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
  • G01N21/17—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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • 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
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

• the invention relates to a calibration probe for calibrating an electronic device for analysing biomolecules by detecting fluorescent signals from a sample probe.
• the invention also relates to methods and uses of the calibration probe.
• Biotechnology, medicine and related technical fields are based on the analysis of biomolecules.
• Electronic devices can analyse biomolecules with high precision and specificity.
• automated electronic devices have been developed for analysing large numbers of samples by routine methods.
• modern DNA sequencers are used for routine analysis of large numbers of DNA probes.
• Protein samples can be analysed by high throughput screening and related methods.
• DNA sequencers are capable of sequencing large numbers of samples labelled with fluorescent dyes in parallel. Recently developed methods, referred to as "next-generation sequencing", have revolutionized sequencing. In such methods, various oligonucleotides of interest are covalently attached to a support. Subsequently, a nucleotide labelled with a fluorescent dye is attached to the growing oligonucleotide chain with DNA polymerase. When the four nucleotides are labelled with different fluorescent dyes, fluorescent signals emitted from a probe can be detected and the type of nucleotide attached to the oligonucleotide can be identified.
• the fluorescent dye is cleaved off and the next synthesis cycle is carried out, in which a new labelled nucleotide is attached to the growing chain.
• the sequence of a growing oligonucleotide chain can be determined in a stepwise manner.
• the working steps are carried out in an automated DNA sequencer device. Fluorescent signals emitted from sample probes with labelled biomolecules are weak, but the signals have to be detected with high precision and specificity. Thus, precise optical equipment, especially cameras and scanning technology, is required for such processes.
• calibration probes are inserted into the electronic device at the same position as the sample probes.
• calibration probes used in the art comprise liquid solutions of fluorescent dyes, often those which are used in the subsequent analysis method. Thereby, the electronic device can be adjusted to properties of the fluorescent dyes, such as wavelength and intensity.
• a calibration probe shall be provided, which is stable and can be used in multiple calibration processes. Further, the calibration probe shall be applicable for calibrating a large number of measuring parameters of an electronic device. The calibration probe shall not only be applicable for calibrating sensitivity or wavelength, but also for determining the structure of the probe. Besides, the calibration should be possible with high precision and specificity. Overall, the calibration probe shall allow calibration under conditions, which are as close as possible to the "real" conditions, under which the biomolecules are analysed. The calibration probe shall be easily available and applicable for the user of such an electronic device.
• Subject of the invention is a calibration probe for calibrating an electronic device for analysing biomolecules by detecting fluorescent signals from a sample probe
• the calibration probe comprises a detection area for detection of fluorescent signals with the electronic device during calibration
• the detection area comprises at least one fluorescent dye attached thereto, wherein the at least one fluorescent dye is attached to first defined regions of the detection area, whilst not being attached to second defined regions of the detection area.
• Calibration is an operation that, under specified conditions, in a first step, establishes a relation between the quantity values with measurement uncertainties provided by measurement standards and corresponding indications with associated measurement uncertainties (of the calibrated instrument or secondary standard) and, in a second step, uses this information to establish a relation for obtaining a measurement result from an indication (definition by the International Bureau of Weights and Measures).
• a “calibration probe” is a probe, which is inserted into the electronic device and from which at least one quantity is determined by the electronic device, with the purpose of adjusting at least one parameter of the electronic device.
• At least one quantity of a calibration probe is known before calibration.
• the quantity may be implicated to the calibration probe in the production process, or may have been determined with a similar reference device.
• the calibration probe is used as a standard and a device to be calibrated is adjusted with the calibration probe regarding that quantity.
• a "sample probe” is a probe comprising a biomolecule, of which at least one property is analysed. The analysis of sample probes is the normal intended use of the electronic device. In the following, the sample probe used for analysis after calibration with the inventive calibration probe is also referred to as "the corresponding sample probe".
• biomolecule is a molecular biological material that is present in a living organism. This does not imply that it is has been obtained from a living organism. A biomolecule could also be produced in vitro, or could be a derivative of a molecule obtained from a living organism.
• the biomolecule is a macromolecule, such as a nucleic acid, protein or carbohydrate. More preferably, the biomolecule is a nucleic acid, most preferably DNA (desoxyribonucleic acid).
• analysing refers to any method, in which at least one property of a biomolecule is analysed.
• the analysis could relate to whether a biomolecule is present or not, or to the amount or location of a biomolecule in a probe.
