, in US 4,662,745 Fluorescence standards for device calibration have been described, which consist of a plate with a fluorescent surface or coating. The fluorescence is based on atoms of rare earth elements, in particular lanthanoids, and their compounds (eg oxides, sulfates etc.), which are present in a brittle layer (glass-like, ceramic or porcelain-like).

WO 01/06227 describes fluorescent Particles described, which either as internal standards for Referencing fluorescence signals or as a marker for labeling and detection of biomolecules become. The fluorescent particles are metal-ligand complexes, preferably Transition metal compounds as a central atom, which in inert form in solid materials, such as inorganic Materials or organic polymers.

Fluorescence standards based on Glasses doped with rare earth elements or metal-ligand complexes often have the disadvantage that they are comparatively narrow Show fluorescence spectra. Such standards are therefore in Areas of application in which the detection over a spectrally broad As with DNA chip technology, the area is not satisfactory used.

Finally, in US 4,302,678 discloses a device calibration standard comprising a fluorescent glass containing uranium oxide as the fluorophore. The standard can only be used in the UV range.

Thus, the present invention based on the task of a new system for the calibration of array fluorescent devices and for the standardization of fluorescence measurements in microarray experiments To provide the disadvantages of known in the art Bypasses systems and in particular a comparability of means Fluorescence measurement enables data from microarray experiments.

This task is carried out in the protection claims featured embodiments solved the present invention.

According to the invention, it was surprisingly found that there are colored glasses in a special way as the standard for fluorescence measurements that in microarray experiments carried out will be suitable.

Thus, according to a first embodiment a microarray carrier comprising a preferred essentially non-fluorescent substrate, and at least one Standard for Fluorescence measurements containing a colored glass are provided.

The term "standard for fluorescence measurements" includes in particular the use as an intensity standard (Reference) for normalizing fluorescence intensities that measured when reading microarray binding tests, and the use as a calibration standard for testing fluorescence measuring devices in the context of microarray experiments (usually so-called array scanners) be used. Furthermore, it is used as a spectral Standard for microarray devices to think - about the property that the fluorescence is stronger at a wavelength or weaker than at a different wavelength.

The term “essentially non-fluorescent Substrate "means according to the invention that the substrate is not at or near the absorption and emission wavelengths of the or the fluorescence standards fluoresce so that the fluorescence of the standard or standards through the substrate is not measurable being affected.

The colored glass present in or on the microarray carrier is preferably a tarnishing glass, more preferably a colored glass, which contains nanocrystals of one or more semiconductor compounds. Such colored glasses have been used in the prior art as absorption partial edge filters due to their selectively decreasing absorption behavior (see, for example, DE-A-20 26 485, US 3,773,530 , DE-A-26 21 741 and DE-A-101 41 101).

The colored or fluorescent Nanocrystals (also referred to as nanocrystallites) are preferred in the used to equip the microarray carrier according to the invention Colored glasses, are distributed colloidally in the glass matrix and result from the specific manufacturing process, in which first the starting components mixed and melted. After that, the resulting one Glass in the desired Mold poured and cooled. First of all manufactured glass is essentially amorphous and is characterized by a defined temperature process of reheating and cooling (tempering) started up. While In this process, the fluorescent nanocrystals generally grow up to one size (diameter) from about 1 to about 10 nm.

More colored glasses that are used to equip the microarray carrier according to the invention can serve one or more fluorescence standard (s) Metal colloid glasses such as Cu or Au-doped glasses. Such metal colloid colored glasses are e.g. available from the applicant and include those of the SSG series, such as SSG 460, SSG 480, SSG 510, SSG 530, SSG 550, SSG 565, SSG 580, SSG 600 and SSG 625. The spectral properties of the metal colloid colored glasses also leave adjust in a manner known to a person skilled in the art.

Color glasses for use in the present invention are preferably those which contain nanocrystals made of Cd semiconductor compounds (one or more or mixed compounds), in particular CdS, CdSe or CdTe. A particularly preferred Cd semiconductor compound is CdSeS. Such colored glasses are in the prior art, for example in DE-A-20 26 485, US 3,773,530 . US 4,106,946 , DE-A-26 21 741 and DE-A-101 41 105 (cf. also EP-A-1 285 890, US-A-2003/0153451 and JP-A-2003146695), the disclosure content of these publications being explicit is included in the present description.

Further colored glasses which can preferably be used according to the present invention are those which contain nanocrystals of class I-III-VI compound semiconductors. Such glasses are described, for example, in DE-A-101 41 101 (see also EP-A-1 288 171) and DE-A-101 41 104 (see also EP-A-1 285 889, US-A-2003 / 0158029 and JP-A-2003119049). The disclosure content of these publications is therefore expressly referred to in the present invention. These glasses contain semiconductor compounds of the form M ^I M ^III Y ^II ₂ (with M ^I = Cu ⁺ and / or Ag ⁺ , M ^III = In ³⁺ and / or Ga ³⁾ as fluorescent centers, ie as dopants for imparting the fluorescent properties ⁺ and / or Al ³⁺ and Y ^II = S ^2– and / or Se ^2– ).

Of course, the colored glasses can be nanocrystals various types of semiconductor connections, for example. Cd semiconductor connections (one or more or mixtures} and semiconductor compounds of the class I-III-VI (also one or more or mixed compositions).

The sum of the compound semiconductors in the glass preferably at least 0.1% by weight, which is the minimum concentration corresponds to the light absorption of the colloidally distributed in the glass Nanocrystals is required. According to preferred embodiments of the present invention the sum of the compound semiconductors in the glass is more than 0.3% by weight, particularly preferably more than 0.5% by weight.

