Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/274—Calibration, base line adjustment, drift correction
  • G01N21/278—Constitution of standards

Definitions

• the invention relates to a device which enables the comparison of the imaging properties and signal sensitivity of fluorescence detection systems and the test-specific referencing of fluorescence signals, and methods for their production.
• Biomedical tests are often based on the detection of an interaction between a molecule or an affinity matrix, the identity or nature of which is known (probe), and an unknown molecule or unknown molecules to be detected (target or target molecule).
• the probes if they are molecules, are often immobilized on supports in the form of a substance library in a known quantity and position.
• Such devices are also called probe arrays or chips.
• a probe array characteristically comprises several so-called array elements, which are the regions of a probe array in which a particular molecular probe is often immobilized in multiple copies. The sum of all occupied array elements thus forms the probe array.
• the immobilization of molecular probes in the form of a substance library on probe arrays enables a sample containing the target molecules to be detected to be analyzed in parallel on several probes at the same time, which enables systematic analysis with high throughput with little time expenditure (high throughput screening , DJ Lockhart, EA Winzeler, Genomics, Gene Expression and DNA Arrays, Nature 2000, 405, 827-836).
• the probes are usually immobilized in a predetermined manner on a suitable matrix, for example described in WO 00/12575 (see, for example, US 5,412,087, WO 98/36827) or synthetically generated (see, for example, US 5,143,854).
• a suitable matrix for example described in WO 00/12575 (see, for example, US 5,412,087, WO 98/36827) or synthetically generated (see, for example, US 5,143,854).
• an interaction between the probe and the target molecule is detected as follows:
• the probe or the probes are fixed in a predetermined manner to a specific matrix in the form of a probe array.
• the targets are then brought into contact with the probes in a solution and incubated under defined conditions. If the probe and the target molecule have an affinity for one another due to complementary properties, a specific interaction takes place during the incubation between the probe and the target. The binding that occurs is significantly more stable than the binding of target molecules to probes that are not specific for the target molecule.
• the system is washed or heated with appropriate solutions or subjected to measures that have a restrictive effect.
• the detection of the specific interaction between a target and its probe can then be carried out by a large number of methods, which generally depend on the type of marker which, depending on the structure of the experiment, before, during or after the interaction of the target molecule with the probe.
• Array has been introduced into the target molecules or into the probe molecules.
• markers can e.g. are fluorescent groups, radioactive labels, enzymes or chemiluminescent molecules, the detection method to be used depends on the type of marker (A. Marshall, J. Hodgson, DNA Chips: An Array of Possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology 1998, 16, 40-44).
• this test principle can be used Interactions between nucleic acids and nucleic acids, between proteins and proteins and between nucleic acids and proteins are examined (for an overview, see F. Lottsspeich, H. Zorbas, 1998, Bioanalytik, Spektrum Akademischer Verlag, Heidelberg / Berlin).
• Antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries can be used as substance libraries that can be immobilized on probe arrays or chips.
• the nucleic acid libraries play by far the most important role, and these are particularly often DNA molecule or RNA molecule libraries.
• the probe array-based analysis of nucleic acid-nucleic acid interactions follows the principles of nucleic acid hybridization technology (AA Leitch, T. Schwarzacher, D. Jackson, IJ Leitch, 1994, in vitro hybridization, Spektrum Akademischer Verlag, Heidelberg / Berlin / Oxford).
• the detection of specific interactions between a probe and a target is usually carried out by fluorescence-optical evaluation, since these are characterized by a high sensitivity, by versatility with regard to the markers that can be used and by the possibility of location- and time-resolved detection of the interaction with comparatively little effort (especially in the Compared to mass spectroscopic methods) as well as by eliminating the radiation exposure that occurs when using radioactive labeling reagents.
• the excitation and detection wavelength range can be set depending on the fluorophores used for labeling.
• the interference must therefore be eliminated or minimized and devices and methods used that allow referencing of the measured fluorescence signals. Such devices are also referred to as the fluorescence drawing standard.
• detectors based on CCD Charge Coupled Device
• CCD Charge Coupled Device
• CCD Charge Coupled Device
• the illustration of the Probe arrays take place either in an exposure or by rasterization using high-resolution optics. Complicated lighting optics and filter systems are necessary in order to minimize or allow auto-fluorescence or system-related optical effects such as lighting homogeneity across the entire probe array.
• Confocal scanning systems allow the evaluation of fluorescence signals from selected levels of a sample. They are based on the selection of the fluorescence signals along the optical axis using pinhole diaphragms, which results in a high adjustment effort for the samples and the establishment of a powerful autofocus system.