• the calibration probe is a vessel or comprises a vessel.
• the vessel is adapted for insertion into the electronic device.
• the vessel comprises an internal surface.
• the vessel could be a flow cell, reaction tube, cuvette or the like.
• the vessel corresponds to the vessel of the sample probe for analysing the biomolecule.
• the calibration probe comprises a detection area.
• the detection area is the site of the calibration probe, in which fluorescent signals are detected by the electronic device.
• the detection area is located on the internal surface of the vessel.
• the detection area only forms a portion of the internal surface, whereas other portions of the internal surface are not part of the detection area.
• the detection area is flat or essentially flat.
• the detection area is analysed by the camera and/or scanner of the electronic device for fluorescent signals. Signals outside the detection area are not analysed.
• the location of the detection area of the calibration probe corresponds to the location of the detection area of the sample probe, i.e. it is located at the same or at least a similar position.
• the calibration probe comprises a fluorescent dye.
• the electronic device detects fluorescent signals emitted from the fluorescent dye.
• the fluorescent dye is attached to the detection area, i.e. to the internal surface of the vessel.
• the dye can be attached covalently or non-covalently, for example by affinity, adsorption and/or ionic interactions.
• the fluorescent dye is covalently attached.
• the fluorescent dye can be attached directly or indirectly, for example through a linker.
• the linker can be a single molecule or a linker entity, such as a particle, especially a bead.
• the calibration probe comprises at least one fluorescent dye.
• the term "at least one" refers to different dyes, i.e. different chemical molecules having different emission spectra.
• the probe comprises more than one fluorescent dye, such as two, three, four, five, six or more fluorescent dyes.
• At least one fluorescent dye is attached to first defined regions of the detection area, whilst not being attached to second defined regions of the detection area.
• a specific fluorescent dye is not distributed equally throughout the detection area. It is present only locally, in the form of a pattern.
• the electronic device can identify first regions of the detection area, in which the specific fluorescent dye is present, and other regions of the detection area, in which it is not present. Thereby, the electronic device can determine the two-dimensional distribution of the specific dye in the detection area.
• each dye is preferably distributed accordingly.
• each dye is distributed in specific regions, but is not present in other regions.
• the regions are preferably unique, i.e. different from the regions in which the other dyes are distributed.
• the regions in which the dyes are present are not overlapping. Then, no region of the detection area comprises more than one type of dye.
• the electronic device is a DNA sequencer.
• a DNA sequencer analyses DNA sequences by performing DNA synthesis and analysis of intermediate products stepwise in an automated process.
• the four nucleotides are labelled with four different fluorescent dyes, which emit different fluorescent signals.
• the nucleotides are the standard building blocks for DNA synthesis comprising the nucleobases A, T, G and C (adenine, thymine, guanine and cytosine).
• the nucleotides are dNTPs (desoxynucleotide triphosphates). Standard methods are also applicable in which modified or different nucleotides are used.
• the four dyes emit blue, green, yellow and red light.
• Standard DNA sequencers based on fluorescent technology are routinely adapted for determining and analysing the respective signals and converting the information into DNA sequence data.
• the DNA sequencer identifies the different fluorescent signals from each dye incorporated into the growing chain and attributes a nucleobase to each signal. Thereby, the sequencer determines the order of the four bases guanine, cytosine, adenine and thymine in the DNA chain.
• the DNA sequencer determines fluorescent signals from the calibration probe comprising the fluorescent dyes.
• the calibration probe comprises the same fluorescent dyes as used in the subsequent DNA sequencing method, for which the device is calibrated.
• the detection area comprises four different fluorescent dyes.
• the DNA sequencer determines four different fluorescent dyes in the corresponding sample probe. According to the invention, the sequencer can be calibrated for detecting different fluorescent signals, but also for detecting different locations of signals emitted from different fluorescent dyes.
• the device sequences DNA based on four different fluorescent signals emitted from a sample probe.
• the sample probe comprises a detection area with an array of sites in which different DNA strands are bound to a support. Each site corresponds to a specific DNA oligomer, which forms the matrix for synthesis of double stranded DNA with DNA polymerase.
• the four different dyes mark the terminal nucleotides of the DNA chain at each specific site. The terminal dyes are detected by the device and cleaved of after each cycle.
• the automated reaction is processes in a flow cell.
• the DNA sequencer is preferably a QIAGEN GeneReaderTM.