Preferred glasses for use according to the invention in the microarray carrier are silicate glasses with higher contents of ZnO and alkali oxide (s), in particular K ₂ O. Such glasses are generally referred to as zinc silicate or alkali zinc silicate glasses.

As mentioned above, lies ZnO also in larger quantities in front. ZnO increased the resistance to temperature changes of the glass. This property is for use as a fluorescence standard of considerable importance since the standard is irradiated with the usual in photoluminometers Radiation sources in general with considerable temperature radiation connected is. ZnO also supports the homogeneous formation of nanocrystals the doping material (i.e. the formation of the fluorescent centers) in the glass and allows thereby a homogeneous crystallite growth of the semiconductor doping in the tempering of the glass. ZnO is therefore for the size distribution of the nanocrystals and therefore for the spectral distribution of the emitted fluorescence of considerable Meaning why the fluorescence spectrum of a desired Standards can also be set using the modulation of the ZnO content is.

Glasses preferably used in accordance with the invention Nanocrystals from one or more semiconductor compounds of the Type I-III-VI show the following composition (in% by weight):

Such glasses and preferred embodiments thereof and their production are described in DE-A-101 41 104 (cf. also EP-A-1 285 889, US-A-2003/0158029 and JP-A-2003119049), whose related Disclosure content expressly included in the present invention is.

Further colored glasses which can preferably be used according to the invention have the following composition: SiO ₂ 50 to 62 ZnO 13.5 to 37 K ₂ O 10 to 25 Na ₂ O 0 to 14 Al ₂ O ₃ 0 to 2 B ₂ O ₃ 3 to 5 F 0 to 1 TiO ₂ 0 to 7 In ₂ O ₃ 0 to 2 SO _3rd 0 to 1 SeO ₂ 0 to 1 C 0 to 1 M ^I M ^III Y ^II ₂ 0.1 to 3 in which: M ^I Cu ⁺ and / or Ag ⁺ M ^III In ³⁺ and / or Ga ³⁺ and / or Al ³⁺ and Y ^II S ^2– and / or Se ^2– means, and wherein at least 0.1 wt .-% of the oxide (M ₂ O ₃ ) or M ^III , which is / are present in M ^I M ^III Y ^II ₂ and at least 0.2 wt .-% of Oxides of the or the Y ^II ₂ , which is present in M ^I M ^III Y ^II ₂ , are present.

Such colored glasses with one or more Semiconductor compounds of type I-III-VI are in DE-A-101 41 101 (cf. also EP-A-1 288 171), on the disclosure content thereof, in particular with regard to preferred embodiments and production methods, express reference is made in the present description.

In the case of the ternary semiconductor systems of the form M ^I M ^III Y ^II ₂ , all edge components, for example CulnS ₂ , CulnSe ₂ , CuGaS ₂ , CuGaSe ₂ , AglnS ₂ , AglnS ₂ , AgGaS ₂ and AgGaSe ₂ as well as all mixed compounds of two - or multi-component systems can be used. Two or more edge components can also be used simultaneously.

Color glasses with dopants of one or more components from the system Culn (Se _1-x S _x ) ₂ with x = 0 to 1, that is to say from the edge components CulnS ₂ and CulnSe _2, and their mixed compounds, are particularly preferred, these components preferably having a content of 0.1 to 0.5 wt .-% are present. Further preferred colored glasses with nanocrystals of semiconductors of the classes I-III-VI contain coloring (fluorescent) components of the form CuAl (Se _1-x S _x ) ₂ with x = 0 to 1, ie from the edge compo nents CuAlS ₂ and CuAlSe ₂ and their mixed compounds. According to a particularly preferred embodiment, these components are present with a content of 0.1 to 0.5% by weight.

Colored glasses with Cd nanocrystals used according to the invention have, for example, the following composition (in% by weight on an oxide basis): SiO ₂ 40 to 60 B ₂ O ₃ 5 to 20 P ₂ O ₅ 0 to 8 Al ₂ O ₃ 1.5 to 6.0 Na ₂ O 4 to 8 K ₂ O 6 to 14 ZnO 4 to 12 BaO 0 to 6 CdO 0.2 to 2.0 S 0.2 to 1.0

Such glasses are in US 3,773,530 described, the disclosure content of which is expressly referred to in the present invention.

Further colored glasses with Cd semiconductor compounds which can be used according to the invention are shown in US 4,106,946 described, the disclosure content of which is fully included in the present invention. As a fluorescence standard, these colored glasses have the following composition (in% by weight): SiO ₂ 40 to 63 Alkali metal oxide (e) 8 to 30 B ₂ O ₃ 0 to 9 P ₂ O ₅ 0 to 2.5 Al ₂ O ₃ 0 to 1.5 ZnO 14 to 30 TiO ₂ 0.9 to 9 F 0 to 2 CdO 0 to 2.0 CdTe 0 to 2.5 ZnS 0 to 1.65 CdS 0 to 2.0 S 0 to 0.9 se 0 to 0.9 wherein the content of CdO, CdTe, ZnS, CdS, S and Se or any combination thereof is at least 0.5% by weight.

Further colored glasses which can be used according to the invention and which contain nanocrystals of one or more Cd semiconductor compounds) have the following composition: SiO ₂ 40 to 63 Alkali metal oxide (e) 8 to 30 B ₂ O ₃ 0 to 9 P ₂ O ₅ 0 to 2.5 Al ₂ O ₃ 0 to 1.5 ZnO 14 to 30 TiO ₂ 0.9 to 9 F 0 to 2 CdO 0 to 2.0 CdTe 0 to 2.5 ZnS 0 to 1.65 CdS 0 to 1.0 S 0 to 0.9 se 0 to 0.9 As ₂ S ₃ and / or As ₂ S ₄ 0 to 1.5 Sb ₂ S ₃ and / or Sb ₂ S ₄ 0 to 1.5 wherein the content of CdO, CdTe, ZnS, CdS, S and Se or any combination thereof is at least 0.5 % By weight.

