EP1877773A1 - Neue apparatur und verfahren zur beschichtung von trägersubstraten für einen analytnachweis mittels affinitäts-nachweisverfahren - Google Patents
Neue apparatur und verfahren zur beschichtung von trägersubstraten für einen analytnachweis mittels affinitäts-nachweisverfahrenInfo
- Publication number
- EP1877773A1 EP1877773A1 EP06724515A EP06724515A EP1877773A1 EP 1877773 A1 EP1877773 A1 EP 1877773A1 EP 06724515 A EP06724515 A EP 06724515A EP 06724515 A EP06724515 A EP 06724515A EP 1877773 A1 EP1877773 A1 EP 1877773A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- carrier substrates
- liquid
- atomized
- coating
- coated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B17/00—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups
- B05B17/04—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups operating with special methods
- B05B17/06—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups operating with special methods using ultrasonic or other kinds of vibrations
- B05B17/0607—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups operating with special methods using ultrasonic or other kinds of vibrations generated by electrical means, e.g. piezoelectric transducers
- B05B17/0615—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups operating with special methods using ultrasonic or other kinds of vibrations generated by electrical means, e.g. piezoelectric transducers spray being produced at the free surface of the liquid or other fluent material in a container and subjected to the vibrations
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
Definitions
- a detection method based on bioaffinity reactions can be carried out both in a homogeneous solution and on the surface of a solid support ("carrier substrate").
- carrier substrate Depending on the specific structure of the method, after binding of the analytes to the corresponding recognition elements and optionally further detection substances and optionally between different Each step of the washing steps is necessary in order to separate the complexes formed from the recognition elements and the analytes to be detected and optionally further detection substances from the remainder of the sample and any additional reagents used.
- methods are used in which the detection of different analytes in discrete sample containers or "wells" of these plates takes place in so-called microtiter plates.
- an adhesion-promoting layer applied to the carrier substrate As suitable for the preparation of the primer layer, a variety of materials are known, for example, unfunctionalized or functionalized silanes, epoxides, functionalized, charged or polar polymers and "self-assembled passive or functionalized mono- or multilayers", alkyl phosphates and phosphonates, multifunctional block copolymers , such as poly (L) lysine / polyethylene glycols.
- coatings of bioanalytical sensor platforms or implants for medical applications are used as carrier substrates with graft copolymers as adhesion-promoting layer, with a polyionic main chain, for example binding to a carrier substrate (electrostatically), and non-interactive "(adsorption-resistant) side chains.
- these non-binding components may be known be selected from the groups of albumins, in particular bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, non-specific or empirical for the analyte or antibodies to be detected non-specific antibodies (especially for immunoassays), detergents - such as Tween 20 -, not with to be analyzed polynucleotides hybridizing, fragmented natural or synthetic DNA, such as an extract of herring or salmon sperm (especially for polynucleotide hybridization assays), or uncharged, but hydrophilic polymers, such as polyethylene glycols or dextranes.
- albumins in particular bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, non-specific or empirical for the analyte or antibodies to be detected non-specific antibodies (especially for immunoassays), detergents - such as Tween 20 -, not with to be analyzed polynu
- the inventive method represents a further development of the spraying method described above, wherein the production of very fine liquid droplets for the inventive method in a preferred embodiment by ultrasound treatment.
- the coating apparatus used for this method comprises in a preferred embodiment a closed container with a holder for horizontal storage of the carrier substrates (based on the surface of the liquid to be atomized) and an underlying ultrasonic generator, which is immersed in the liquid to be atomized.
- the inventive method is characterized in that the droplets produced are substantially smaller than in the case of the spray process.
- a very dense fog is generated over the liquid to be atomized, which is evenly distributed in a preferred embodiment by turbulence using a weak additional nitrogen stream in the container, the container is preferably closed except for gas inlets and gas outlets.
- Fig. 1 shows schematically a coating apparatus according to the invention.
- Fig. 2 shows the geometry of an array of measurement areas with 12 different applied samples in a two-dimensional array (“microarray”) and a linear array of 6 arrays on a common carrier substrate.
- 3A-3C show the fluorescence signals of microarrays, wherein the free surfaces of the associated carrier substrates were passivated by means of different coating methods, in each case with enlargements of the marked image sections underneath (A: dipping method, B: spraying method, C: nebulizing method according to the invention).
- FIG. 4A shows the mean values and standard deviations of the background signal intensities, which were respectively determined between all spots of the microarrays, wherein the free surfaces of the associated carrier substrates were passivated by means of different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention).
- 4B shows the mean values and standard deviations of the fluorescence intensities of all reference spots (for explanation, see in the exemplary embodiment) of the microarrays, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention).
- 5A shows the averaged intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulisation method according to the invention) and the microarrays afterwards with solutions of the antibody Al (anti-p53) and then each incubated for detection by fluorescence detection with Alexa 647 fluorine anti-rabbit Fab fragments.