• any first region of the detection area in which a specific fluorescent dye is present, no other fluorescent dye is present.
• the regions in which different fluorescent dyes are present are not overlapping.
• Each site of the detection area comprises only one of the dyes or no dye at all.
• the camera of the electronic device will then only determine a single fluorescent signal at any position.
• the local presence of only one fluorescent dye is advantageous, because it simulates the corresponding sample probe used for analysing biomolecules after calibration.
• such a DNA sample probe emits only a fluorescent signal from one specific fluorescent dye in any region of the detection area. Since the detection area of the calibration probe and sample probe are highly similar, such a calibration process is very precise.
• the calibration probe comprises first defined regions, which are surrounded by the second defined regions.
• the first defined regions form spots, islands or other discrete structures amongst the second defined regions.
• the calibration probe comprises at least one fluorescent dye.
• the fluorescent dye is a fluorophore, which is a fluorescent chemical compound. Fluorescence is the emission of electromagnetic radiation by a substance that has absorbed another electromagnetic radiation.
• the electromagnetic radiation emitted from the dyes could have emission wavelengths and/ or an emission peak, preferably the main emission peak, between 200 nm and 3000 nm, which covers the US, visible and IR-A and IR-B range.
• an emission peak, more preferably the main emission peak, of a dye, more preferably of all dyes is in the range of visible light between 380 nm and 780 nm.
• the fluorescent dyes are attached to the detection area.
• the dye can be attached directly or indirectly to the internal surface of the calibration probe.
• the dye could be covalently attached to the surface, if the surface and the dye comprise chemical moieties which can be covalently linked. Such methods are known in the art and applicable corresponding groups could be amine and carboxyl.
• the surface has been activated, i.e. has been equipped with a reactive group for specifically binding the dye. Methods are known in the art for activating surfaces and making them suitable for covalent attachment of organic small molecules.
• the surface comprises azide groups, to which the dye is directly or indirectly attached. Such a covalent link can be established with reagents and kits available under the trademark AziGrip from SuSoS AG, DE.
• the dyes are attached to the surface through an intermediate entity.
• the entity could be a molecular linker.
• the linker could be a larger, particulate entity, preferably beads.
• multiple molecules of the same fluorescent dye are attached to larger entities, preferably beads, which are covalently attached to the internal surface of the detection area. This is advantageous, because multiple fluorescent dyes can be accumulated locally in defined regions of the detection area, thereby providing a more intensive and pronounced local fluorescent signal. When distributing such larger entities, preferably beads, in the detection area, they may form the first regions.
• all the beads in a calibration probe have substantially the same size.
• only one type of fluorescent dyes is attached to each bead. This is advantageous, because in the calibration process the electronic device can focus on the beads as local areas emitting fluorescence from a single dye.
• the calibration probe can be adapted as close as possible to the corresponding sample probe.
• the intensity of fluorescent signals can be adjusted by controlling the amount of the fluorescent dye attached to each bead.
• the amount of a dye could be controlled in the preparation process by diluting the dye with a non-fluorescent molecule which binds to the same site.
• some of the functionalities on the bead surface will be linked to non- fluorescent molecules and the bead exhibits reduced luminosity.
• the luminosity of the calibration probe can be fine-tuned. It is important to control the signal strength of the test sample in order to prevent the camera sensor from reaching signal saturation at normal illumination level.
• the beads form a monolayer.
• the beads are arranged in form of a layer in the detection area on the internal surface.
• the monolayer is aligned in parallel to the surface.
• the camera of the electronic device can scan and analyse the two-dimensional distribution of distinct signals emitted from the single bead.
• the entire detection area will only emit signals from single beads and thus the electronic device determines only signals from a single dye (or no dye at all). This is advantageous, because in the calibration process precise and unambiguous adjustment of the electronic device to each single dye and to a two-dimensional pattern is possible.
• the beads are magnetic.
• Magnetic beads can be arranged easily in a monolayer when applying a magnetic force underneath the detection area. Besides, the working steps during attachment of the dyes to the beads, such as activation, binding and washing, can be carried out conveniently with magnetic beads.
• the detection area comprises beads to which no fluorescent dye is attached ("dead beads"). Dead beads without a fluorescent dye form regions in the detection area, which do not emit a fluorescent signal. Thus, a first region in which a specific dye is present can be identified and distinguished more clearly, because stray light is reduced.