Regarding these glasses and preferred embodiments as well as manufacturing process is expressly referred to DE-A-26 21 741 referenced, their related Full disclosure amount is included in the present invention.

According to a further preferred embodiment, colored glasses are used as the fluorescence standard, which have the following composition (in% by weight on an oxide basis): SiO ₂ 30 to 75 K ₂ O 5 to 35 B ₂ O ₃ > 4 to 17 ZnO 5 to 30 F 0.1 to 1 CdO 0.1 to 1 S + Se + Te 0.1 to 1.5

The above colored glasses are in DE-A-101 41 105 (cf. also EP-A-1 285 890, US-A-2003/0153451 and JP-A-2003146695), the disclosure content of which, in particular regarding preferred embodiments and manufacturing process of the colored glasses described there, completely in the present description is included.

Specific compositions, in particular from in the red, orange and yellow range of the visible spectrum fluorescent tarnish glasses can can be found in the publications mentioned above.

In order to improve the glass quality, one or more customary refining agents, for example Na ₂ SO ₄ , KNO ₃ and the like, can be added in customary amounts to the colored glass batches which are used for producing fluorescence standards according to the invention. The purification promotes the inner glass quality with regard to freedom from bubbles and streaks.

The spectra of those absorbed by the standard and emitted radiation, i.e. the spectral properties of the Standards, hang especially the bandgap of the semiconductor compounds that make up the nanocrystals) from: the smaller the band gap, the longer wavelength the radiation emitted. A common one Criterion for the optical properties of the colored glasses, which are characterized by a low Transmission in the short-wave range and high transmission in the higher Wavelength range distinguish is the so-called edge wavelength (also as edge position or Called steep edge position). It corresponds to the wavelength at which the spectral pure transmittance is half the Maximum transmission reached. For the cadmium chalcogenides can For example, the following values of the spectral properties can be specified (Wavelength the one for the Standard characteristic absorption partial edge = band gap / Planck's Constant):

The given in Tab. 1 above Values for the band gap the semiconductor are in reality due to the low Size of the crystallites occurring quantum effects to larger values postponed.

Thus determines the type of semiconductor connection and the relative concentrations of those building the semiconductor compound Components the size of the bandgap within of the nanocrystals and therefore fundamentally the spectral properties of the present in the microarray carrier according to the invention Standards.

The size of the nanocrystals also determines the bandgap of the semiconductors. The larger the crystallites, the smaller the band gap and the longer-wave ("red") the emitted radiation becomes. The formation of the colloidally distributed nanocrystallites in the glass matrix is during the "start-up process" mentioned process of color formation influenced by the glass toughness, the supersaturation, the solubility product and the growth rate of the fluorescence centers. The first crystallites can already form during the cooling of the glasses. Most of the time, however, the glass initially produced is essentially amorphous. The actual start-up process takes place through a further temperature treatment (tempering) in the temperature range of glass transition temperature (T _g ) + ≤ 150 K. The start-up temperature and time (i.e. the amount and duration of the temperature treatment) influence the crystallite size. Shorter annealing leads to smaller crystallites and shorter fluorescence wavelengths. Using these parameters, a person skilled in the art can thus finely control the spectral properties of the fluorescence standards present in the microarray carrier according to the invention for a given nanocrystal composition. According to the invention, a crystal size (diameter) of etwa approximately 10 nm, in particular approximately 3 to approximately 10 nm, is preferred, since above approximately 10 nm undesirable scattering effects occur. Colored glasses used according to the invention can thus be excited in the wavelength range between approximately 320 nm and approximately 1200 nm, preferably between approximately 480 nm and approximately 750 nm. Accordingly, the absorption maxima of the colored glasses according to the invention lie in these areas. The emitted fluorescence can be between UV values and the near IR range. The fluorescence maximum of the colored glass is therefore preferably between approximately 380 nm and approximately 850 nm, more preferably between approximately 500 nm and approximately 770 nm.

Colored glasses to be used according to the invention, the Cd semiconductor compounds are particularly suitable to Fluorescence emissions in the wavelength range to provide from 400 to 850 nm. By varying the above parameters the spectral range can be set in the desired range.

Cd-containing colored glasses according to the invention, the a shorter wave Fluorescence (about 400 to about 500 nm) are also known as yellow glasses designated and are, for example, under the designations GG 400, GG 420, GG 435, GG 455, GG 475 and GG 495 available from the applicant. Fluorescence in the range of e.g. for example, about 500 to about 600 nm. with the so-called orange glasses OG 515, OG 530, OG 550, OG 570 and OG 590 realized by the applicant become. In the longer wave Range (about 600 to about 850 nm) are e.g. which are referred to as red glasses glasses RG 9, RG 610, RG 630, RG 645, RG 665, RG 695, RG 715, RG 780, RG 830 and RG 850 are available, available from the applicant are. As a particularly inventive preferred example is the orange glass OG 550 with the one in Tab. 3.2 specified composition.