• Such systems are highly complex in the technical solution and the necessary components, which include lasers, pinhole diaphragms, (cooled) detectors (e.g. PMT, avalanche diodes, CCD systems), high-precision mechanical translation elements and optics, have to be integrated with each other with considerable effort can be optimized (described in US 5,459,325, US 5,192,980, US 5,834,758).
• Detection devices using fluorescence drawing standards necessary.
• a calibration of detection systems by means of fluorescence drawing standards is carried out in order to be able to make statements regarding the sensitivity of the spatial and temporal resolution and the geometric image errors, such as the field curvature of the respective system.
• the calibration of detection devices with regard to their temporal resolving power is necessary because to distinguish the actual (often long-lived) fluorescence signal from (often short-lived) autofluorescence signals, the signals must be measured over a longer period of time.
• CCD detectors When using CCD detectors with the help of standards e.g. the linearity and sensitivity of the detector in the fluorescence wavelength range used, the spatial and temporal resolution as well as the field curvature (flat field determination) of the detector are determined. Confocal detection systems must be calibrated with regard to the areas that are stimulated or contribute to the overall intensity.
• the calibration of different fluorescence detection systems using fluorescence drawing standards is also very important because only such a calibration allows a comparison of fluorescence signals from experiments that were measured with different detection systems but also devices of a system (cross-system or cross-device comparison).
• WO 01/06227 describes the production of a fluorescence drawing standard based on micro- or nanoparticles and their use for the calibration of both fluorescence detection systems and for referencing fluorescence intensity signals in fluorometric assays. These standards are also not suitable for calibrating the
• Fluorescence detection systems with regard to their spatial resolution. A cross-device comparison of signal intensity data obtained from experiments based on probe arrays is only possible to a limited extent.
• long-term emitting marker substances can be used as standard, the signal of which is detected by time-resolved detection methods.
• These are often phosphorescent chelates of rare earth metals (especially those of Europium or Terpium).
• these substances have the disadvantage that they can only be excited with UV light sources.
• the chelates used in aqueous form are often unstable.
• the object of the present invention is to provide devices which reference fluorescence signals with respect to the measured intensity and / or calibrate fluorescence detection systems with regard to their sensitivity, their spatial and temporal
• Another object of the present invention is to provide devices which easily allow a cross-device and cross-system comparison of fluorescence signals from experiments, e.g. can be probe array based experiments.
• Another object of the present invention is to provide methods for producing such fluorescence standards.
• the objects are achieved in that at least one polymer layer is applied to a substantially non-fluorescent carrier in defined areas in such a way that these areas fluoresce after corresponding irradiation, some of the applied polymer layers differing in terms of their thickness and / or composition.
• Fluorescence drawing standards can thus be used for the calibration of different fluorescence detection systems with regard to their spatial and temporal resolution, with regard to their geometric and dynamic properties and with regard to their sensitivity.
• the fluorescence standards according to the invention can be used to calibrate different ones Fluorescence detection systems are used with regard to their dynamic properties.
• the polymer layers of devices according to the invention are applied in defined areas, the shape and size of which can be predetermined and reproducibly set.
• fluorescence drawing standards which are also referred to as structured fluorescence drawing standards, can be used for the calibration of different fluorescence detection systems with regard to their spatial resolution.
• the fluorescent drawing standards according to the invention are suitable for cross-device and cross-detection and / or cross-test evaluation and referencing of fluorescence signals, which e.g. can be measured in probe array based experiments.
• the polymer layers applied in the defined areas of devices according to the invention can be one or more
• the polymer layers consist of at least one fluorescent polymer or of a polymer mixture, at least one polymer component of the mixture being fluorescent.
• Preferred fluorescent polymers include, for example, positive and / or negative photoresists based on epoxy resins such as SU8 and Novo lacquers and / or PMMA and / or photosensitive polyimide and / or benzocyclobutene.
• Polymers which are suitable for the production of fluorescence standards according to the invention, ie which show fluorescence after appropriate irradiation, are also known from US Pat. No. 6,091,488 or US Pat. No. 4,482,424.
• the polymer layers of devices according to the invention can additionally contain fluorescent substances which are not polymers. Since these substances are embedded in the polymer layers, their fluorescence properties are influenced by environmental factors (e.g.
• Such fluorescent substances preferably comprise chromophores, organic dyes such as e.g. Azo dyes, triphenylmethane dyes, porphynine dyes and / or inorganic dyes such as e.g. metallic dyes and especially lanthanides.
• organic dyes such as e.g. Azo dyes, triphenylmethane dyes, porphynine dyes and / or inorganic dyes such as e.g. metallic dyes and especially lanthanides.
• inorganic dyes such as e.g. metallic dyes and especially lanthanides.
• Such substances also include perylene derivatives as described in H. Langhals, J. Karolin, L. B.-A.