• the amount of dead beads without a fluorescent dye could be from 5 to 50%, preferably form 10 to 30%, or about 20%, of the total number, or weight percent, of all beads in the detection area.
• all beads are present in equal amounts (or weight percent, if all are the same basic type). In a standard DNA synthesis process, this would mean that the amount (or weight% ratio) of beads labelled with each dye is 20% each, and 80% in total, whereas the amount of dead beads is 20%.
• the beads may form the first regions in the detection area, from which a fluorescent signal from one specific dye is emitted.
• the size of the beads is adapted to the size of the sites from which a specific signal is emitted from the sample probe in the corresponding analysis process.
• the average diameter of the beads could be from 100 nm to 10 ⁇ , preferably form 200 nm to 5 ⁇ or from 500 nm to 2 ⁇ .
• the calibration probe comprises the same fluorescent dyes as the corresponding sample probe.
• numerous fluorescent dyes are known and also commercially available for labelling biomolecules.
• the dyes could be organic dyes, which typically comprise aromatic systems and/or conjugated double bonds. Alternatively, they could be inorganic dyes, such as fluorescent nanocrystals.
• any fluorescent dye is applicable for the calibration probe, which is applicable for staining biomolecules, such as DNA or proteins, in the corresponding analysis process.
• organic fluorescence dyes could be based on fluorescein, rhodamines, coumarin, carbopyronins, oxazines, cyanine or derivatives thereof.
• Fluorescent dyes for labelling biomolecules are well- known in the art and commercially available.
• applicable dyes, especially for labelling DNA are commercially available under the trademark ATTO Dyes from ATTO-TEC GmbH, DE.
• the fluorescent dye comprises reactive groups or other binding sites for covalent or non-covalent attachment to the calibration probe or to a linker, for example to beads or a surface.
• Commercially available dyes are often provided with such reactive groups or binding sites.
• the dyes can be attached to the detection area or the linker by known methods.
• the dyes may comprise biotin or amino groups, whereas the beads may comprise streptavidin or carboxy groups on the surface. A covalent link between amino groups and carboxy groups is especially preferred.
• Organic fluorescent dyes are applicable in the calibration probe. However, it was observed that fluorescent small organic molecules often have a tendency to bleaching, especially when multiple calibrations steps or cycles are carried out with the same calibration probe. The relatively high light density of such organic dyes may have to be compensated by decreasing the amount of dyes and the exposure to electromagnetic radiation in the calibration process. However, small amounts of dyes and low signal intensity may not be in line with the conditions for subsequent analysis of biomolecules. Overall, organic fluorescent dyes are applicable for the inventive calibration probe, but the lifetime and performance of such calibration probes could still be improved.
• the fluorescent dyes are nanocrystals. Surprisingly, it was found that the problems with organic dyes are solved when using fluorescent nanocrystals. Calibration probes with nanocrystals were found to be stable in multiple calibration cycles, because no significant deterioration due to bleaching was observed. The signal intensity can be adjusted to a required strength and radiation, such that standard conditions for analyzing biomolecules with the corresponding sample probe can be simulated.
• fluorescent nanocrystals are inorganic crystals.
• a nanocrystal has at least one dimension of less than 100 nm, preferably of less than 50 nm.
• the average crystal diameter is below 100 nm or below 50 nm. The average diameter can be determined from microscopic pictures by evaluating a large number of randomly selected crystals, such as 100 or 500 crystals, in a random direction.
• the nanocrystal is a quantum dot.
• This is a semiconductor nanocrystal having an average diameter of less than 10 nm, which has fluorescence properties. The color of the fluorescence is determined by the size of the crystals.
• the nanocrystals comprise inorganic metal sulfides, especially of cadmium, selenium and/or zinc.
• the crystals comprise reactive groups or binding sites for attachment to the probe, for example to beads or a surface. Typically, these are the same as outlined above for the fluorescent dyes.
• Applicable fluorescent nanocrystals are known in the art and commercially available, for example under the trademark Trilite from Cytodiagnostics, CA, or under the trademark QDots from ThermoFisher Scientific, US. Various nanocrystals are available for emission of fluorescent signals at various defined wavelengths.
• the detection area is covered, such that the fluorescent dyes are shielded and protected from the environment.
• the cover could be a solid and/or a liquid cover.
• the solid cover could be a polymer film or a polymer matrix.
• the detection area is embedded in a polymer matrix.
• the polymer matrix is a solid matrix.
