WO2006114249A1 - 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-nachweisverfahren Download PDFInfo
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- WO2006114249A1 WO2006114249A1 PCT/EP2006/003726 EP2006003726W WO2006114249A1 WO 2006114249 A1 WO2006114249 A1 WO 2006114249A1 EP 2006003726 W EP2006003726 W EP 2006003726W WO 2006114249 A1 WO2006114249 A1 WO 2006114249A1
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- carrier substrates
- liquid
- atomized
- coating
- coated
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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
- Silanizations can be carried out, for example, both in the gas and the liquid phase, for example by means of dipping processes. While the coating processes in the gas phase, with sufficiently large sized reaction vessels (in comparison to the size of the carrier substrates to be coated), generally lead to good homogeneity of the deposited layer, layers deposited from the liquid phase often show strong spatial inhomogeneities, for example trace traces Application of dipping method.
- the step of applying passivation layers occurs after application of the In most cases non-heat-resistant detection elements for analyte detection, usually from the liquid phase.
- a dipping process is typically used.
- the carrier substrate is dropped into a vessel which is filled with a solution of the analytes or their detection substances or other binding partners "chemically neutral", ie this non-binding compounds ("passivation") to the entire surface of the carrier substrate as possible to wet quickly and simultaneously with the passivation solution.
- the carrier substrate is left in the passivation solution for a predetermined period of time (“incubated"), so that the compounds used for surface passivation can be adsorbed on the substrate surface.
- An advantage of this conventional method is that it can be carried out without any further aids and does not require special requirements for the abilities of the laboratory personnel.
- a disadvantageous feature of this method is a relatively high risk of "blurring" of spots during the moment of immersion of the support substrate by passivation solution flowing along the substrate surface, thereby desorbing and sweeping away material from the discrete measurement areas (immobilized specific binding partners) and can occur in the Environment in the direction of the relative flow direction of the passivation solution (based on the substrate surface) are not adsorbed again in not completely covered with PassivitationsENSen areas.
- spots depends, among other things, on the surface density of the immobilized specific binding partners in the discrete measurement areas and the composition of the passivation solution, in particular on the solubility of the specific binding partners in this passivation solution.
- Feature density ie, with a small distance between adjacent spots
- the quantitative analysis of the assay signals from an array of measurement areas strongly affect or even exclude, due to the resulting inhomogeneous distribution of the background signals from the areas between the discrete measurement areas.
- immobilized material is even transported from one spot to an adjacent spot, this undesirable effect can lead to a meaningful analysis of the assay signals is no longer possible.
- Another disadvantage of this method is the inherent need for relatively large volumes of passivation solution and associated relatively high costs.
- the use of spraying methods is known, for example with the aid of atomizers, in which the passivation solution is applied in the form of small liquid droplets on the substrate surface until a continuous liquid film has formed on this surface a saturated atmosphere of the liquid vapor (ie at 100% agitfeuchtmaschine in the case of an aqueous passivation solution) for a predetermined period of time, in turn so that the compounds used for surface passivation can be adsorbed on the substrate surface.
- the support substrates during the implementation of this passivation process are horizontal (relative to the substrate surface to be coated) stored.
- the results of the spraying process are also not optimal. Due to the ejection of the droplets over one Nozzle or an atomizer, the droplets have a momentum more or less strong against this surface impulse at the moment of impact on the surface to be coated. This is associated with the risk of splashing into even smaller droplets when hitting the surface, so that the edges of the measuring areas (spots) to be generated are generally not generated well-defined. In addition, spray processes generally produce relatively large droplets with a large variation in droplet size.
- the coating apparatus according to the invention developed for carrying out this method is characterized by a very simple construction, in which cost-effective commercially available components can be used, and also simple operability.
- the method according to the invention, to be carried out with a coating apparatus according to the invention according to one of the embodiments described below, is suitable for applying both adhesion-promoting and passivation layers on any but preferably planar carrier substrates for the detection of analytes in affinity detection methods.
- 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.
- the inventive method is also characterized by the possibility of simultaneous coating of a large number of carrier substrates in a common, appropriately sized container and ease of automation and is easily feasible even for untrained personnel.