• Johansson Spectroscopic properties of new and convenient Standards for measuring fluorescence quantum yields, J. Chem. Soc, Faraday Trans., 1998, 94, 2919-2922 and S. Kalinin, M. Speckbacher, H. Langhals, LB-A. Johansson, A new and versatile fluorescence standard for quantum yield determination, Phys. Chem. Chem. Phys., 2001, 3, 172-174 may be mentioned. In particular, it can e.g. B.
• the wavelength range of the fluorescence occurring in the defined ranges after corresponding irradiation can be adjusted by the choice of the composition of the polymer layers.
• the polymer layers applied to a non-fluorescent carrier in defined areas are characterized by a broadband intrinsic fluorescence, so that they show fluorescence signals in a wavelength range of greater excitation in the visible spectral range and in the near IR and UV range after narrowband excitation.
• the polymer layers in FIG a narrow-band inherent fluorescence in the defined areas, the wavelength range of which depends on the composition of the polymer layers.
• the polymer layers consist of only one polymer, so that the wavelength range of fluorescence depends only on this polymer.
• the polymer layers consist of one or more non-fluorescent polymers and / or one or more additional fluorescent substances, so that the wavelength range of the fluorescence of these devices according to the invention depends on the type and combination of the polymers and / or the fluorescent substances ,
• the intensity of the fluorescence caused by a polymer layer in a defined area after corresponding irradiation can be set in a variety of ways. According to the invention, the intensity of the fluorescence caused in the defined areas after corresponding irradiation can be adjusted by the composition of the polymer layers and / or the thickness of the polymer layers. According to the invention, the thickness of a polymer layer in a defined area includes both the thickness of the individual polymer layers in one area, if several in one area
• Polymer layers are applied, as well as the thickness, which results from the sum of the thickness of the individual polymer layers in an area.
• the intensity is set in a defined range by the successive application of
• fluorescence drawing standards according to the invention can be produced which have polymer layers of uniform composition but different layer thicknesses in all defined areas.
• preferred embodiments of the invention can be used to produce fluorescence drawing standards in which the intensity of the fluorescence caused in an area after corresponding irradiation is proportional to the thickness of the polymer layer applied in the respective area.
• the intensity of the fluorescence in the defined areas of calibration standards according to the invention can also be adjusted by changing the composition of the polymer layers.
• the type and number of polymer components in one is determined by the composition of the polymer layers
• polymer layers of uniform thickness are applied in different defined areas, the polymer layers differing only in the amount of additional fluorescent particles per unit area.
• fluorescence drawing standards according to the invention can be produced, in which the intensity of the fluorescence caused in the areas with corresponding irradiation is proportional to the amount of fluorescent particles in the various defined areas, given the same layer thickness.
• the resulting intensity can be adjusted quasi linearly by changing the layer thickness at a constant concentration of the fluorescent particles in the polymer.
• a further possibility for adjusting the intensity of fluorescence drawing standards produced according to the invention is to add physical treatment methods such as radiation and temperature treatment (annealing) to the polymer layers according to the invention during the production process submit.
• physical treatment methods such as radiation and temperature treatment (annealing)
• annealing annealing
• these treatment methods change the degree of crosslinking or crosslinking of the polymer layers and accordingly the fluorescence properties of the polymer layers.
• crosslinking degree or degree of crosslinking is familiar to the person skilled in the art.
• Adjustment of the intensity of polymer layers by methods for changing the degree of crosslinking, especially polymers that have a thermosetting crosslink are suitable, e.g. the mentioned photoresists like SU8.
• fluorescence drawing standards can be produced, which are characterized by a wide range of both the intensities and the wavelength ranges according to the corresponding radiation occurring
• fluorescence drawing standards according to the invention can be used in many ways for the calibration of fluorescence detection systems with regard to their spatial and temporal resolution as well as their dynamic and geometric properties.
• Such devices according to the invention can also be used as fluorescence standards, which easily allow a comparison of fluorescence signals between different detection systems, but also devices of a system.
• the fluorescence properties are subject to changes over the irradiation time due to the chemical and physical properties of the polymers and additional fluorescent substances used, so that the resulting intensity decreases after a certain period of use (bleaching, see also Miehler, 1992, plastic micromechanics: morphology, deformation and fracture mechanisms., Hanser, Kunststoff Vienna).
• the polymer layers deposited in defined areas can be optimized in terms of their intensity changes over time by means of suitable production protocols (for example, so-called tempering protocols) such that these changes are linear and consequently the intensity ratios of the resulting fluorescence of the individual defined areas of the structured standard remain constant.