• it could be a gel, i.e. it could comprise a liquid, preferably water, distributed throughout the polymer matrix.
• the polymer is selected to have relatively low background fluorescence.
• the solid polymer matrix comprises a polymer selected from epoxy polymers and acrylates, such as polymethylmethacrylate (PMMA). Water should be removed before application of the polymer matrix, for example by adding and removing a volatile solvent, followed by evaporation. Then, the moisture free detection area can be covered with a polymer resin, followed by curing.
• the resin could be a two-component epoxy resin or an acrylate resin, such as PMMA dissolved in butyl acetate.
• the detection area is covered with a buffer and sealed with a cover, for example with a polymer film or glass cover.
• the detection area should be washed before sealing to remove unbound molecules or particles.
• the calibration probe may have any shape which is applicable for calibrating the electronic device.
• it has the shape of the corresponding sample probe.
• the calibration and sample probe are flow cells. This is advantageous, because during calibration fluids can be led through the calibration probe, and real working steps and cycles of the electronic device can be simulated.
• the sample probe is a flow cell, whereas the calibration probe is not a flow cell. In this embodiment, calibration is carried out without passing fluids through the calibration probe. This is advantageous, because the system does not require fluids and is relatively simple.
• the electronic device is a DNA sequencer and the detection area comprises four different fluorescent dyes,
• the fluorescent dyes are attached to beads, which are attached to the detection area, whereas only one of the fluorescent dyes is attached to each bead, wherein the beads form a monolayer, and the detection area comprises beads to which no fluorescent dye is attached.
• Subject of the invention is also a method for calibrating an electronic device electronic device for analysing biomolecules by detecting fluorescent signals from a sample probe, comprising the steps of
• the parameter is selected from sensitivity parameters, motor parameters, linearity range parameters, dynamic range parameters or noise determination parameters.
• Sensitivity parameters relate to the intensity of the signal.
• Motor parameters relate to the movement of the camera in the scanning process.
• the linear range is the range of input or output values for which an output signal is produced that is a direct, linear function of the input signal.
• Dynamic range parameters relate to the ratio between the largest and smallest values of the fluorescent signals.
• Noise determination parameters relate to suppression of unwanted signals.
• Subject of the invention is also a method for analysing biomolecules by detecting fluorescent signals from a sample probe, the method comprising
• sample probe which preferably comprises the same fluorescent dyes as the calibration probe
• Another subject of the invention is the use of the inventive calibration probe for calibrating an electronic device for analysing biomolecules by detecting fluorescent signals from a sample probe.
• the calibration probe is adapted to the sample probe.
• the calibration probe is prepared such that structural features and components are adapted to the sample probe as far as possible, such as the intensity and type of fluorescent signals, the distribution, size and shape of first and second regions, and the like. If the sample probe comprises beads, especially in a monolayer arrangement, the calibration probe may likewise comprise corresponding beads in a similar arrangement, such as a monolayer.
• a calibration probe for calibrating an electronic device for analysing biomolecules is provided, which allows calibration with high precision and specificity.
• the probe is especially suitable for DNA sequencers based on fluorescent technology.
• the electronic device can be adjusted easily and precisely to conditions, under which biomolecules are analysed.
• the fluorescent signals from the calibration probe are close to those emitted under real measurement conditions during examination of biomolecules.
• a calibration probe can be provided with a high stability, which can be stored for a long time and used in a large number of calibration processes. Especially when provided with nanocrystals, the probe is resistant to bleaching. It is not necessary any more to prepare solutions with fluorescent dyes for every calibration process. It is a specific advantage that the calibration probe can be provided without fluids.
• the probe can be produced by the manufacturer of the electronic device and can be adapted internally to a standard electronic device before transmission to a user.
• the calibration probe is applicable for optimal calibration of an electronic device with respect to scanning two-dimensional samples and surfaces.
• the probe can be used for calibrating various parameters of the electronic device with great precision.
• the intensity and distribution of fluorescent signals can be conveniently adapted for each calibration probe.
• Figure 1 shows schematically and in exemplified form a schematic view of a test sample
• Figure 2 shows fluorescence images of a test sample at different excitation wavelengths using the four ATTO dyes: a) green fluorescence at excitation with blue light, b) yellow fluorescence at excitation with green light; c) orange fluorescence at excitation with yellow light; d) dark red fluorescence at excitation with red light.