- A dipping method
- B spraying method
- C nebulisation method according to the invention
- 5B shows the average intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention) and the microarrays afterwards with solutions of antibody A2 (anti-phospho-p53) and then each incubated for detection by fluorescence detection with Alexa 647 fluorine anti-rabbit Fab fragments.
- A dipping method
- B spraying method
- C nebulizing method according to the invention
- the first object of the present invention is an apparatus for coating carrier substrates for detecting one or more analytes in an affinity detection method, comprising:
- liquid to be atomized is to be understood as the total amount of liquid within the coating apparatus according to the invention, to which the pulses of the actuator act to atomize the liquid, with the consequence of the conversion of a part of this liquid into mist.
- the generation of the mist above the liquid to be atomized is effected by the action of ultrasound within that liquid. Accordingly, it is preferred that said actuator serves to generate ultrasound.
- said actuator comprises the membrane of an ultrasonic generator.
- the uniformity and high homogeneity of the layer to be produced is of utmost importance.
- simple, commercial nebulizers such as those used primarily in terrarium, but must also be expected with the occurrence of large drops or even splashes from the liquid to be atomized.
- the coating apparatus comprises a droplet separator.
- This droplet separator is to be arranged in the volume of space between the surface of the liquid to be atomized and the holder on which the carrier substrates to be coated are stored during the coating process.
- a mist eliminator to be used may be impermeable to vapor and mist (for example, if the mist eliminator is a closed solid). It may be advantageous if the mist eliminator has the geometric shape of a concave mirror. For example, a watch glass (having a concave surface) may be used as a mist eliminator.
- the carrier substrates are coated during storage in the holder during the coating process on its side facing away from the surface of the liquid to be atomized / surface, wherein a coating on other surfaces must not be excluded.
- a coating apparatus in a coating method according to the invention to produce geometrically structured coatings by optionally sequential atomization of one or more optionally different liquids.
- the prerequisite for producing reproducible coated regions on the carrier substrates in their geometry is that areas of the carrier substrate that are not to be coated in each case are covered fluidically by a corresponding suitable mask, so that no mist droplets reach the areas not to be coated.
- the coating apparatus additionally comprises at least one gas inlet.
- the apparatus may additionally include one or more outlets for venting gas and / or mist.
- said provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the vicinity of said carrier substrates comprise a ventilator with which the generated mist and, if appropriate, additional gases introduced into the container of the apparatus are fluidized to achieve a better mixing and thus elimination of inhomogeneities of the mist distribution.
- the coating apparatus additionally comprises provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or one or all walls of the liquid container. It is also preferred that the holder of the coating apparatus for receiving and / or storing the carrier substrates during the coating process is thermostatable.
- the coating apparatus additionally comprises provisions for controlling and / or regulating the pressure within the liquid container during the coating process.
- the coating apparatus according to the invention additionally nebulizes provisions for collecting and recycling / recycling on the walls of the liquid container Liquid includes.
- the coating apparatus additionally comprises provisions to facilitate the cleaning of the liquid container.
- the provisions may include a hydrophobic coating on the surface of said container walls, both for recirculation to the inner walls of the liquid container and liquid to be atomized, and for ease of cleaning.
- Such provisions may also relate to the geometric shape, for example, by corners are avoided in which liquid can collect and is difficult to remove again, or at least rounded.
- the carrier substrates to be coated are stored substantially horizontally in the holder of the coating apparatus.
- essentially horizontal deviations of up to +/- 10 ° from a horizontal bearing should be included.
- the coating apparatus additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and surfaces of the carrier substrates to be coated.
- the liquid container is closed except for optional gas inlet and optional additional gas and / or mist outlets.
- the liquids to be atomized are low-viscosity liquids having a viscosity of less than 3 cP. In particular, this may be act aqueous solutions. However, the liquids to be atomized may also be organic, in particular alcoholic solutions.
- the carrier substrates to be coated are substantially planar.
- the term "essentially planar” should be understood to mean that said carrier substrates comprise a plane in which, apart from a possibly existing three-dimensional structure (such as side walls of sample containers to be provided on the carrier substrate surface), the surface to be coated is located to substantially parallel second plane in which the opposite surface of the carrier substrates is located, wherein under the name “substantially parallel” deviations of up to +/- 10 ° of parallelism are included.
- substantially planar carrier substrates with both smooth and rough surfaces to be coated are to be understood.
- the carrier substrates to be coated may consist of a single (self-supporting) layer, such as. As glass slides, or even of several layers.
- At least one layer of the carrier substrates to be coated in the propagation direction of an irradiated excitation light or measurement light is substantially optically transparent.
- optical transparency of a material or of a carrier substrate should be understood to mean that the run length of a light propagating in said material or in said carrier substrate or in the (high-index) waveguiding film of a carrier substrate (see below) as optical waveguide is at least is a portion of the visible spectrum (between 400 nm and 750 nm) greater than 2 mm, provided that this run length is not limited by structures for changing the propagation direction of said light, for example, the run length of optically visible light, ie the distance on the path of the Light in the corresponding material, until the light intensity decreases to a value 1 / e of the intensity of origin when the light enters this material, on the order of several centimeters (eg in thin-film waveguides, see below) up to meters or kilometers (in Trap of optical fiber n for the optical signal transmission).