- the liquid volumes to be used for coating the carrier substrates are of similar order of magnitude as in the case of the spray method.
- 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.
- said actuator is immersed in the operating state in liquid to be atomized.
- the actuator is completely within the liquid to be atomized.
- the intensity and frequency of the ultrasound acting on the liquid to be atomized can be regulated and / or measured by means of suitable precautions.
- 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.
- a drop separator to be used may also be permeable to drops up to a defined size, for example with a diameter of less than 200 ⁇ m. 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 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 provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the surroundings of said carrier substrates , For example, it may be helpful if a gas is admitted into the container of the apparatus (ie into the air or gas or mist space), which mixes with the generated mist and / or swirls with it.
- 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 additionally provides for the rotation of the carrier substrates around an axis perpendicular to the mounting plane. Due to an inherent property of the inventive method, namely a substantially spatially non-directional deposition, the droplet formation and thus application of the compounds contained for surface coating takes place not only on the free surfaces of the carrier substrates, but for example on the walls of the liquid container of the inventive coating apparatus.
- 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 at least one in the propagation direction of an irradiated excitation light or measurement light substantially optically transparent layer of carrier substrates to be coated comprise a material which is selected from the group consisting of silicates, for.
- silicates for example, glass or quartz, transparent thermoplastic molding, sprayable or millable plastics, such as polycarbonates, polyimides, acrylates, in particular polymethyl methacrylates, polystyrenes, cyclo-Olef ⁇ npolymere and cyclo-olefin copolymers.
- 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.
- 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.
- Corresponding waveguiding regions are to be understood as “continuous waveguiding regions” which extend over substantially the entire region of the part of the surface of a carrier substrate used for analyte detection without an interruption of the high-refractive, waveguiding layer.
- 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 during the detection step of an affinity detection method with said carrier substrates in optical interaction with one or more optical coupling elements for coupling excitation or measuring light from one or more light sources, said optical coupling elements being selected from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with focusing lenses arranged in front of a waveguiding layer of the carrier substrates, preferably cylindrical lenses, and grating coupler.
- 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.
- 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 . It is also preferred that the second optically transparent layer (b) of the carrier substrates to be coated comprises a material from the group consisting of silicates, for.
- 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.
- planar optical thin-film waveguides which are suitable as carrier substrates are described, for example, in international patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 01/79821, WO 01/88511, WO 01/55691 and WO 02/79765.
- special carrier substrates usually referred to as sensor platforms, and methods to be carried out therewith for analyte detection, as well as the content of this application are hereby incorporated in their entirety as part of the present invention.
- Characteristic of a preferred group of embodiments of coating apparatuses 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 medium detection of one or more excited luminescences.
- the carrier substrates to be coated enable 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.
- 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
- Embodiments are inserted, - contained in the liquid container of said coating apparatus
- 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 intensity and frequency of the ultrasound acting on the liquid to be atomized can be regulated and / or measured by means of suitable precautions.
- 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 comprises provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the surroundings of said carrier substrates.
- the coating apparatus additionally comprises at least one gas inlet via which a gas is introduced into the liquid container, which gas mixes with the generated mist.
- the apparatus may additionally include one or more outlets for venting gas and / or mist.
- 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 coating apparatus additionally includes provisions for controlling and / or regulating the pressure within the Liquid container during the coating process and the pressure during the coating process is controlled and / or varied.
- the carrier substrates during the coating process about an axis be rotated perpendicular to the mounting plane.
- 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 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.
- liquid deposited on the walls of the liquid container be collected and returned to the liquid to be atomized.
- 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.
- 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.
- the at least one in the propagation direction of an irradiated excitation light or measurement light substantially optically transparent layer of carrier substrates to be coated for example, comprise a material which is selected from the group consisting of silicates, for.
- silicates for example, 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.
- 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 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 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.