The colored glasses used according to the invention can be produced, for example, essentially by methods known to a person skilled in the art (see, for example, DE-A-20 26 485, US 3,773,530 . US 4,106,946 , DE-A-26 21 741, DE-A-101 41 105, DE-A-101 41 101 and DE-A-101 41 104). Such manufacturing processes begin with the melting of the glass composition (the steps of melting the batch, refining, homogenizing and conditioning are recorded here). The melting takes place in ceramic crucibles (harbors) at temperatures of approximately 1100 to approximately 1550 ° C., preferably in the range of approximately 1200 to 1360 ° C. The bright melting (refining) is preferably carried out at a somewhat lower temperature, for example at about 1300 to about 1340 ° C. for about 1 to about 2 hours. After the resting phase (usually about 3 to about 4 hours), the temperature is usually reduced to about 1230 ° C., the melt being homogenized. The casting is typically carried out at a desired shape at about 950 to 1000 ° C. The glass is then cooled, the nanocrystallites according to the invention being able to form completely or partially. Possibly. the glass can also be rolled. According to the invention, the color or fluorescence centers are preferably formed in the glass matrix essentially by the subsequent tempering of the glass, that is to say reheating and cooling, for example at temperatures from approximately 500 to approximately 720 ° C., preferably at approximately 560 to approximately 720 ° C. , for about 1 hour to about 2 weeks, and slowly cooling to room temperature (for example cooling by about 1 to about 10 K per hour in electric or gas-fired ovens). As already explained above, given the composition of the glass melt and thus the resulting crystallites, their size can be controlled in particular by the duration and amount of the temperature treatment, which makes it possible to set the desired spectral properties of the colored glass relatively finely, the excitation and emission wavelength of the resulting standards deviates from the desired values by no more than about 6 nm, more preferably no more than about 3 to about 5 nm.

With the method described above, the standards for the microarray carrier according to the invention are usually produced as solid glasses. A solid glass standard for the microarray carrier of the present invention can be attached to a substrate, for example made of glass. It is preferably one or more essentially non-fluorescent glasses. Methods known per se such as “low-temperature bonding”, “fusion in a glass slot”, gluing and “singeing” can be mentioned as suitable joining methods. For example, the (usually non-fluorescent) carriers used in the field of biochip or microarray technology can be used as carriers. Substrates, preferably those made of glass, are equipped with a fluorescence standard according to the invention. This can advantageously be done by part of the (glass) substrate used, for example. removed by sawing off and replaced by an equally large fluorescence standard with the desired spectral properties. This makes it possible to adhere to the specified standard dimensions (usually microarray carriers or biochips with dimensions of approximately 75 × 25 × 1 mm) that are used in microarray technology. Of course, the removed substrate or generally sample piece can be replaced by several standards with different compositions, so that the finished product contains standards with different spectral properties. Preferably one or more standards will be present in one or both end regions (in particular on the narrow side (s) of an object with a rectangular surface, as is the case with conventional microarray carriers or biochips) of the otherwise non-fluorescent substrate , In the case of conventional microarray carriers or biochips, the dimensions of the product made of substrate, preferably non-fluorescent glass, plus solid colored glass (and possibly applied coatings and probe molecules), in particular of about 75 mm × 25 mm × 1 mm, are retained.

The colored glass used according to the invention can also as thinner Film with a thickness of a few nanometers to a few micrometers, preferably about 3 nm to about 20 μm, which is on a Substrate, e.g. an essentially non-fluorescent glass substrate, is applied. In this embodiment can the glass film by known deposition methods, such as Sol-gel process, screen printing process (with organic or inorganic Binders) or sputtering, if necessary with subsequent tempering steps, getting produced. Such a fluorescence standard in the form of a Glass films can also be used in several flat Individual segments are structured on the substrate. The structuring of the thin film standard can e.g. directly by screen printing or by masking of the substrate. Furthermore, a structuring of the Glass films by etching techniques possible. With such structured standards, further test options can be used for microarray fluorescence measuring devices, e.g. the areal Resolving power provided become.

Furthermore, it is possible to use fluorescence standards according to the invention as thin glass manufacture and as thin glass flakes with a thickness of, for example, about 50 to about 200 μm, typically about 100 μm, to a suitable one Apply substrate, preferably non-fluorescent glass. This embodiment can, for example, by sticking the thin glass will be realized.

In principle, the fluorescence standard in the microarray carrier serve two functions. The standard can firstly be used to check the respective microarray fluorescence device used become. Here, with the help of the colored glasses in particular the uniformity, the linearity and the areal Resolution of the meter getting tested. Furthermore, the device can be timed Variability over e.g. days, Weeks, months or years to be checked. The microarray carrier with the fluorescence standard, secondly serves to standardize uncalibrated Fluorescence intensities which allows a general comparability with microarray experiments measurements obtained becomes. The equipment of the microarray carrier according to the invention with one or more colored glass fluorescence standard (s) allows simultaneous Standardization of those measured in the sample (s) of the biochip Fluorescence signals.

One made using the colored glasses according to the invention Standard can advantageously with different fluorescence or fluorescence intensities to be provided. The intensity emitted by the standard essentially depends on the intensity the excitation radiation and the amount of the excited material. The excitation intensity (e.g. the intensity of a laser) is a purely external influence, whereby excitation and fluorescence intensity usually linearly related. Therefore, by a change the excitation intensity the fluorescence intensity be modulated and so e.g. the linearity of the detection can be tested.