• Such fluorescence drawing standards according to the invention can be used for the calibration of fluorescence detection devices with regard to their temporal
• Fluorescence drawing standards according to the invention can also be produced as preferred embodiments, the fluorescence yield of which can be described in a linear or sufficiently predeterminable manner over the irradiation duration and the aging process.
• Such fluorescent standards can e.g. for referencing fluorescence signals from experiments that were carried out at different times. They also allow the cross-device and cross-system comparison of fluorescence signals.
• the polymer layers are applied in defined areas of different shape and / or size on a substantially non-fluorescent carrier.
• these defined areas have a square, rectangular and / or circular shape, the side lengths or diameters of which are between 500 nm and 5 mm.
• Such structured fluorescence drawing standards according to the invention can be used for the calibration of different fluorescence detection systems with regard to their spatial resolution.
• spatial resolution is to be understood as the separability of two fluorescent dots shown.
• fluorescence standards according to the invention can be produced, the defined areas of which have dimensional tolerances in the 10 nm range, such fluorescence standards can be used in microscopic techniques, e.g. confocal 3-D microscopy can be used as a standardization instrument for lateral and axial distances.
• the defined areas are applied to the carrier in array form, i. H. that several defined areas of the same size and shape are grouped into an array element, whereas areas of a different shape and / or size are grouped into other array elements.
• the thickness of the polymer layers can be set such that the maximum thickness of the various polymer layers is significantly less in an area than the minimum depth of focus of commercially available confocal detection systems.
• such fluorescence drawing standards should have a preferred layer thickness between a few nanometers and a maximum of 50 micrometers, the layer thicknesses should preferably be between a few nm and 20 ⁇ m, between 100 nm and 10 ⁇ m, particularly preferably between 200 nm and 5 ⁇ m.
• the depth of focus is to be understood as the area along the optical axis in which a detection of fluorescence signals is possible.
• fluorescence drawing standards can be used for the calibration of physical structures and devices, for the detection of fluorescence signals and for the referencing of fluorescence signals, appropriately labeled substances and groups of substances.
• the polymer layers are applied to essentially non-fluorescent, preferably optically transparent supports.
• these supports consist of glass, particularly preferably of quartz glass and borofloat glass, or of optically transparent, non-fluorescent polymeric panes, particularly preferably of polycarbonate, PMMA and / or foils.
• optically non-transparent materials are also suitable as carriers, which essentially have no inherent fluorescence.
• the fluorescence drawing standards are connected to further carrier systems.
• These carrier systems also consist of essentially non-fluorescent materials. These are preferably optically transparent materials such as glass, particularly preferably quartz glass, but also optically non-transparent materials such as plastic and / or metal, which have essentially no inherent fluorescence.
• this carrier system is a commercially available slide, as it is for z. B for the
• Immunofluorescence microscopy is used.
• Cells or tissue sections can be immobilized on such supports and evaluated directly by comparing the signals against the fluorescence standard according to the invention.
• the fluorescence drawing standards according to the invention can also be integrated, for example, directly on the supports on which substance libraries are or will be stored in the form of a probe array. Such fluorescence drawing standards allow direct referencing of fluorescence signals and thus enable the evaluation of test-specific examinations.
• the surface of the supports in at least the areas which are to contain the substance library should be functionalized by amino, carboxy, aldehyde or epoxy groups. Further functional groups for linking substance libraries are known to the person skilled in the art.
• the supports on which the fluorescence drawing standards or the substance libraries are stored in the form of probe arrays are to be selected such that they are essentially non-fluorescent and, if required, optically transparent.
• Preferred materials for the production of such supports are glass, particularly preferably quartz glass, borofloat glass and / or polymers and / or silicon. It is also possible to choose materials which are not optically transparent, but which are essentially non-fluorescent.
• these probe arrays can be functionalized by substance libraries based on proteins, peptides or nucleic acids.
• the protein substance libraries with which the fluorescence standards according to the invention are functionalized are preferably antibody libraries, receptor libraries, receptor-ligand libraries and / or hormone libraries.
• the peptide libraries with which fluorescence standards are functionalized according to the invention are usually pharmaceutically or biologically active peptides, antigen libraries and / or receptor-ligand libraries and / or hormone libraries.
• the nucleic acid libraries with which fluorescence drawing standards are functionalized according to the invention are preferably DNA molecule libraries and / or RNA molecule libraries. It is particularly preferably mRNA libraries, rRNA libraries, genomic DNA libraries and / or cDNA libraries and or plasmids.
• fluorescence drawing standards according to the invention can be integrated directly into the fluorescence detection systems, so that online calibration of the devices is possible. Since the properties of the fluorescence standards according to the invention by the
• the fluorescence drawing standards according to the invention can also be used as a universal external tool to calibrate several different detection systems in the manner described.