- the propagation length of an in of the waveguiding layer guided light by a decoupling diffractive grating are limited to a few microns.
- this limitation of the run length is due to the structuring, and not by the material properties of the structure.
- such a lattice waveguide structure is to be referred to as "optically transparent.”
- support structures or “layers that are substantially optically transparent” are to be understood as meaning the intensity of a support substrate or layers transmitted light by less than 50%.
- the support substrates to be coated comprise a thin metal layer, preferably of gold or silver, optionally on an underlying intermediate layer with refractive index preferably ⁇ 1.5, wherein the thickness of the metal layer and the possible intermediate layer are selected such that a microwavenplasmon at the wavelength of an incident excitation light and / or at the wavelength of a generated luminescence can be excited.
- the thickness of the metal layer is preferably between 10 nm and 1000 nm, more preferably between 30 nm and 200 nm.
- luminescence in this application refers to the spontaneous emission of photons in the ultraviolet to infrared range after optical or non-optical, such as electrical or chemical or biochemical or thermal excitation.
- chemiluminescence, bioluminescence, electroluminescence and in particular fluorescence and phosphorescence are included under the term “luminescence”. Fluorescence and phosphorescence are particularly preferred forms of luminescence.
- Optical waveguides are particularly well suited as a carrier substrate for analyte detection in an affinity detection method, since waveguiding combines the formation of a so-called "evanescent" field at the interfaces of the high-refractive-wave waveguiding layer with the adjacent layers (which may also be air) with lower refractive index
- the penetration depth of this evanescent field into the environment is limited to dimensions less than the wavelength of the guided light (eg, 200 nm to 400 nm) such that interactions of analyte molecules or detection molecules or molecular moieties (such as fluorescent labels ) with this evanescent field excited and observed spatially highly selectively on a surface of the waveguide and interference signals from the far field, eg., From the depth of a sample medium, can be largely excluded.
- the continuous or discrete waveguiding regions to be coated on the carrier substrates comprise a surface of the carrier substrates to be coated.
- the carrier substrates to be coated planar waveguide optical waveguide having a substantially optically transparent, waveguiding layer (a) on a second, also substantially optically transparent layer (b) having a lower refractive index than layer (a) and optionally also in Substantially optically transparent intermediate layer (b ') between layer (a) and layer (b) also with a lower refractive index than layer (a).
- the lower the layer thickness the greater the sensitivity up to a lower limit of the layer thickness.
- the lower limit value is determined by the termination of the light pipe when the value falls below a value which depends on the wavelength of the light to be led, as well as by an increase in the propagation losses in the case of very thin layers with further layer thickness decrease. It is preferred that the product of the thickness of the layer (a) and its refractive index is one tenth to one whole, preferably one third to two thirds, of the wavelength of an excitation light or measurement light to be coupled into the layer (a).
- the coupling via prisms which are preferably added to the waveguide without interstice or connected to the waveguide via a refractive index-matching liquid. It is also possible to introduce the excitation light via an optical fiber to the optical waveguide and to couple in via an end face or to couple the light coupled into another waveguide into the waveguide by bringing both waveguides close to each other such that their evanescent fields overlap and therewith Energy transfer can take place.
- the discrete or continuous waveguiding regions of the carrier substrates to be coated are in contact with one or more grating structures (c), which enable the coupling of excitation light or measuring light into waveguiding layers of said carrier substrates, and / or to one or more grating structures (c '), which enable the decoupling of excitation light or measuring light from waveguiding layers of said carrier substrates are, wherein in the case of simultaneously present on a carrier substrate grating structures (c) and (c') may have the same or different grating periods.
- Said lattice structures are preferably relief gratings of any desired profile, for example of rectangular, triangular, sawtooth, semicircular or sinusoidal profile, or of phase or volume gratings with a periodic modulation of the refractive index in the essentially planar layer (a ).
- grating structures (c) are formed as surface relief gratings.
- the grating structures (c) and / or (c ') may be mono- or multi-diffractive and have a depth of 2 nm-100 nm, preferably 10 nm-30 nm, and a period of 200 nm-1000 nm, preferably 300 nm - 700 nm, have.
- the ratio of the land width of the grid lines to the grating period may be between 0.01 and 0.99, with a ratio between 0.2 and 0.8 being preferred.
- Glass or quartz, transparent thermoplastic moldable or millable plastics for example polycarbonates, polyimides, acrylates, especially polymethyl methacrylates, polystyrenes, cyclo-olefin polymers and cyclo-olefin copolymers.
- Another object of the present invention is a method for coating carrier substrates for the detection of one or more analytes in an affinity detection method, characterized in that said to be coated carrier substrates in a holder of a coating apparatus according to the invention according to one of
- Liquid is fogged up and - From the generated mist, a deposition of substances contained in the nebulised liquid (compounds) on the carrier substrates to be coated takes place, wherein the carrier substrates in any contact with the surface to be nebulized
- Liquid are located.