According to the color glasses as Fluorescence standards for Microarray carrier through suitable choice of the concentration or absolute amount of the excited and thus fluorescent centers with different emission intensities become. After the standards have been calibrated, they can be used to: the measuring range or the linearity of the detection of an unknown equipment to consider. The Concentration of the fluorescent centers can in particular by change the glass composition and / or the conditions chosen in the glass production (e.g. temperature and duration of the tempering) can be modulated. If the colored glass according to the invention as thin Layer, in particular as a glass film, the intensity of the emitted Radiation can also be changed by varying the layer or film thickness. This possibility is based on the fact that with a thin layer, for example a glass film above thickness, not all the stimulating light but only one Part of it is absorbed. The non-absorbed part is transmitted. With a thicker film you can therefore more fluorescence centers are excited, which is why Schichtbzw. Film thickness the intensity of that emitted by the standard Light can be controlled. Thus, by using the colored glass according to the invention the concentration of the fluorescent centers (nanocrystallites of one or more semiconductor compounds) and / or the film thickness that the radiation emitted by the standard (at given excitation intensity) in an intensity range which is the range of the fluorescence intensities of the corresponds to the samples to be measured.

Microarray carriers according to the invention which provided with one or more fluorescent standards made of colored glass are, can therefore adapted to the respective measurements in two ways become. On the one hand, as described above, the spectral range of the glass, i.e. Absorption and emission properties, in particular Absorption maxima and fluorescence maxima can be set in such a way that the spectral parameters in a similar Range, as are the corresponding parameters of a microarray measurement fluorescent materials used, especially organic and / or biochemical fluorophores. Preferably soft the spectral properties, such as fluorescence maxima and / or absorption maxima, by no more than 20%, more preferably no more than 10%, especially preferably not more than 5%, in particular not more than 1%, of the fluorescence maxima or absorption maxima, which fluorescence centers, mostly organic and / or biochemical fluorophores, that are used in a given microarray measurement. On the other hand the standard, if it is, for example, a thin layer, can be given excitation intensity a fluorescence intensity have, which is in the range of the medium intensity, measured on a given microarray system. Preferably gives way to the intensity the radiation emitted by the colored glass (for a given excitation intensity) by no more than 20%, more preferably not more than 10%, particularly preferred not more than 5%, especially not more than 1%, of the middle fluorescence intensity from that measured in a typical measurement in the microarray range becomes.

That in its optical properties Targeted in a wide range compared to other standards changeable Colored glass for use in the present invention can therefore used in a variety of ways as a fluorescence standard in microarray experiments become. The color glass also contributes to this versatility in many different forms, e.g. as solid glass, glass film or thin glass, can be produced.

First of all, all are suitable embodiments very good as a reference for comparing the intensity of a measured in a sample Signal. You can also exams long-term stability microarray fluorescence measuring devices, in particular array scanners, be made. This is particularly the temporal and chemical practically unchangeable stability the optical properties of the colored glasses from of central importance.

As already indicated above, the areal resolving power of the array measuring device can be tested with a structured colored glass standard, which is preferably present as a glass film or thin glass on a non-fluorescent substrate. For example. For this purpose, the standard is structured into individual segments with dimensions of approximately 9 μm ² to approximately 12 mm ² , the surfaces taking on, for example, square shapes of approximately 3 μm × 3 μm or round with diameters of approximately 3 μm to approximately 2 mm can be present. Furthermore, existing color glasses for checking the uniformity of fluorescence measurements on two-dimensional array measurement surfaces can be used as large-area standards, here preferably as solid glass, glass film or thin glass.

Microarray carriers according to the invention also serve colored glasses as standards for testing of linearity of microarray devices equipped with fluorescence spectrometers. By suitable choice of the composition of the fluorescence standard achieved that intensity of the emitted radiation (given the excitation intensity) Ranges can be varied, which enables the linear range of the item to be checked Determine array measuring device.

Another area of application is in the so-called “Cross Talk "in an array meter used according to the invention Colored glasses. Here it becomes due to the known, essentially in terms of time allows no fluctuations in the inferior fluorescence spectrum, the accuracy of the delimitation of different fluorescence detection channels from one another to consider.

A particularly advantageous point of view when using the inventive, with colored glass fluorescence standards equipped microarray carrier or Biochips is the possibility the comparison of fluorescence measurements in different laboratories. Because the intensities due to an increased Effort is usually not measured in absolute units, represents a common color glass standard, preferably containing Nanocrystals of one or more semiconductor compound (s), one Reference, with the measurement results of different laboratories (those with different devices were collected) can be compared.

Finally, with known quantitative Composition of the standard according to the invention a conversion of the fluorescence signal in efficiencies (e.g. fluorescence per molecule and area).

As already explained above, according to the invention, in one Microarray carriers used colored glasses with Spectral properties that can be changed in a wide range getting produced. Therefore, the material to be examined can be found in stimulated a suitable spectral range, the fluorescence measured and be compared to the standard.

Microarray carriers according to the invention with colored glass standards can advantageously be used in quality control. Here the material to be tested is usually excited in the UV range at wavelengths below 400 nm, for example at approximately 320 ± 15 nm or below, and at wavelengths around 400 nm, for example 380 ± 15 nm nm, but possibly also below or above it, the emitted (fluorescent) radiation is detected. According to a preferred embodiment of the present invention, the colored glass standard to be used in the microarray carrier is set in accordance with the spectral properties required in this area of application by suitable selection of the glass composition and temperature treatment (tempering). A person skilled in the art can readily provide the respective colored glass with the desired optical properties on the basis of known nanocrystallite or metal colloid colored glasses with the aid of routine tests and his general specialist knowledge. This applies to all embodiments of the present invention.

A particularly preferred area of application The color glasses described here are in the standardization of fluorescence signals that are detected in microarray measurements and in the calibration of measuring devices used in this area of application, in particular Array scanners. Accordingly, a microarray carrier according to the invention is also used at least one fluorescence standard comprising one or more Color glass / color glasses described above, preferably with microarray systems connected, which are applied in the biological-chemical field. Particularly noteworthy are laser-based systems, which preferably for high throughput processes are designed. Such systems are commonly used in materials testing, drug discovery ("high throughput screening ", HTS) and binding tests (e.g. DNA or protein microarrays) used.