• Fluorescence standards according to the invention can also be integrated in closed chamber systems (e.g. PCR and or hybridization chambers).
• Fluorescence standards according to the invention can be produced by methods in which the layers fluorescent after irradiation are applied in a defined manner to the carrier according to the invention in defined areas. Such methods are known, for example, from semiconductor technology (US 6,091,488).
• the polymer layers of fluorescence standards according to the invention can preferably by photolithographic methods, by spotting Dry etching, by ion implantation, by printing processes, by rolling, by injection molding or by surface embossing can be applied to the carrier.
• the polymer layers according to the invention can be applied to the support by chemical and / or physical methods for coating.
• the polymer layers can be applied from the gas phase (e.g. by CVD or oxidation), from the liquid phase (e.g. by electrolytic, electrochemical and other wet chemical processes) or from the solid phase (e.g. by oxidation).
• the application can be from the gas phase or from the plasma (e.g. by PVD, sputtering or vapor deposition), from the liquid phase (e.g. by spin-on processes, by painting, spraying or dipping) or from the solid phase (e.g. by lamination ) respectively.
• Combinations of these methods or subsequent modifications such as ion implantation can also be used.
• ion implantation e.g. PECVD
• the respective methods and corresponding configurations are known to the person skilled in the art (see, for example, W. Menz, J. Mohr, Microsystem Technology for Engineers, VCH, 1997).
• the polymer layers according to the invention can be structured by various methods which are known to the person skilled in the art. These can be biochemical methods, such as, for example, the enzymatically mediated selective structuring (described in WO98 / 08086), but also chemical and / or microtechnical methods. These include selective etching of the functional layer against a mask that is itself insensitive to the etcher (e.g. liquid or dry etching). Methods are also applicable which bring about selective changes in properties by radiation, such as, for example, by irradiation with UV or lasers. This includes photolithography. Other methods include self-assembling layers. These methods can be done using a Masking, but also by means of “directly rubbing” devices (spotting).
• Printing technology processes such as spotting or offset printing or other processes such as rolling, stamping, injection molding or surface embossing processes can also be used.
• Different embodiments of the methods are known to the person skilled in the art (see, for example, W. Menz, J. Mohr, Microsystem Technology for Engineers, VCH, 1997).
• the polymer layers are applied by negative or positive photolithographic methods, particularly preferably by negative photolithographic methods. Negative photolithographic processes are particularly preferred in which the polymer layers become insoluble in the developer after irradiation. In contrast, unexposed areas remain soluble in the developer and can be removed.
• photolithographic methods for structure transfer using a photoresist are known to the person skilled in the art.
• Chemical and physical processes which allow the application of a homogeneous functional layer on the carrier are preferred. Particularly preferred methods include CVD, PVD and spin-on methods.
• the parameters by means of which the thickness of the polymer layers can be set in the case of photolithographic processes (but also other processes) include the processing parameters (process parameters) such as the circulation speed, the duration of the production process, the viscosity of the polymer, the temperature and / or the air humidity , When using photopolymers, the processing parameters also include the radiation dose, the parameters of the development process and the annealing process.
• processing parameters such as the circulation speed, the duration of the production process, the viscosity of the polymer, the temperature and / or the air humidity .
• the shape and size of the defined areas in which the polymer layers are applied can be determined by using a mask and / or shadow masks, for example by using a mask on a 1: 1 scale. Depending on the cutout of the mask are with it According to the invention, different geometrical shapes and sizes can be set for the defined areas. Fluorescence standards according to the invention can also be produced according to this method, in which the polymer layers deposited in different defined areas have an array shape. Structured chrome on glass and / or quartz is preferably used as the shadow mask.
• fluorescence drawing standards according to the invention can be produced, in which polymer layers with different compositions and / or thicknesses are applied in different areas.
• the degree of crosslinking (and thus the intensity) of the polymer layers can also be specifically changed in defined areas.
• the bleaching behavior of fluorescence standards according to the invention can also be set in a targeted manner.
• the fluorescent drawing standard is preferably applied to the carrier system by gluing, by position adjustment or by a vacuum system.
• the fluorescence drawing standard can be connected to the carrier system on which the substance libraries are located.
• the fluorescence drawing standards can first be produced on supports on a waver scale, and the correspondingly isolated fluorescence drawing standards can then be functionalized by storing substance libraries in array form.
• the fluorescence standards according to the invention can be used in different ways for the calibration of fluorescence detection systems.
• the device properties can be defined by using fluorescence drawing standards according to the invention for device-specific calibrations. For example, the corresponding shape and size of the defined areas in which the polymer layers which are fluorescent after appropriate irradiation are deposited can be used to determine the spatial resolution.