- the generation of the mist above the liquid to be atomized is effected by the action of ultrasound within that liquid. Accordingly, it is preferred that said actuator serves to generate ultrasound.
- said actuator comprises the membrane of an ultrasonic generator and the atomization of liquid occurs by means of ultrasonic waves generated therein.
- said actuator is immersed in the operating state in liquid to be atomized.
- the actuator is completely within the liquid to be atomized.
- the coating apparatus comprises a droplet separator which prevents the contact of splashes and large droplets from the liquid to be atomized with the carrier substrates to be coated.
- a "large" drop is to be understood as meaning a drop with a diameter of more than 200 ⁇ m, whereby the mist eliminator may be impermeable to gas and mist, for example, the mist eliminator may be a closed solid
- a watch glass having a concave surface
- a mist eliminator to be used may also be permeable to drops up to a defined size. This can be technically realized, for example, by virtue of the fact that the mist eliminator comprises a fine-meshed net, with the mesh spacing of which the maximum size of drops to be passed is determined.
- the coating apparatus additionally comprises provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or individual or all walls of the liquid container and the temperature of the nebulizing Liquid and / or single or all walls of the liquid container is controlled and / or varied during the coating process. It is also preferred that the holder of the coating apparatus for receiving and / or storing the carrier substrates is thermostated during the coating process.
- the carrier substrates during the coating process about an axis be rotated perpendicular to the mounting plane.
- a particular variant of the method according to the invention is characterized in that geometrically structured coatings are produced by optionally sequential atomization of one or more optionally different liquids using masks to be applied to the carrier substrates to be coated with a coating apparatus according to the invention.
- the prerequisite for the production of coated regions reproducible in their geometry on the carrier substrates is that areas of the carrier substrate which are not to be coated are covered in a fluid-tight manner by a suitable mask, so that no mist droplets reach the areas not to be coated.
- the carrier substrates to be coated are stored substantially horizontally during the coating process in the holder of the coating apparatus.
- the coating apparatus additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and surfaces of the carrier substrates to be coated, and so that a well-defined distance between said liquid and the liquid surfaces to be coated for the period of the coating process is set.
- the liquid container of the coating apparatus is closed except for optional gas inlet and optional additional gas and / or mist outlets.
- the liquids to be atomized are low-viscosity liquids having a viscosity of less than 3 cP.
- these may be aqueous solutions.
- the liquids to be atomized may also be organic, in particular alcoholic solutions.
- the carrier substrates to be coated are substantially planar.
- the carrier substrates to be coated may consist of a single (self-supporting) layer, such as. As glass slides, or even of several layers.
- the carrier substrates to be coated comprise a thin metal layer, preferably of gold or silver, optionally on an underlying intermediate layer with refractive index preferably ⁇ 1.5, wherein the thickness of the metal layer and the possible intermediate layer are selected such that a microwavenplasmon at the wavelength of an incident excitation light and / or at the wavelength of a generated luminescence can be excited.
- the carrier substrates to be coated comprise optical waveguides comprising one or more layers.
- the carrier substrates may be formed throughout as optical waveguides or comprise discrete waveguiding regions.
- the carrier substrates to be coated planar waveguide optical waveguide having a substantially optically transparent, waveguiding layer (a) on a second, also substantially optically transparent layer (b) having a lower refractive index than layer (a) and optionally also in Substantially optically transparent intermediate layer (b ') between layer (a) and layer (b) also with a lower refractive index than layer (a).
- the discrete or continuous waveguiding regions of the carrier substrates to be coated can be brought into optical interaction with one or more optical coupling elements for coupling excitation or measuring light from one or more light sources during the detection step of an affinity detection method with said carrier substrates, wherein said optical coupling elements are selected from the group comprising prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, face couplers with focusing lenses arranged in front of one waveguide layer of the carrier substrates, preferably cylindrical lenses, and grating couplers.
- the discrete or continuous waveguiding regions of the carrier substrates to be coated are in contact with one or more grating structures (c), which enable the coupling of excitation light or measuring light into waveguiding layers of said carrier substrates, and / or to one or more grating structures (c '), which enable the decoupling of excitation light or measuring light from waveguiding layers of said carrier substrates are, wherein in the case of simultaneously present on a carrier substrate grating structures (c) and (c') may have the same or different grating periods.
- the refractive index of the first optically transparent layer (a) is larger than 1.8. It is also preferable that the first optically transparent layer (a) comprises a material selected from the group consisting of silicon nitride, TiO 2 , ZnO, Nb 2 O 5 , Ta 2 O 5 , HfO 2 , and ZrO 2 , particularly preferably TiO 2 , Ta 2 O 5 or Nb 2 O 5 .
- the second optically transparent layer (b) of the carrier substrates to be coated comprises a material from the group consisting of silicates, for.
- silicates for.