• the fluorescent flow cell is a special flow cell used for sequencing DNA with the QIAGEN GeneReaderTM platform. Instead of magnetic beads containing DNA, the FFC contains 4 kinds of magnetic beads that each had been labelled with a specific fluorescent dye. The FFC also contains 20% non-labelled beads, making it an "80% fluorescent" flow cell. The purpose of these FFCs is to have a tool to calibrate the GeneReader instruments. Material needed for one FFC
• ATT0532 the dyes have a rather poor solubility in water or in Phosphate Buffered Saline (PBS buffer).
• PBS buffer Phosphate Buffered Saline
• ATT0532 can be dissolved in Millipore water alone. All others have to be dissolved in 20 % DMSO in water. The concentration of the stock solution for all dyes is 1 mg/ml. All dyes except ATT0532 were first dissolved in 200 ⁇ DMSO, then after complete dissolution, 800 ⁇ PCR grade water was added and the solutions were vortexed for 10 seconds. The dye stock solutions have to be stored in the dark at -20°C in the freezer.
• the desired aliquot of the stock solution is diluted 1 :100 after thawing with PBS buffer and vortexed for 5 seconds.
• the work solutions are stored in the freezer at -20°C after use. While processing the beads with the work solutions, the work solutions should be kept in the dark to avoid bleaching.
• Example 3 Labelling the beads
• the process how to label a set of beads is the same for all 4 dyes; only the amount of dye work solution varies. In the following, a process how to label approximately 120 million beads is described, which would be enough to fill one flow cell to "100% fluorescent" if only one set of labelled beads would be used. Since 4 sets of beads with different labels are used and also 20% non-labelled beads, the amount that is created is sufficient for 5 flow cells.
• Wash 1 200 ⁇ of 1 x PBS buffer is pipetted into the tube and mixed thoroughly about 20 times with the pipette set to 100 ⁇ to completely re-suspend the beads. Then the magnet is put to the tube again for 1 -2 minutes and the supernatant is removed again.
• Wash 2 Repeat all the steps of wash 1 .
• Labelling the beads Re-suspend the beads with 50 ⁇ 1 x PBS buffer and then add the following amounts of the desired fluorescent dye. Only one of the dyes per batch of labelled beads is added. Dyes may never be mixed to prevent formation of beads that carry more than one dye.
• ThermoFisher Scientific US, under the following trademarks:
• the non-biotinylated nanocrystals were biotinylated prior to use with a biotinylation kit (D- succinimidylester) from Life Technologies/ThermoFisher Scientific, US, according to the manufacturer ' s instructions. Then, the fluorescent nanocrystals were coupled via biotin to the streptavidine magnetic beads in PBS buffer in a method as described in example 3 above. After the coupling, the labelled beads were injected into a flow cell and left to crosslink for 2h.
• a biotinylation kit D- succinimidylester
• a flow cell prepared as described in example 6 above was emptied of all liquid and flushed once with methanol, then flushed once with butylacetate and then filled with a 15% solution of polymethylmethacrylate (PMMA, from Goodfellow Cambridge Ltd, GB) in butylacetate. The flow cell then was left standing for 3-5 days until all the butylacetate was evaporated. The PMMA resided in the flow cells and formed a solid transparent polymer matrix which covered the beads.
• PMMA polymethylmethacrylate
• the nanocrystal calibration probe prepared according to example 7 was used to calibrate DNA sequencers of the QIAGEN GeneReaderTM platform. It is advantageous if different instruments are adjusted equally. This means that the signal ratio for all color channels is always the same and that each channel of a specific color yields the same fluorescence read-out signal to the camera. With the calibration probe as a reference, instruments were calibrated to the same readout intensities. This was possible by adjusting the LED intensity of each channel to the desired signal level. Adjusting the LED intensity to a standard is important, because the light intensity of LEDs is variable.
• this calibration flow cell was used to mimic a biological sample in multiple test runs, in which the DNA sequencer was operated as in a normal workflow mode but without liquid reagents. During the test run, mechatronic, visual and algorithmic operations were checked. This saved the time consuming, error-prone and expensive preparation of and operation with instable liquid dye solutions. The complete tests under operating conditions could be performed without the need to prepare samples anew or refill reagents.
• the calibration probe could be applied for multiple test runs with different DNA sequencer instruments without substantial deterioration of the probe and change of the fluorescent signals.

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• 2016
  • 2016-11-14 EP EP16795052.6A patent/EP3377876A1/de not_active Withdrawn

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