USE OF A CALIBRATION STARGET

Description

FIELD OF TECHNOLOGY

[0001] The present invention relates to a calibration target and its use for calibrating an optoelectronic device for analyzing biomolecules by detecting fluorescence signals from a sample. STATE OF THE ART

[0002] In the field of medical diagnostics and biotechnology, systems for analyzing biomolecules by detecting fluorescence signals from a sample are an important tool. Such systems contain optical detectors that detect the light emitted by a sample. One example of an application for such systems is gene sequencing.

[0003] For example, the four bases found in DNA—adenine (A), guanine (G), cytosine (C), and thymine (T)—are labeled with fluorescent dyes specific to each base. When the sample treated in this way is excited with laser light, these dyes emit different wavelengths that can be assigned to the respective bases. By detecting the intensity of this emission, resolved by location and wavelength, information can be obtained down to the precise structure of DNA.

[0004] To achieve this, however, it is crucial to calibrate the optical detectors—that is, to expose them to light of a known frequency and intensity in order to adjust the detector signal to a reference value. This calibration may be necessary occasionally, during service, or before each use of the system to achieve results with the required accuracy.

[0005] Instead of a sample to be examined, a calibration target is usually used for calibration. This target is either permanently installed in the respective system or is introduced into the system for the purpose of calibration.

[0006] A module, similar to the one used for the actual measurement process in the respective system, is often used as a calibration target. These modules have channels and reservoirs through which the samples to be examined are pumped. Filling these with a known mixture of the same dyes used for the measurement produces defined signals at different fluorescence wavelengths in the detectors when excited with laser light at one or more excitation wavelengths.

[0007] A disadvantage of these calibration targets is the short service life of the dyes. These can bleach or otherwise degrade due to laser light irradiation, thus emitting less light intensity after a while. They are then unusable for calibration purposes. A known countermeasure is to ensure the exchange of aged dyes by constantly moving the dye mixture in the channels (e.g., through Brownian motion or pumping). However, this is a relatively complex process for obtaining a stable calibration target.

[0008] To prevent dye degradation, WO 2017084998 A1 describes a calibration target based on a fluorescent, nanocrystalline solid. Metal sulfides are mentioned as suitable solids in this context. However, due to the relatively narrow-band emission of these nanocrystals, four different materials are required to cover the wavelengths to be calibrated. Furthermore, such nanoparticles often exhibit a relatively large Stokes shift, making it impossible to calibrate detectors that require sensitivity near the excitation wavelength.

[0009] The documents US 2008 / 038835 A1, US 2007 / 190566 A1, DE 2010446 U1 and US 2013 / 126757 A1 disclose further calibration targets based on a fluorescent solid.

[0010] Document US 2010 / 323178 A1 describes a transparent, conductive layer based on a matrix with embedded graphene. This layer is used to manufacture sensors based on the electrical conductivity of graphene.

[0011] Document CN 104 596 994 A describes a doped titanium dioxide / graphene oxide composite film and a process for its preparation, as well as its use for the semi-quantitative in situ characterisation of the amount of protein adsorption on the film surface, in vitro cell culture and tissue engineering. SUMMARY OF THE INVENTION

[0012] It is therefore an object of the invention to provide the use of an improved calibration target for calibrating an optoelectronic device for analyzing biomolecules.

[0013] This object is achieved by using a calibration target according to claim 1. Advantageous details also emerge from the claims dependent on claim 1.

[0014] The use of a calibration target for calibrating an optoelectronic device for analyzing biomolecules by detecting fluorescence signals from a sample is disclosed. The calibration target comprises a solid fluorescent layer arranged on a substrate and excitable by laser light. The fluorescent layer comprises an optically inactive, transparent matrix with a refractive index greater than 2 and a carbon-based component excitable to emit light. The component is graphene, graphene oxide, or reduced graphene oxide.

[0015] It has been shown that a calibration target constructed in this way is particularly stable over the long term, even when excited to emit light using high laser power. Such a calibration target can be permanently installed in a system and used for calibration as needed.

[0016] Materials can be found for the transparent matrix and the component that can be excited to emit light that ensure good coupling of the excitation light and broadband fluorescence, so that detectors can be calibrated for several different wavelengths.