- glass or quartz transparent thermoplastic molding, sprayable or millable plastics, such as polycarbonates, polyimides, acrylates, in particular polymethyl methacrylates, polystyrenes, cyclo-olefin polymers and cyclo-olefin copolymers.
- Characteristic of a preferred group of embodiments of the coating method according to the invention is that the carrier substrates to be coated enable the detection of one or more analytes in an affinity detection method by means of detection of one or more excited luminescences.
- the carrier substrates to be coated enable the detection of one or more analytes in an affinity detection method by means of detection of changes in the effective refractive index in the near field (evanescent field) on a surface of said carrier substrates.
- a group of embodiments of the method according to the invention is characterized in that the layer to be deposited on the carrier substrates is an adhesion-promoting layer.
- said adhesion promoting layer has a thickness of less than 200 nm, more preferably less than 20 nm.
- the primer layer may comprise a chemical compound from the groups comprising silanes, functionalized silanes, epoxides, functionalized charged or polar polymers, and "self-assembled passive or functionalized monolayers or multilayers", thiols, alkyl phosphates and phosphonates, multifunctional block copolymers, such as poly (L) lysine / polyethylene glycols.
- the method according to the invention is characterized in that one or more specific binding partners are immobilized on the surface of the carrier substrates for the detection of one or more analytes in an affinity detection method (binding the binding partner from a supplied solution to the immobilized binding partner).
- These specific binding partners can be applied to an adhesion-promoting layer applied by means of the coating method according to the invention or else directly to the uncoated surface of the carrier substrates, with areas of the surface remaining free of specific binding partners preferably being provided with a passivation layer in a subsequent coating step according to the method according to the invention below).
- the specific binding partners immobilized on the surface of said carrier substrates are biological or biochemical or synthetic recognition elements for the specific recognition of one or more analytes present in a sample supplied.
- different such specific recognition elements are present in each case in as highly pure a form as possible, generally in different discrete measurement ranges, so that different analytes from the sample generally bind to measurement regions with different recognition elements.
- Such arrays of measurement areas are also referred to as "capture arrays".
- Characteristic of a further widely applicable embodiment of the method according to the invention is therefore that the specific binding partners immobilized on the surface of said carrier substrates are the one or more analytes themselves, which are embedded in a native sample matrix or modified with one or more processing steps Form of the sample matrix are immobilized.
- Said binding partners i. the self-immobilized analyte to be detected or detected in a supplied sample and / or their immobilized or supplied in a supplied detection reagent biological or biochemical or synthetic Erkenuungs shame can be selected from the group which proteins, such as monoclonal or polyclonal antibodies and antibody fragments, peptides, enzymes , Glycopeptides, oligosaccharides, lectins, antigens for antibodies, proteins functionalized with additional binding sites ("tag proteins", such as "histidine tag proteins") and nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (eg. PNA), aptamers, membrane-bound and isolated receptors and their ligands, cavities produced by chemical synthesis for incorporation of molecular imprints, natural and artificial polymers, etc.
- proteins such as monoclonal or polyclonal antibodies and antibody fragments, peptides, enzymes , Glycopeptides
- said specific binding partners applied to the surface of the carrier substrates can be immobilized in discrete measuring areas (spots) which can have any geometry, for example circular, oval, triangular, rectangular, polygonal, etc., wherein a single measuring area has identical or different specific binding partners may contain.
- albumins in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, alien or empirical for the analyte (s) to be detected and / or their Binding partners non
- the present invention therefore relates to a method according to the invention according to one of the aforementioned embodiments, which is characterized in that the layer deposited on the carrier substrates is a passivation layer which is located between the spatially separated measuring areas or in unoccupied partial areas within these measuring areas
- Analytes and / or to its binding partners "chemically neutral" compounds after the generation of these measuring ranges is applied and preferably, for example, comprises compounds from the groups which albumin, especially bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, alien or empirical for the or the analytes to be detected and their binding partners nonspecific antibodies (especially for immunoassays), detergents - such as Tween 20 -, do not hybridize with polynucleotides to be analyzed end, fragmented natural or synthetic DNA, such as extracts of herring or salmon sperm (especially for polynucleotide hybridization assays), or even uncharged but hydrophilic polymers, such as polyethylene glyco
- a further subject of the present invention is a carrier substrate for the detection of one or more analytes in an affinity detection method comprising an adhesion-promoting layer, characterized in that said adhesion-promoting layer is produced by a coating method according to the invention according to one of said embodiments.
- a carrier substrate for the detection of one or more analytes in an affinity detection method comprising a passivation layer covering the carrier substrate at least in partial regions, characterized in that said passivation layer is produced by a coating method according to the invention according to one of said embodiments.
- FIG. 1 A schematic representation of a coating apparatus according to the invention is shown in FIG. 1.
- the areas of unprotected areas of specific binding partners are to be "passivated” by carrier substrates prepared for an affinity detection method, i.e. a "passivation layer” is applied in these areas.