Microarray applications are commonly used in the visible range of light up to the near IR emitting fluorescent dyes used, in which suitable derivatives of which are coupled to the molecules to be investigated become. In general, this is known in the prior art Fluorescence marker used. Such fluorescent markers are in the Trading, e.g. at Amersham Bioscience (Piscataway, NJ, USA; http://www.amershambioscience.com), Molecular Probes (Eugene, OR, USA; http://www.probes.com), Sigma-Aldrich (Munich, Germany; http://www.sigmaaldrich.com), Atto-Tec (Siegen, Germany; http://www.atto-tec.de) etc., available. Under the corresponding Internet sets of those known to a person skilled in the art Companies can the spectral and chemical properties of the respective fluorophores be retrieved.

In the field of microarrays (labeling of biomolecules, but also, where desired, of smaller organic molecules, e.g. compounds to be tested pharmacologically), Texas red, fluorescein, in particular fluorescein isothiocyanate (FITC), rhodamine and corresponding derivatives, and fluorescent markers from CyDye are often used ^® series (Amersham Bioscience). The essential spectral properties of these preferably used fluorophores are given as examples in Table 2.

Particularly preferred excitation wavelengths which are selected in microarray applications are around 530 nm, for example 532 nm, and around 630 nm, for example 635 nm (in each case usually ± 15 nm). The fluorescence is preferably detected at wavelengths around 580 nm and / or 680 nm, typically at 580 nm ± 15 nm and 680 nm ± 15 nm.

Therefore, the present in the microarray carrier according to the invention Standard preferably equipped with spectral properties, which is the case with microarray experiments fluorophores used correspond. Of course you can too Solid glass standards (as such or attached to suitable objects, preferably made of glass), glass films or thin glasses be designed that the end product as the standard for various spectral ranges and / or emission intensities can serve. So two can or more different standards made of solid glass (e.g. glued) and on substrates made of glass or plastic (see here also the above explanations), added become. It can also two or more different standards made of thin glasses on a suitable substrate be applied. The same naturally applies to glass films. So they can Signals from two or more different fluorescent markers during one individual measurement process can be standardized if the array measuring device accordingly is designed and emitted radiation in two or more different Detect channels can. Such measurements of double and multiple markings will be common in biochip technology carried out.

The provision of durable and consistent fluorescence standards with well-defined spectral Properties are particularly desirable in biochip technology. Just here the standards known in the prior art lack the Longevity, shelf life, chemical resistance, photo resistance and the consistent optical properties (both via the Time as well as from standard to standard) for those present in the microarray carrier according to the invention Colored glasses, the properties of which a person skilled in the art, as explained above, in can control specific areas, are characteristic.

Other specific embodiments of the carrier the present invention is known to a person skilled in the art. The carrier will preferably suitable coatings known in the prior art, e.g. Have silane derivatives, polymer coatings, including hydrogels, to anchor the probe molecules to be applied to the substrate. The microarray carrier can also accommodate multiple arrays (multiple arrays) be designed.

The fluorescence standard preferably forms (it can, as described above, there are also several) part of the substrate, i.e. one or more areas of the surface of the microarray carrier (on which in the case of a biochip, the probe molecules are applied). The standard (s) is preferably in one or more End areas) of the substrate. In a preferred form of Microarray carrier as already stated above, it is preferably objects made of glass, with dimensions of about 75 mm × 25 mm × 1 mm (i.e. a rectangular surface with 75 mm × 25 mm). In the case of one or more standards made of solid glass, this can be done the carrier be designed such that a corresponding end region from Substrate removed, for example cut, and the section so removed by a corresponding standard made of solid glass is replaced (gluing, scorching Etc.). Further preferred embodiments make microarray carriers where the standard (s) are at least as glass films or as thin glass in some areas on the substrate (preferably not at the corresponding wavelengths of the standard (s) fluorescent) is / are applied. Preferably are these again one or more edge or end areas) of the Carrier. On the surface of the substrate, which is intended to accommodate the array molecules there is advantageously no fluorescence standard.

Another subject of the present Invention is a biochip comprising one as defined above Microarray substrate, on which a microarray of probe molecules is applied. For this count protein microarrays, DNA microarrays, Saccharide microarrays, etc. It can several microarrays can also be applied to a biochip (multiple Arrays).

The figure shows:

1 shows exemplary embodiments ((A) to (D)) of a microarray carrier according to the invention in supervision.

A microarray carrier according to the invention ( 1 ) typically includes a substrate (generally glass) ( 2 ) on which at least one area ( 3 ) is provided to accommodate a microarray. In a biochip according to the invention, probe molecules are applied in a microarray arrangement in the microarray area. In the performance form according to 1A is in an end region (in the present case a narrow side of a rectangular embodiment) of the microrarray carrier ( 1 ) a fluorescence standard ( 4a ) applied from a colored glass (e.g. as thin glass or glass film) or in the form of a solid glass to the substrate ( 2 ) glued on. In the case of a substrate with dimensions of approximately 75 × 25 × 1 mm, such a region can comprise a surface of, for example, approximately 2 × 25 mm. If this is a solid glass standard, it will have corresponding dimensions of approximately 2 × 25 × 1 mm, whereby this can only form part of the microarray carrier of 75 × 25 × 1 mm or be attached to a substrate of this dimension (e.g. can be glued). In the embodiment according to 1B the colored glass fluorescence standard forms part of the narrow side of the microarray carrier. Such an embodiment is advantageously suitable for determining the orientation of the microarray carrier or the corresponding biochip. In a further embodiment ( 1C and 1D ) two fluorescence standards (e.g. standards of different absorption / emission spectra or standards of different intensities) are provided, the standards according to 1C are present on a narrow side of the support (for example two solid glass standards glued to one another or two thin glasses or glass films glued or otherwise applied to one another on the non-fluorescent substrate). The different fluorescence standards can also be on opposite sides of the support, as in 1D shown, are present, which in the embodiment shown is the narrow sides of the microarray carrier with a rectangular surface. Of course, more than two fluorescence standards can also be provided, the arrangement of which is shown in FIGS 1 shown variants may differ.