- discrete measurement ranges be created by spatially selective application of specific binding partners on said carrier substrates, preferably using one or more of the group of methods which include "ink jet spotting", mechanical spotting, “micro contact printing”, fluidic Contacting of the areas for the measuring ranges to be created with the compounds to be immobilized by their supply in parallel or crossed microchannels, under the effect of pressure differences or electrical or electromagnetic potentials, as well as photochemical and photolithographic immobilization processes.
- 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.
- Another object of the present invention is a carrier substrate according to one of the aforementioned embodiments for use in human and / or animal diagnostics.
- 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 finest droplets produced by the action of ultrasound and rising above the liquid level are additionally swirled by means of a small stream of nitrogen, which is introduced into the container via the inlet (5), in order to distribute the resulting mist as homogeneously as possible throughout the entire container produce.
- 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 human colon cancer cell line (HT29) is used These adherent cells are incubated in modified McCoy's 5A medium at 37 ° C in conventional culture plastic bottles (Greiner Bio-One). St. Gallen, Switzerland, Cat No: 658170) Similar cell cultures of different culture flasks are then either exposed to UV light for 10 minutes or treated with 10 ⁇ M doxorubicin.As a comparative sample to these treated cell cultures, an otherwise similar cell culture is used remains untreated and will serve as a negative control in the analytical detection method. After treatment, the different cell cultures are washed with 10 ml PBS (phosphate buffered saline, cooled to 4 ° C).
- PBS phosphate buffered saline
- the cells are detached from the bottom of the culture flasks by adding lysis buffer containing 7 M urea, 2 M thiourea and complete (protease inhibitor, Roche AG, 1 tablet / 50 ml) and at the same time completely lysed, whereby all protein-containing cell constituents are spontaneously denatured and solubilized ,
- the cell lysate thus obtained is centrifuged for 5 minutes at 13,000xg in a benchtop centrifuge (Eppendorf, Hamburg, Germany) to separate insoluble cell components (e.g., DNA and cell membrane fragments).
- the supernatant is collected and used for the following measurements, the total protein concentration typically being between 5 mg / ml and 10 mg / ml.
- the described treatments of the HT29 cell cultures lead to damage of the DNA, namely UV irradiation and the like. by chain breaking or by formation of thymine dimers and by doxorubicin addition by its intercalation between adjacent bases of the DNA. This has the consequence that within damaged cells certain signal paths are activated or deactivated, e.g. may result in programmed cell death (apoptosis).
- signal paths are activated or deactivated, e.g. may result in programmed cell death (apoptosis).
- marker proteins responsible for activating or deactivating signaling pathways are certain key proteins (called "marker proteins”) that regulate one or more signaling pathways through phosphorylation at one or more different sites.
- 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
- planar optical thin-film waveguides as carrier substrates are dropped vertically into a vessel filled with passivation solution (polystyrene tube), so that the entire surface of the carrier substrates is wetted as quickly and simultaneously as possible. After one hour of incubation at room temperature, the carrier substrates are thoroughly rinsed under running ultrapure water (Millipore) and then dried in a stream of nitrogen (quality 50). For each thin-film waveguide of the dimensions mentioned as the carrier substrate, a volume of about 25 ml of passivation solution is required.
- the passivation solution is sprayed onto the carrier substrates here by means of a chromatography atomizer (Glas Keller Cat. No. 12.159.603, Basel, Switzerland) and a pressure of approximately 3.5 bar, until a continuous layer is obtained on the surface to be coated Liquid film has formed.
- the distance between the exit nozzle of the atomizer and the carrier substrate surface is approximately 30 cm.
- the carrier substrates thus treated are incubated in a closed container at 100% humidity for one hour at room temperature, then thoroughly rinsed under running ultrapure water (Millipore) and finally dried in a stream of nitrogen (quality 50).
- a volume of about 3 ml passivation solution is needed.
- 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 arrays are washed with assay buffer (two times each with 200 ⁇ l) to remove non-specifically bound fluorescently labeled Fab fragments. Thereafter, the prepared analytical platforms are stored in the ZeptoREADER TM (see below) until the detection step by excitation and detection of resulting fluorescence signals.
- the fluorescence signals from the various arrays of measurement ranges are sequentially automatically measured with a ZeptoREADER TM (Zeptosens AG, CH-4108 Witterswil, Switzerland).