- the inventive apparatus in this exemplary embodiment comprises a desiccator (1) with a volume of about 2 1 as a container for the liquid to be atomized and the volume of mist to be generated over the liquid, a holder (2) for receiving the carrier substrates to be coated Ultrasonic atomizer ("Lucky Reptile Mini-Nebulizer", Reptilica, D-90431 Nuremberg, Germany) as an actuator (3) for liquid atomization, a watch glass as a droplet separator (4) and a gas inlet (5) and an outlet (6) for gas and / or generated fog.
- the ultrasonic generator mounted on the bottom of the desiccator was poured into polydimethylsiloxane (PDMS) just below the vibrating vibrating diaphragm, requiring only the application of a thin layer of fluid to be atomized is.
- PDMS polydimethylsiloxane
- the holder To be coated planar optical thin-film waveguides as carrier substrates, with the external dimensions of 14 mm wide x 57 mm long x 0.7 mm thick (for more details see below), in the holder (2) during the coating process at a distance of about 8 cm the liquid surface stored horizontally (with respect to the liquid surface).
- the holder is formed in the present example as a perforated carrier made of plastic, so that through these holes Excess liquid separated from the mist can flow off.
- the holder can accommodate ten thin-film waveguides as carrier substrates with the dimensions mentioned.
- the watch glass as a droplet is glued in the present example to the underside of the holder (2) and shields the substrates to be coated against splashes from the nebulizing solution (coating solution).
- the generated, very homogeneously distributed mist deposits on the carrier substrates, with in the present example on the top (with respect to the storage in the coating apparatus) arranged high-refractive wave-guiding layer (a), in the form of very small droplets, and already within 5 minutes to 10 Minutes forms on the tops of these substrates a thin, continuous liquid film.
- the carrier substrates are removed from the coating apparatus, thoroughly rinsed with flowing ultrapure water (Millipore) and finally dried in a stream of nitrogen.
- a volume of about 2 ml of passivation solution (liquid to be atomized) is required for a thin-film waveguide of the stated dimensions as the carrier substrate.
- the carrier substrates used for an affinity detection method to be carried out later are planar optical thin-film waveguides, each with the outer dimensions 14 mm wide x 57 mm long x 0.7 mm thick.
- These support substrates each comprise a glass substrate (AF 45) and a 150 nm thin, high refractive layer of tantalum pentoxide applied thereon.
- grating period: 318 nm, grating depth: (12 +/- 2) nm are modulated at a distance of 9 mm, which are to serve as diffractive gratings of the light coupling into the high refractive layer.
- a monolayer of mono-dodecyl phosphate (DDP) formed as an adhesion-promoting layer by spontaneous self-assembly (“soap assembly”) is applied hydrophobic bonding layer provided carrier substrates are each 6 identical microarrays of 144 discrete measuring areas (spots), in turn, in an array of 16 rows and 9 columns, with an inkjet spotter (model NP 1.2, GeSiM, Grosserkmannsdorf, Germany) applied is created by applying a single droplet of approximately 350 pL volume to the chip surface.
- DDP mono-dodecyl phosphate
- the analytes to be detected themselves are to be immobilized in a subsequent affinity detection method on the prepared carrier substrates, embedded in a native sample matrix or in a form of sample matrix (cell lysate) prepared with a few sample preparation steps. These forms of the samples are also referred to below as “nature-identical samples.”
- the detection step should then take place after addition of further detection reagents.
- a signaling pathway via a marker protein is the tumor suppressor protein p53, which via its degree of phosphorylation controls cell division, apoptosis and certain repair mechanisms for damaged DNA. These signaling pathways are often disturbed in cancer cells at one or more sites by mutations or lack of one or more marker proteins in their regulation, which may ultimately be responsible for uncontrolled growth.
- the detection and determination of the relative levels of p53 and P-p53 is performed by using highly specific antibodies that bind to these proteins, which directly act as analytes in the recovered and treated cell lysates the carrier substrates (preferably after application of a primer layer as described above) to be immobilized.
- each microarray contains further measurement areas with Cy5 fluorescently labeled bovine serum albumin (Cy5-BSA) immobilized therein, which are used to refer to local differences and / or temporal variations of the excitation light intensity during the measurement ("reference spots"). Cy5-BSA is applied in a concentration of 0.5 nM in 7 M urea, 2 M thiourea (labeling rate: about 3 Cy5 molecules per BSA molecule).
- FIG. 2 The geometry of the arrangement of the measurement areas in a two-dimensional array and a linear arrangement of six (identical) arrays on a carrier substrate are shown in FIG. 2.
- An array of measuring ranges for these examples comprises in each case an arrangement of measuring regions with 12 different samples applied in 4 replicates, the 4 identical measuring regions each being arranged in a common column perpendicular to the direction of propagation of the light guided during the detection step in the waveguiding layer of these carrier substrates ,
- the reproducibility of the measuring signals within the array of measuring ranges is to be determined with the help of the 4 identical measuring ranges.