[0017] Further advantages and details of the present invention will become apparent from the following description of various embodiments with reference to the figures. The invention is explained and described using gene sequencing, but the calibration target according to the invention is suitable for other types of optoelectronic devices for analyzing biomolecules by detecting fluorescence signals from a sample, such as automated fluorescence measuring microscopes, which can be used to investigate viral distribution in tissue, metabolic processes, or protein interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] This shows Figure 1 shows an emission spectrum from gene sequencing, Figure 2 shows a first embodiment of the calibration target, Figure 3 shows a second embodiment of the calibration target, Figure 4 shows a third embodiment of the calibration target. DESCRIPTION OF THE EMBODIMENTS

[0019] The Figure 1 shows a schematic of the emission spectrum that can occur during DNA sequencing. The intensity I of the light is plotted in arbitrary units against the wavelength λ of the light.

[0020] If the sample to be examined is excited with laser light L at a wavelength of approximately 510 nm, the dyes used to label the four bases found in DNA emit light of different wavelengths. This fluorescence-based emission of light occurs at different wavelengths specific to the respective bases. The four emission peaks are therefore labeled with the names of the respective bases: G, A, T, and C. Photodetectors with corresponding color filters are used, for example, to detect these peaks G, A, T, and C.

[0021] To calibrate these detectors, a calibration target is used, which is inserted into the gene sequencing system instead of a sample carrier.

[0022] Such a calibration target T is in the Figure 2 shown. An opaque layer 2 of chromium, which is interrupted in some places, is arranged on a transparent glass substrate 1. Instead of chromium, other opaque materials can be used, such as aluminum, tantalum, molybdenum, or tungsten. This creates a reference structure (e.g., dots, lines, crosses, etc.) that can be used in a detector D to evaluate the position of the calibration target T or to focus the optics, for example, by analyzing the edges of the reference structure.

[0023] The calibration target T also has a fluorescent layer 3 applied to the chromium layer 2, so that the openings of the chromium layer 2 are also filled with the fluorescent material.

[0024] An optoelectronic evaluation unit contains a source of laser light L and one or more photodetectors D for detecting the light emitted by the fluorescent layer 3.

[0025] The Figure 3 The second embodiment shown for a calibration target T represents a slight modification of the first embodiment. Here, the fluorescent layer 3 is applied such that the openings in the chromium layer 2 are not filled with fluorescent material. The excitation light L nevertheless reaches the fluorescent layer 3.

[0026] According to the Figure 4In the third embodiment shown, the opaque chromium layer 3 can also be omitted if the fluorescent layer 3 itself is structured, so that bright and dark areas can be evaluated as a reference structure in the detector D.

[0027] In all three embodiments, a fluorescent layer 3 is used, which, when excited by laser light L, emits broadband fluorescent light, covering all wavelengths to be detected by the detector D. This allows the spectral sensitivities of these detectors D to be calibrated.

[0028] The fluorescent layer 3 is preferably inorganic to prevent degradation upon excitation with high laser power. Thus, TiO2 or Ta2O5 can be used as a transparent, non-luminescent matrix with embedded graphene oxide. Graphene oxide is an inorganic, luminescent, or fluorescent material.

[0029] To maximize the interaction of the irradiated laser light L with the fluorescent medium in the matrix, the matrix is ​​selected from a high-refractive index (approximately n > 2) material with a colloidal structure, such as TiO2 or Ta2O5, which was obtained by a sol-gel process.

[0030] Similar to organic dyes, the fluorescent material should exhibit a low Stokes shift and the broadest possible emission bandwidth to address all detection channels. Furthermore, it must exhibit fluorescence at relatively long wavelengths, e.g., 532 nm. Graphene oxide fulfills all of these requirements particularly well. However, other carbon-based materials such as graphene or reduced graphene oxide are also generally suitable.

[0031] A reasonable composition of the fluorescent layer 3 is based on a proportion of the fluorescent material in the entire layer 3 of 1 to 50 percent by weight, preferably 5 to 20 percent by weight.