- the planar optical thin-film waveguide is adjusted as a carrier substrate (according to 2.1.) To fulfill the resonance condition for the light coupling via a grating structure (c) in the waveguiding tantalum pentoxide layer and to maximize the available in the measuring ranges excitation light.
- a user-selectable number of images of the fluorescence signals from the respective array is generated by each array, wherein different exposure times can be selected.
- 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 mean signal intensity from the measurement areas is determined using image analysis software (ZeptoVIEW TM, Zeptosens AG, CH-4108 Witterswil) allows to semi-automatically evaluate the fluorescence images of a large number of arrays of measurement areas.
- 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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CN200680023014.9A CN101208599B (zh) | 2005-04-26 | 2006-04-22 | 用于涂覆通过亲和力-测定法用于被分析物检测的底物基质的新的设备和方法 |
DK06724515.9T DK1877773T3 (en) | 2005-04-26 | 2006-04-22 | NEW APPARATUS AND PROCEDURE FOR THE COATING OF CARRIER SUBSTRATES FOR ANALYTICAL DETECTION BY THE AFFINITY DETECTION PROCEDURE |
ES06724515.9T ES2525324T3 (es) | 2005-04-26 | 2006-04-22 | Nuevo aparato y procedimiento de recubrimiento de sustratos portadores para la detección de analitos mediante un procedimiento de detección por afinidad |
EP06724515.9A EP1877773B1 (de) | 2005-04-26 | 2006-04-22 | Neue apparatur und verfahren zur beschichtung von trägersubstraten für einen analytnachweis mittels affinitäts-nachweisverfahren |
US11/919,054 US9050615B2 (en) | 2005-04-26 | 2006-04-22 | Apparatus and method for coating substrates for analyte detection by means of an affinity assay method |
JP2008508125A JP5002584B2 (ja) | 2005-04-26 | 2006-04-22 | 親和性検定方法による分析試料検出用の基質をコーティングするための新規な装置および方法 |
AU2006239534A AU2006239534B2 (en) | 2005-04-26 | 2006-04-22 | Novel equipment and method for coating substrates for analyte detection by way of an affinity assay method |
CA002605750A CA2605750A1 (en) | 2005-04-26 | 2006-04-22 | Novel apparatus and method for coating substrates for analyte detection by means of an affinity assay method |
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US (1) | US9050615B2 (de) |
EP (1) | EP1877773B1 (de) |
JP (1) | JP5002584B2 (de) |
CN (1) | CN101208599B (de) |
AU (1) | AU2006239534B2 (de) |
CA (1) | CA2605750A1 (de) |
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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 |
US10703843B2 (en) | 2014-12-08 | 2020-07-07 | University Of Virginia Patent Foundation | Compositions and methods for bonding glues, adhesives, and coatings to surfaces |
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 |
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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 |
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- 2006-04-22 ES ES06724515.9T patent/ES2525324T3/es active Active
- 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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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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DE102014112625A1 (de) * | 2014-09-02 | 2016-03-03 | Schmid Energy Systems Gmbh | Großflächen-Ultraschallverdampfer |
EP3235574A1 (de) | 2016-04-19 | 2017-10-25 | Camag AG | Derivatisierungsgerät und -verfahren |
Also Published As
Publication number | Publication date |
---|---|
JP2008539399A (ja) | 2008-11-13 |
CA2605750A1 (en) | 2006-11-02 |
US20090311773A1 (en) | 2009-12-17 |
EP1877773A1 (de) | 2008-01-16 |
ES2525324T3 (es) | 2014-12-22 |
EP1877773B1 (de) | 2014-10-15 |
AU2006239534A1 (en) | 2006-11-02 |
AU2006239534B2 (en) | 2012-05-31 |
CN101208599B (zh) | 2014-12-10 |
US9050615B2 (en) | 2015-06-09 |
JP5002584B2 (ja) | 2012-08-15 |
DK1877773T3 (en) | 2015-01-19 |
CN101208599A (zh) | 2008-06-25 |
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