- the analytical platform according to the invention in this example comprises 6 similar arrays of measuring ranges, as shown in FIG. 2.3. Passivation of the free areas between and within the measuring ranges
- the support substrates After application of the "naturally identical" samples and Cy5-BSA, the support substrates are dried in dust-free room air before the free, uncovered hydrophobic surface areas of the support substrates in a further step to minimize undesired non-specific binding of detection reagents, in this case antibodies and / or fluorescently labeled molecules, are saturated (passivated) with bovine serum albumin (BSA).
- detection reagents in this case antibodies and / or fluorescently labeled molecules
- a second assay step is carried out using an Alexa Fluor 647-labeled anti-rabbit Fab fragment (Molecular Probes, cat no Z-25308 , Leiden, The Netherlands), which binds to the aforementioned antibodies Al and A2.
- This fluorescently labeled Fab fragment is, starting from the commercially available stock solution, in a dilution of 1: 500 in assay buffer applied to the arrays (each 30 ul) and then for 1 hour at Room temperature incubated in the dark.
- the excitation wavelength in the measurements for the present example is 635 nm
- the detection of the fluorescent light with a cooled camera at the fluorescence wavelength of Cy5 using an interference filter (transmission (675 ⁇ 20) nm) for the suppression of scattered light at the excitation wavelength is positioned in front of the lens of the camera.
- the generated fluorescence images are automatically stored on the disk of the control computer. Further details of the optical system (ZeptoREADER TM) are described in International Patent Application PCT / EP01 / 10012, which is hereby incorporated in its entirety as part of this application.
- the raw data of the individual pixels of the camera represent a two-dimensional matrix of digitized measured values, with the measured intensity as the measured value of a single pixel corresponding to the area imaged on it on the sensor platform.
- a two-dimensional (coordinate) network is placed over the pixels (pixel values) in such a way that the partial image of each spot falls into an individual two-dimensional network element.
- each spot is assigned a circular area of interest (AOI) with a user-definable radius (typically 120 ⁇ m), which can be adjusted as well as the location of the individual AOIs individually as a function of the signal intensity of the individual
- the average radius signal intensity of each spot determines the arithmetic mean of the pixel values (signal intensities) within a selected evaluation range.
- the background signals are determined from the measured signal intensities between the spots.
- four further circular areas are defined per spot as evaluation areas for background signal determination, which are preferably arranged in the middle between between adjacent spots.
- the average background signal intensity is determined, for example, as the arithmetic mean of the pixel values (signal intensities) within an AOI selected for this purpose.
- the average net signal intensity from the measurement areas (spots) is then calculated as the difference between the local average gross and local mean background signal intensity of the respective spot.
- the referencing of the net signal intensity of all spots is done with the help of reference spots (Cy5-BSA) of each array of measuring ranges.
- the net signal intensity of each spot is divided by the average of the net signal intensities of the adjacent reference spots of the same row (arranged parallel to the propagation direction of the light guided in the evanescent field sensor platform).
- 3A shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the measurement areas were passivated by means of the dipping method (according to 2.3.1.).
- the signal intensity within each individual reference spot and between different reference spots (Cy5-BSA) is distributed very evenly and homogeneously, and the edge of the almost perfectly circular spots is sharply delimited from the background (see detail image).
- the measuring ranges of the immobilized cell lysates are characterized by tail-like "smears", which is particularly noticeable at high signal intensities. As described above, these "smears" are caused during the moment of immersion of the carrier substrates provided with the spots into the passivation solution.
- sample components dissolved from the measurement areas by the passivation solution which are adsorbed along the flow in the opposite direction in the immediate vicinity of such a measurement area on the free, not yet passivated carrier substrate surface, before they even contain the BSA contained in the passivation solution can be passivated. Since these sample portions, which have been removed from the measurement areas and resorbed in the neighborhood, always contain a certain content of analyte to be detected, a corresponding fluorescence signal is visible on reading at said locations.
- 3B shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the spots were passivated by means of the spray method (according to 2.3.2.).
- the signals from the reference spots are comparable in terms of their shape and uniformity or homogeneity as well as their intensity with those of a microarray after use of the dipping method.
- the signals from the measurement areas with immobilized cell lysates are also comparable in their intensity to the corresponding measurement signals from the microarrays which had been subjected to the immersion process.
- the cell lysate spots do not show the "smudges" described above, but only smaller "outgrowths” of lesser fluorescence intensity, which are evidently arranged approximately randomly around the intended spots. These are most likely caused by the local dissolution and outflow of non-bound cell lysate at the edges of the measurement areas, since the impinging small spray droplets of the passivation solution on impact with the surface have a non-negligible impulse perpendicular to the coating surface, which can lead to the generation of splashes.
- 3C shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the measurement areas were passivated by means of the method according to the invention by fogging passivation solution, as described under 1. Striking here, compared to the passivated with the other methods described microarray, the high quality with comparably good homogeneity and shape of reference spots and Zellysatspots. "Smudges" or "outgrowths" of the cell lysate spots can be avoided here due to the fact, apart from the influence of gravity, essentially undirected and pulse-free application of the passivation in the form of fine mist droplets, the size of which is well below that produced by spraying droplets.