The following examples explain the present invention closer, without restricting them.

Examples

example 1

Production of a fluorescence standard for one Microarray substrate as solid glass With the components according to Table 3.2 below the orange glass OG 550 (see Example 10), the nanocrystals of the form CdSeS. The components are placed in a crucible at about 1300 ° C (about 1300 mm × 1300 mm × 200 mm) melted down (so-called port melt) and then poured into molds as well as rolled. The glass is then slowly cooled. at this cooling process few CdSeS aggregates form in an otherwise amorphous and colorless glass matrix. While the subsequent annealing at about 600 ° C for 1 to 3 days CdSeS nanocrystals. The end product contains about 0.4% by weight of nanocrystals in the glass matrix. While sublimate or decompose the melting process of the glass components more than 80% of the Cd chalcogenides, which is why the Cd, S and Se components in excess be used.

Examples 2 to 22

Composition of tarnish glasses as Fluorescence standards for Microarray carrier Compositions of visible, red, orange and yellow areas Spectrum fluorescent tarnish glasses is shown in the following Tab. 3.1 to 3.3 specified. Except for the red glass RG 9 the numbers in the name of the glasses in each case the so-called edge wavelength (Wavelength at which the transmission of the glass reaches half of the maximum value) on. glasses with the same initial composition, but different spectrals Properties can by varying the tempering conditions (temperature and duration) Routine trials will be provided.

The colored glass OG 550 (see example 10) is particularly suitable due to its spectral properties for use as a fluorescence standard for microarrays.

1

Microarray substrate

2

substratum

3

Microarray area

4a / b

Colored glass fluorescence standard

Claims (30)