• Fluorescence drawing standards according to the invention which have polymer layers of different thicknesses but of uniform composition in several defined geometric areas, as a result of which the intensity of the fluorescence in the different areas, which is produced after corresponding irradiation, is proportional to the polymer layer thickness, allow calibration of a corresponding detection system with regard to its dynamic properties.
• the intensity range in which a CCD detector should work linearly can be defined in this way.
• fluorescence drawing standards according to the invention can also be used to adjust the sensitivity of the detection system, ie to determine which minimum or maximum fluorescence intensities a detection system should still evaluate as a signal.
• Fluorescence drawing standards according to the invention in which the intensity of the fluorescence, which is produced after corresponding irradiation in different geometric areas with polymer layers of different thicknesses and / or composition, decays in a predeterminable and controllable manner, can be used to draw conclusions about the device-specific bleaching of fluorescence signals.
• fluorescence drawing standards according to the invention can be used to calibrate the geometric properties of detection systems, in particular to correct the curvature of the image field (flatfield determination) in CCD detectors.
• geometric properties are generally understood to mean the local resolution, the image scale, the field curvature and other geometric errors which result from the optical construction.
• fluorescence drawing standards Since the fluorescence properties of fluorescence drawing standards according to the invention do not depend on external factors and can be predetermined and reproducibly set, they can be used to calibrate different devices of the same detection principle (cross-device calibration) or devices of different detection principle (cross-system calibration).
• the calibration can be carried out to standard values that are used, for example, in work groups and laboratories, to which fluorescence signals from experimental measurements are then related. This means that the signals that are obtained when evaluating, for example, a probe array-based experiment with different detection systems are directly comparable (cross-test comparison).
• Fluorescence drawing standards according to the invention which show a wide range with regard to the wavelength and intensity of the fluorescence produced in the defined areas after irradiation of the polymer layers, can also be used for test-specific referencing of fluorescence signals. This is possible because the test-specific signals e.g. can be related to the fluorescence of a defined area, the properties of which in the area of
• Test signal This enables the test signals to be normalized.
• the standardized experimental data obtained using fluorescence drawing standards according to the invention significantly eliminates the errors which result from the use of different detectors or different settings of the detectors. They can therefore be compared directly with one another. Fluorescence drawing standards according to the invention thus enable cross-test and cross-device comparison of
• Fluorescence signal intensities that have been obtained when carrying out probe array based experiments and tests.
• the settings of scanners are usually adapted to the corresponding measurement results. If the resulting fluorescence signals are weak in a probe array-based experiment, the laser power or the sensitivity of the detector (eg the bias when using a PMT system or the integration time when using a CCD system) are increased. In addition, the devices are subject to device-specific fluctuations. This is particularly critical if results from different time periods are to be compared, since the power of the lasers used can change dramatically, for example with laser scanners. A comparison of fluorescence intensity signals, which, when performing similar probe array-based experiments, however were measured at different times with the fluorescence standards according to the invention without any problems ("time to time” comparison), since the bleaching behavior of the standard can be used to normalize the device fluctuations. With the aid of the fluorescence standards according to the invention, detection systems can also be used with regard to their
• Aging properties e.g. the change in lamp wattages are calibrated.
• Detection systems can be calibrated, the results of probe array-based experiments can be analyzed across devices and systems and thus across laboratories.
• Devices which are characterized in that polymer layers, which are uniform in terms of their layer thickness and composition, are applied to an essentially non-fluorescent carrier in one or more defined areas can also be used for referencing fluorescence signals. Such devices are particularly suitable if they have several defined areas for the device and cross-system calibration of fluorescence detection systems with regard to their spatial resolution.
• the polymer layers of such devices can have the same composition as stated above, i. H. they can contain fluorescent polymers, polymer mixtures with at least one fluorescent polymer and / or additional fluorescent substances.
• the intensity and the wavelength range of the fluorescence caused in the areas after corresponding irradiation can be predetermined and reproducibly adjusted by the choice of the composition of the polymer layers, the layer thickness and by changes in the degree of crosslinking. All other properties such as bleaching behavior, size and shape of the defined areas can also be set in the manner described above. Such devices can also be produced by all of the methods described above.
• Such devices can generally be used for the qualitative and quantitative referencing of fluorescence signals, particularly preferably for referencing fluorescence signals from probe array-based tests and / or for calibrating fluorescence detection systems.
• Fluorescence detection systems are used in terms of their spatial resolution. Since the polymer layers applied in one or more defined areas are uniform in terms of their composition and layer thickness, they are not suitable for calibrating the dynamic properties of fluorescence detection systems. Also calibration of detection systems by comparing the ratios of different ones Intensities that occur after irradiation from different areas are not possible with these devices.