- the efficiency of passivation of the surface free of components from the immobilized sample i. the degree of suppression of nonspecific binding by means of the BSA contained in the passivation solution can be determined semiquantitatively from the signal intensity measured in the areas free of spots (between the spots, "background signals") nonspecific binding of the fluorescently labeled detection reagents (Alexa 647 anti-rabbit Fab) used in the assay to the BS A-free surface give a higher signal than a surface continuously coated with BSA.
- Figure 4A shows the averages and standard deviations of the background signal intensities determined between all spots of the free carrier substrate surfaces passivated by the three different methods with the microarrays generated thereon.
- the standard deviation of the background signal intensities after application of the spray method or the nebulization method according to the invention is in each case significantly lower than after use of the dipping method for surface passivation, which led to a standard deviation of the background signal intensities of 34%. It is concluded that the uniformity or homogeneity of the coating after application of the spraying or misting process is higher than after use of the dipping process.
- 4B shows the mean values of the fluorescence intensities of all reference spots of the microarrays, wherein the free surfaces of the associated carrier substrates were in turn treated with the three different coating methods.
- FIG. 5 A shows the average intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, the carrier substrate surfaces of which were each treated with the different passivation methods and which are subsequently treated with solutions of the antibody Al (anti-p53) (FIG. 5 A, top ) and A2 (anti-phospho-p53) (Figure 5A, bottom) and then each incubated for detection by fluorescence detection with Alexa 647 Fluor anti-rabbit F ab fragments.
- the measured fluorescence signal intensities correlate with the respective relative content of analytes contained in a cell lysate (corresponding to Zellysatkonzentration; higher signal corresponding to a higher analyte concentration, the correlation obviously being nonlinear in nature).
- Fig. 5B shows that the content of phospho-p53 in the UV-light-treated sample is also markedly increased compared to the control sample, while the content of phospho-p53 in the doxorubicin-treated sample, despite a massively increased total concentration of p53 only slightly above (in the case of lysate concentrations from 0.2 mg / ml to 0.4 mg / ml) or even below (in the case of the lysate concentration of 0.1 mg / ml) that of the control sample.
Abstract
Description
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EP (1) | EP1877773B1 (de) |
JP (1) | JP5002584B2 (de) |
CN (1) | CN101208599B (de) |
AU (1) | AU2006239534B2 (de) |
CA (1) | CA2605750A1 (de) |
DK (1) | DK1877773T3 (de) |
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GB2485564A (en) * | 2010-11-18 | 2012-05-23 | Rolls Royce Plc | Luminescent surface coating for inspection |
EP2726852B1 (de) | 2011-06-30 | 2020-11-25 | Koninklijke Philips N.V. | Mehrfache untersuchungen einer probe |
WO2015105114A1 (ja) | 2014-01-10 | 2015-07-16 | コニカミノルタ株式会社 | イムノアッセイ法およびイムノアッセイシステム |
DE102014112625A1 (de) * | 2014-09-02 | 2016-03-03 | Schmid Energy Systems Gmbh | Großflächen-Ultraschallverdampfer |
US10703843B2 (en) | 2014-12-08 | 2020-07-07 | University Of Virginia Patent Foundation | Compositions and methods for bonding glues, adhesives, and coatings to surfaces |
CH712376A1 (de) | 2016-04-19 | 2017-10-31 | Camag | Derivatisierungsgerät und -verfahren. |
CN106198952A (zh) * | 2016-07-01 | 2016-12-07 | 清华大学 | 一种抑制核酸分子对传感界面非特异性吸附的封闭方法 |
CN107807133A (zh) * | 2017-11-08 | 2018-03-16 | 苏州安路特汽车部件有限公司 | 一种雾化渗透探伤装置 |
CN115147617B (zh) * | 2022-09-06 | 2022-11-22 | 聊城集众环保科技有限公司 | 基于计算机视觉的污水处理智能监控方法 |
CN115837944A (zh) * | 2023-02-16 | 2023-03-24 | 太原理工大学 | 一种电厂烟气注入采空区防灾封存的气体监测装置 |
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- 2006-04-22 JP JP2008508125A patent/JP5002584B2/ja not_active Expired - Fee Related
- 2006-04-22 CN CN200680023014.9A patent/CN101208599B/zh not_active Expired - Fee Related
- 2006-04-22 CA CA002605750A patent/CA2605750A1/en not_active Abandoned
- 2006-04-22 EP EP06724515.9A patent/EP1877773B1/de not_active Not-in-force
- 2006-04-22 US US11/919,054 patent/US9050615B2/en not_active Expired - Fee Related
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CA2605750A1 (en) | 2006-11-02 |
US20090311773A1 (en) | 2009-12-17 |
ES2525324T3 (es) | 2014-12-22 |
EP1877773B1 (de) | 2014-10-15 |
AU2006239534A1 (en) | 2006-11-02 |
WO2006114249A1 (de) | 2006-11-02 |
AU2006239534B2 (en) | 2012-05-31 |
CN101208599B (zh) | 2014-12-10 |
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