Microarray carrier ( 1 ) comprising a preferably essentially non-fluorescent substrate ( 2 ) and at least one standard for fluorescence measurements ( 4a / b ), which contains a colored glass. Microarray carrier ( 1 ) according to claim 1, wherein the colored glass is a tarnishing glass. Microarray carrier ( 1 ) according to claim 1 or 2, wherein the colored glass contains nanocrystals of one or more semiconductor compounds). Microarray carrier ( 1 ) according to one of claims 1 to 3, wherein the semiconductor compounds) are selected from Cd semiconductor compounds and semiconductor compounds of the form M ^I M ^III Y ^II ₂ , where M ^I Cu ⁺ and / or Ag ⁺ M ^III In ³⁺ and / or Ga ³⁺ and / or Al ³⁺ and Y ^II S ^2– and / or Se ^2– , with the proviso that the sum of the semiconductor compounds in the glass is at least 0.1% by weight. Microarray carrier ( 1 ) according to claim 3 or 4, wherein the colored glass comprises a Cd chalcogenide, in particular CdS, CdSe or CdTe, or a mixed compound thereof. Microarray carrier ( 1 ) according to claim 5, wherein the colored glass comprises CdSeS. Microarray carrier ( 1 ) according to claim 3 or 4, wherein the colored glass has the following composition (in% by weight): SiO ₂ 30 to 75 K ₂ O 5 to 35 TiO ₂ 0 to 5 F 0.01 to 10 M ^I M ^III Y ^II ₂ 0.1 to 3 where: M ^I denotes Cu ⁺ and / or Ag ⁺ M ^III In ³⁺ and / or Ga ³⁺ and / or Al ³⁺ and Y ^II S ^2– and / or Se ^2– , and, if appropriate, conventional ones Refining agents in usual quantities. Microarray carrier ( 1 ) according to claim 3 or 4, wherein the colored glass has the following composition (in wt .-%): SiO ₂ 50 to 62 ZnO 13.5 to 37 K ₂ O 10 to 25 Na ₂ O 0 to 14 Al ₂ O ₃ 0 to 2 B ₂ O ₃ 3 to 5 F 0 to 1 TiO ₂ 0 to 7 In ₂ O ₃ 0 to 2 SO ₃ 0 to 1 SeO ₂ 0 to 1 C 0 to 1 M ^I M ^III Y ^II ₂ 0.1 to 3 where: M ^I Cu ⁺ and / or Ag ⁺ M ^III In ³⁺ and / or Ga ³⁺ and / or Al ³⁺ and Y ^II S ^2- and / or Se ^2- , and wherein at least 0.1 wt. -% of the oxide (M ₂ O ₃ ) of the M ^III , the / the is in the M ^I M ^II ₂ ^III Y / are present and at least 0.2 wt .-% of the oxide of Y or ^II _2, / die / be present in M ^I M ^II ₂ ^III Y are present, and optionally conventional Refining agents in usual quantities. Microarray carrier ( 1 ) according to claim 7 or 8, wherein the colored glass contains nanocrystals of a semiconductor compound of the form Culn (Se _1-x S _x ) ₂ , with x = 0 to 1. Microarray carrier ( 1 ) according to claim 7 or 8, wherein the colored glass contains nanocrystals of a semiconductor compound of the form CuAl (Se _1-x S _x ) ₂ , with x = 0 to 1. Microarray carrier ( 1 ) according to one of claims 3 to 6, wherein the colored glass has the following composition (in wt .-% on an oxide basis): SiO ₂ 40 to 60 B ₂ O ₃ 5 to 20 P ₂ O ₅ 0 to 8 Al ₂ O ₃ 1 , 5 to 6.0 Na ₂ O 4 to 8 K ₂ O 6 to 14 ZnO 4 to 12 BaO 0 to 6 CdO 0.2 to 2.0 S 0.2 to 1.0 and, if appropriate, customary refining agents in customary amounts. Microarray carrier ( 1 ) according to one of claims 3 to 6, wherein the colored glass has the following composition (in% by weight): SiO ₂ 40 to 63 alkali metal oxide (s) 8 to 30 B ₂ O ₃ 0 to 9 P ₂ O ₅ 0 to 2 , 5 Al ₂ O ₃ 0 to 1.5 ZnO 14 to 30 TiO ₂ 0.9 to 9 F 0 to 2 Cd0 0 to 2.0 CdTe 0 to 2.5 ZnS 0 to 1.65 CdS 0 to 2.0 S 0 to 0.9 Se 0 to 0.9, the content of CdO, CdTe, ZnS, CdS, S and Se or any combination thereof being at least 0.5% by weight, and optionally conventional refining agents in customary amounts. Microarray carrier ( 1 ) according to one of claims 3 to 6, wherein the colored glass has the following composition (in% by weight): SiO ₂ 40 to 63 alkali metal oxide (s) 8 to 30 B ₂ O ₃ 0 to 9 P ₂ O ₅ 0 to 2 , 5 Al ₂ O ₃ 0 to 1.5 ZnO 14 to 30 TiO ₂ 0.9 to 9 F 0 to 2 CdO 0 to 2.0 CdTe 0 to 2.5 ZnS 0 to 1.65 CdS 0 to 1.0 S 0 to 0.9 Se 0 to 0.9 As ₂ S ₃ and / or As ₂ S ₄ 0 to 1.5 Sb ₂ S ₃ and / or Sb ₂ S ₄ 0 to 1.5 where the content of CdO, CdTe, ZnS, CdS, S and Se or any combination thereof is at least 0.5% by weight, and, if appropriate, customary refining agents in customary amounts. Microarray carrier ( 1 ) according to one of claims 3 to 6, wherein the colored glass has the following composition (in wt .-% on an oxide basis): SiO ₂ 30 to 75 K ₂ O 5 to 35 B ₂ O ₃ > 4 to 17 ZnO 5 to 30 F. 0.1 to 1 CdO 0.1 to 1 S + Se + Te 0.1 to 1.5 and, if appropriate, customary refining agents in customary amounts. Microarray carrier ( 1 ) according to one of claims 3 to 14, wherein the nanocrystals have a diameter of not more than about 10 nm, preferably between about 3 and about 10 nm. Microarray carrier ( 1 ) according to claim 1 or 2, wherein the colored glass is a metal colloidal glass. Microarray carrier ( 1 ) according to claim 15, wherein the metal colloid glass is a Cu or Au-doped colored glass. Microarray carrier ( 1 ) according to one of claims 1 to 17, wherein the colored glass has an absorption maximum at a wavelength between about 320 nm and about 1200 nm, preferably between about 480 and about 750 nm. Microarray carrier ( 1 ) according to claim 18, wherein the colored glass has an absorption maximum at a wavelength of about 320 ± 15 nm, 532 ± 15 nm or 645 ± 15 nm. Microarray carrier ( 1 ) according to one of claims 1 to 19, wherein the colored glass has a fluorescence emission maximum at a wavelength between about 380 and about 850 nm, preferably between about 500 and about 770 nm. Microarray carrier ( 1 ) according to claim 20, wherein the colored glass has a fluorescence emission maximum at a wavelength of about 380 ± 15 nm, 580 ± 15 nm or 680 ± 15 nm. Microarray carrier ( 1 ) according to one of claims 1 to 21, wherein the standard ( 4a / b ) is available as solid glass, glass film or thin glass. Microarray carrier ( 1 ) according to claim 22, wherein the solid glass standard ( 4a / b ) part of the substrate ( 2 ) forms. Microarray carrier ( 1 ) according to claim 23, wherein the solid glass standard ( 4a / b ) at least one end region of the carrier ( 1 ) trains. Microarray carrier ( 1 ) according to claim 22, wherein the glass film or the thin glass in regions on the substrate ( 2 ) is applied. Microarray carrier ( 1 ) according to claim 25, wherein the glass film or the thin glass in at least one end region of the substrate ( 2 ) is applied. Microarray carrier ( 1 ) according to one of claims 1 to 26, comprising two or more fluorescence standards ( 4a . 4b ). Microarray carrier ( 1 ) according to claim 27, wherein the fluorescence standards ( 4a . 4b ) have different fluorescence spectra and / or intensities. Microarray carrier ( 1 ) according to one of claims 1 to 28, wherein the fluorescence standard (s) (s) ( 4a / b ) has a fluorescence maximum / fluorescence maxima and / or a fluorescence intensity / fluorescence intensities which are less than 20%, preferably less than 10%, more preferably less than 5%, in particular less than 1%, of the fluorescence wavelength / the fluorescence wavelengths or the mean fluorescence intensity / the mean fluorescence intensities, which are different for a microarray carrier ( 1 ) microarray experiment can be used / measured. Biochip comprising a microarray carrier ( 1 ) according to one of claims 1 to 29 and one or more microarrays of probe molecules applied to the carrier.