[0032] The thickness of the glass substrate 1 (another transparent substrate can be used instead of glass) is typically in the range of 0.05 mm to 4 mm. The opaque chromium layer 2 typically ranges from 20 nm to 1000 nm, and the fluorescent layer 3 ranges from 500 nm to 20,000 nm.

[0033] The respective layers can be easily produced using known manufacturing processes, e.g., spin coating, dip coating, or spray coating with solution; PVD processes such as magnetron co-sputtering, arc evaporation, high-energy pulse magnetron sputtering, thermal or electron beam evaporation; or PECVD.

[0034] In the previously described embodiments, a transparent substrate 1 was used to guide the laser light L through the substrate 1 onto the fluorescent layer 3. Alternatively, it is also possible to use an opaque substrate. In this case, the laser light L must be irradiated from the side facing away from the substrate. A reference structure made of structured chromium must then be arranged between the fluorescent layer 3 and the laser light source.

[0035] Some manufacturing processes for such calibration targets T are given as examples: Spin-on process: 20 ml of toluene is mixed with 5 wt.% PMMA and stirred at 45°C for 5 hours. 1 ml of titanium tetraisopropoxide (TTIP) is added dropwise. 5 ml of the resulting solution is spun onto a glass wafer, which has previously been coated with a structured chromium layer using reactive ion etching (RIE), at 1500 rpm for 30 s. The coated wafer is annealed at 300°C for 5 hours to create fluorescent carbon structures. The calibration targets T are then cut out using laser cutting. Spin-on process: 20 ml of toluene is stirred dropwise with 1 ml of titanium tetraisopropoxide (TTIP). 5 ml of alkylamine-functionalized graphene oxide in toluene (e.g., Sigma Aldrich Art. No. 809055-50ML) are stirred with the solution. 4 ml of the resulting solution is spun onto a glass wafer (D263 bio) that has previously been coated with a structured chromium layer using a lift-off process at 1000 rpm for 30 s.The coated wafer is annealed at 250°C for 5 hours to crystallize the matrix. The calibration targets T are then separated using a dicing saw. PVD process: An 8" quartz wafer is coated with a structured chromium layer using sputter deposition, lithography, and RIE. This wafer is placed in a PVD system with two magnetron sputter targets and coated with a 10 wt.% graphite Ta2O5 coating with a thickness of 200 nm using co-sputtering of Ta2O5 and graphite. After removal from the wafer, the wafer is separated using laser perforation and breaking. PACVD process: An 8" glass wafer is coated with a structured titanium layer using e-beam evaporation and lithography. This is placed in a PACVD system and coated with a 20 percent C / TiO2 coating using RF plasma discharge in argon, octadiene, and TTIP. The wafer is then annealed in a furnace at 320 °C for 3 hours to crystallize the matrix.After removal, the wafer is separated using laser cutting.

Claims

1. Use of a calibration target for calibrating an optoelectronic device for analysis of biomolecules by detecting fluorescence signals from a sample, wherein the calibration target has a solid fluorescence layer (3) which is disposed on a substrate (1) and is excitable by laser light (L), wherein the fluorescence layer (3) has an optically inactive transparent matrix having a refractive index greater than 2 at a wavelength of 510 nm, wherein the matrix has a colloidal structure comprising titanium dioxide or tantalum pentoxide and a carbon-based component embedded therein which is excitable for emission of light, wherein the carbon-based component is graphene, graphene oxide or reduced graphene oxide.

2. Use of a calibration target according to Claim 1, characterized in that the substrate (1) is a glass.

3. Use of a calibration target according to either of the preceding claims, characterized in that the fluorescence layer (3) is composed entirely of inorganic components.

4. Use of a calibration target according to any of the preceding claims, characterized in that a structured opaque layer (2) partly shields the fluorescence layer (3) from the laser light (L) and thus forms a reference structure.

5. Use of a calibration target according to Claim 4, characterized in that the opaque layer (2) consists of chromium.

6. Use of a calibration target according to any of Claims 1-3, characterized in that a reference structure is formed by structuring of the fluorescence layer (3).

7. Use of a calibration target according to any of the preceding claims, characterized in that the proportion of the component excitable for emission of light in the fluorescence layer (3) is between 1 and 50 per cent by weight, preferably between 5 and 20 per cent by weight.