• Such devices can e.g. can be used to compare experimentally determined fluorescence signal data with standard values and thus allow the cross-device and cross-system comparison of fluorescence signals. These devices also allow the calibration of fluorescence detection systems with regard to their sensitivity and their device-specific bleaching behavior at the intensities and wavelength ranges defined by the composition and thickness of the polymer layers.
• the example shows how a universal fluorescence drawing standard according to the invention is produced on a polymer basis by means of negative photolithography.
• a fluorescence drawing standard according to the invention is produced which, in different areas of the same shape and size, has polymer layers of different thicknesses but essentially the same composition.
• the intensities of the fluorescence caused in the areas after corresponding irradiation are thus proportional to the layer thickness.
• Materials that show high transparency and low self-fluorescence in the desired fluorescence spectrum are used as supports.
• borofloat glass 40 from Schott (BF 40, diameter: 100 mm, thickness: 0.7 mm) was chosen as the carrier material.
• the actual functional material (these are the
• polymer layers should have a strong inherent fluorescence.
• polymer SU8-10 dissolved in PGMEA (organic solvent from Sigma), was chosen as the polymer which fulfills the properties mentioned.
• PGMEA organic solvent from Sigma
• Self-fluorescence and the ability to be photostructured have the great advantage that the chosen polymer can be structured using photolithographic processes.
• the carrier material was annealed (180 ° C, 20 min). This removed any adsorbates (often H 2 O) that hinder the good adhesion of the SU8 polymer layer to the substrate. To reinforce this, the surface of the substrate was modified with 3-glycydoxypropyltrimethoxysilane.
• a first layer with a thickness corresponding to the desired fluorescence or sensitivity was then applied to the substrate (see Figure 1).
• the spin-on method was used for this, by selecting the parameters (duration: 30s, rotational speed: 5000 rpm, acceleration: 100 rpm s for 10 s and 1000 rpm s for 20 s, degree of dilution with PGMEA) the desired thickness or the fluorescence was set.
• the polymer was distributed homogeneously on the substrate and the solvent was driven off.
• the subsequent tempering above the glass point (95 ° C, 15 min, so-called pre-bake) forms and homogenizes the layer.
• the polymer layers were then structured using conventional microphotolithography.
• the solubility in the developer (in this case PGMEA) was changed by irradiating the photopolymer with UV light (15 min at approx. 300-450 nm with 15 mW / cm 2 , so-called exposure).
• SU8-10 shows a tone-negative behavior, ie the UV-exposed areas polymerize and unexposed areas remain soluble in the developer. Irradiation was carried out by mask projection using lithography masks, which had corresponding thin chrome structures on quartz. This quartz mask had the desired lateral geometry of the standard, which was imaged in the polymer on a 1: 1 scale. The smallest structures in the mask therefore also determine the smallest structures of the standard or of the smallest resolution test to be detected on the standard.
• 3rd layer SU8-10 (MicroChem Inc.) dissolved in PGMEA (Sigma) 20% w / w
• the SU8-10 photopolymer was dissolved in various weight ratios in PGMEA. As a result, the solutions have different viscosities, which means that polymer layers with different thicknesses can be produced.
• the Solvent is later largely driven out (more than 95%), so that the polymer layers have essentially the same composition.
• the polymer structure was then developed by immersion in PGMEA (Sigma) and annealed again (120 ° C., 30 min, so-called hard bake).
• step-like geometric structures emerged ( Figure 1).
• the step heights then correspond to a corresponding fluorescence intensity, the lateral geometries to the resolving power.
• FIG. 2A shows an inventive fluorescence drawing standard in array form, which was produced microlithographically on borofloat glass (Schott) using the method from Example 1.
• the standard consists of 9 array elements, all of which contain fluorescent polymer layers in a square shape.
• the squares of the 9 array elements have 3 intensity levels, which result from 3 different thicknesses.
• Different integration densities of molecular arrays are simulated by different structure sizes and distances.
• the standards were produced on a wafer scale and separated into chips.
• the standard is suitable for determining the spatial resolution, the geometric errors, the imaging optical, electromechanical and information technology systems as well as the sensitivity of the respective device at the time of the measurement.
• the standard can be used for cross-device evaluation of the fluorescence signals from probe array-based experiments.
• the described fluorescence standard was read out in various reader systems as a 16bit - *. Tif Images exported and analyzed using special software (IconoClust from Clondiag).
• the differently sized fluorescent structures on the fluorescence drawing standard can be used to correct geometric deviations of the different detection systems.
• the field curvature of an optical system can be
• the settings of the detection systems e.g. with regard to the laser or lamp power, the integration time per spot / subarray or the signal amplification / adjustment of the detector to a specific value of the resulting fluorescence of the standard.
• the long-term drift of a detection apparatus which can be caused by the aging processes of the lasers, lamps or detectors used, are determined.
• fluorescence signals which are measured with different detection systems when evaluating a probe array-based experiment, can be compared directly with one another.
• FIG. 2B shows a fluorescence standard according to the invention in array form, which was produced by spotting on borofloat glass (Schott).
• fluorescent standards were produced on various supports by producing polymer mixtures SU8 (MCC Inc.) and Novolack (AZ 1514 Clariant) in a mass ratio of 1: 2, 1: 3, 1: 5 and applying them to the glass substrates.
• slides were cleaned and the polymer mixtures using a needle spotting tool (Microgrid 2 / Biorobotics) mocked unslotted spider needle (Solidpins / Biorobotics).
• the polymer mixtures were applied with a piezo-driven dispenser head (diameter 100 ⁇ m, heated nozzle from microdrop). The dispenser nozzle was heated (55 ° C).
• Pre-Bake 95 ° C, 15min Exposure: 1min, 15mW / cm 2 , flood exposure
• Post-Bake 95 ° C, 15min Hard-Bake: 120 ° C, 60min
• the standard produced in this way consists of circular areas (spots) of uniform dimensions, the polymer layers of which differ both in terms of their thickness and their composition.
• the standard therefore has a wide spectrum of intensities and wavelength ranges for the fluorescence that can be produced in the spots.
• the standard came in the same way as that used in Figure 4A.
• Figure 3 shows how a fluorescence standard according to the invention is applied to a slide by gluing.
• an annealing protocol can be selected so that the intensity of the fluorescence caused in the defined areas after multiple irradiation decays linearly.
• Figure 4A shows the bleaching behavior of the three layers without thermal treatment. It is a non-linear bleaching behavior.
• Figure 4B shows the bleaching behavior of the three layers after thermal treatment. It is a linear bleaching behavior.
• the samples were subjected to a thermal treatment in the oven for two hours at 180 ° C. during the manufacturing process and then for 40 minutes at a wavelength between 520 and 600 nm at 40 mW / cm 2 irradiated.
• the intensity behavior changes in such a way that the thickness ratio is no longer directly proportional to the resulting intensity ratio, but the intensity ratios of the structures of different thicknesses are now constant when irradiated (see Figure 4C).
• Fluorescence drawing standards were produced as in Example 1, in which polymer layers with different thicknesses are applied in the defined areas.
• Fluorescence drawing standards according to the invention which likewise had a probe array, were used to standardize probe array-based experiments. The fluorescence drawing standard can then be taken into account in the readout process before or after the biochemical interaction reaction has been carried out (see Figure 6A).
• Fluorescence standards according to the invention were applied to epoxidized slides (company QMT) by ultrasonic drilling and gluing, which had polymer layers in the defined areas with the same polymer thickness but different content with respect to the polymer components. These areas thus show different intensities after appropriate irradiation.
• a series of PCR products aceA, acs, amiB, ampG, argC, atpA, creB, icdA, napH, rpoA, rpoH, rpoS
• Specially purified specific cDNA labeled with fluorescent dyes (Cy3 or Cy5 from Amersham) was immobilized on the PCR spots by hybridization.
• the samples were then read using a confocal laser scanner (Affymetrix, Packard) and the results were analyzed using special software (IconoClust from Clondiag). The results were evaluated with reference to the different intensities of the different areas of the fluorescence drawing standard.
• the three bars of a cDNA stand for referencing the signal data of the hybridization to three different areas of the standard, which have different intensities.
• the results were standardized to the different intensity levels and taking into account the content of the Polymer layers calculated on one level.
• the graph ( Figure 6B) shows that such a back calculation is permissible since the same results are obtained for each cDNA, regardless of the area to which the measurement of the cDNA is related. If the content of the polymer layers is known, it is therefore permissible to use each area for referencing, which increases the universality of the standard.
• the measurements were carried out with a confocal biochip scanner Scanarray 4000 (Packard) with 100% laser power and 85% PMT gain.
• Fluorescence standard according to the invention in the form of an array, produced microlithographically on boro float glass.
• Fluorescence standard according to the invention in array form produced by spotting on borofloat glass.
• the top line shows the d3 / d2 ratio
• the bottom line the d2 / dl ratio from Figure 4A.
• Fig. 5 Measurement results of a scan for different dyes. The intensity is given as a gray value. These standards are suitable for standardizing results in different wavelength ranges.
• Fig. 6A Inverse scan of a spotted probe array. In the upper part you can see the fluorescence drawing standard (1). Below this is the probe array (2). These are spotted PCR products after hybridization with Cy3 / Cy5-labeled cDNA.

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