CH703278A1 - Device and platform for multiplex analysis. - Google Patents

Abstract

The design and performance of an analytic apparatus and correspondingly designed microarray platform devices are described for easier multiplex analysis of analytes in liquids. The apparatus, in combination with the microarray platform devices, is designed to perform bioanalytical assays and to detect the presence of organic molecular analytes, possibly in microarray format. A plurality of microarray platform devices, including one or more channel-type reaction chambers, may be analyzed simultaneously. In preferred aspects, the microarray platform devices are laminated microstructured microarray platforms, providing integrated channel-based reaction chambers, having capture molecules printed and immobilized in open channel structures prior to lamination. The microstructured microarray platforms are designed for use in conjunction with the apparatus, resulting in short dosing times, low reagent volumes, and performance at temperatures above and below room temperature.

Description

Field of the invention

The present invention relates to an apparatus and devices for the manufacture and development of microarrays, as well as their application and their use in the broadband analytical field, including drug development and quality control, or as as part of prognostic and diagnostic test systems.

Background of the invention

[0002] Various biological and chemical assays have been developed to detect the presence of compounds of interest in complex multicomponent samples. Microarray devices in general, and especially microarrays containing biopolymers as capture ligands, such as proteins, polysaccharides, nucleic acids, as well as low molecular weight compounds, have a wide range of applications. research, diagnosis and analysis. Microarray devices measure and quantify molecular interactions after chemical, biochemical or immunological interaction of surface-immobilized capture ligands with their corresponding target molecules. These biochemical, biochemical or chemical interaction assays are based on exposure of an unknown sample to one or more known reagents and can record the progress or measure the reaction result. It is often desirable to expose a sample to multiple reagents, to react multiple reagents with dilutions of a single sample, or to perform a particular assay with a given sample in a specific time and location.

The in vitro diagnostic industry has considered, for example, a diagnostic "protein chip" in which a microarray of antibodies or capture ligands is printed on a specimen. In theory, after such a chip has been inoculated with a sample of a patient (eg a drop of blood, urine or saliva), the proteins should bind to very specific antibodies or capture ligands and should be quickly measured to determine if they are at normal or abnormal levels. Such technology should enable health professionals to predict or diagnose health status early, and administer appropriate medication and / or treatment quickly and with greater confidence.

Many of the technical hurdles associated with microarray technology including networks of nucleic acids, proteins, carbohydrates, or cells have been overcome, and solutions have been proposed or presented to immobilize the microarrays. capture ligand, nonspecific binding suppression, biological activity preservation, fluid transport, timely reagent delivery, rapid analytical processing, and signal amplification.

The current limitations of microarrays are the time and effort required to develop appropriately designed analytical assays, and to achieve robust and reliable high quality results in a short time, particularly with diagnostic assays at the d use made ad hoc.

[0006] Current microarray technology uses microarray devices and platforms that are customized in specific product formats (eg the Affymetrix gene chip, or the Nanogen gene chip, Randox analytics platform) or platforms. open, designed for general use, on the basis of glass slats or plastic slats to standard dimensions. While product-specific formats have overcome some of the limitations of reproducible processing with expensive, large machines capable of processing large numbers of platforms simultaneously, simple and efficient systems designed for fast, addressable processing single platform, or for fast multiplex platform analysis, are not available.

Loeffler et al. (EP 171,761), Tanaami Takeo (JP 2004 226,068) and Sigrist et al. (EP1,300,194) describe multicomponent lamella processing chambers designed for microarray lamellae. Fluidic systems and fluidic networks, as well as methods for using them to promote bio-interactions, have been described by Lee et al. (US Patent Application 2004 0 258 571). McNeely et al. (US 2004 0 037 739) describe a method and a system for providing a fluidic interface to lamellae carrying microarrays of biomolecules or other samples immobilized thereon. U.S. Patent No. 5,797,898 and U.S. Patent No. 6,123,861 disclose microchip devices that release drug molecules from reservoirs having reservoir caps that actively or passively disintegrate. Kroy et al. (US Patent No. 5,252,294) disclose micromechanical structures having closed cavities for use in the storage and handling of substances. U.S. Patent No. 5,548,673 to Cottingham discloses an independent multi-chamber reactor for carrying out hybridization assays in a sealed unit, wherein some reagents are provided by coating the walls of the chambers, and other reagents are provided. introduced into the open chambers before starting the reaction.

In view of the foregoing, there is a need for corresponding devices and devices for diagnostic and analytical use in the initiation and control of biochemical, immunological or chemical reactions, for carrying out rapid bioanalytical detection. , for measurements in an area or volume of micrometric scale, compatible with microarray technologies. It would also be desirable to have methods for producing and using these devices and the corresponding easy-to-handle devices.

Definitions

In this specification, the following terms should be interpreted in accordance with the corresponding definitions presented hereinafter "Apparatus". The word apparatus relates to an analytical use system for analytically housing and processing a selected quantity of microarray platform devices, wherein the microarray platform devices are positioned and screwed between a structured tablet type plate and a plate forming cover, the latter allowing access and connection to an inlet port and an outlet port. Below the tablet-type plate is a heating pad mounted on a plate, ultimately allowing temperature adjustment in the microarray platform reaction chamber at ambient temperature and at a higher temperature. The entire heating pad and plate, tablet plate and cover plate assembly, with the microarray platform devices inserted, is mounted on a pedestal that houses the electrical wiring and the connections to the heating pad and a unit. electric controller, the latter not forming part of the invention.

"Device. In the following, the word device is used in the context of the microarray platform, said microarray platform device consisting of a first microarray substrate and a second microarray substrate, each substrate having first and second surfaces. The microarray platform device is applicable as an analytical tool in combination with the apparatus described herein. The microarray platform and the corresponding apparatus form a set to be used for analytical and bioanalytical purposes.

"Microarray platform". Microarray platforms, in general, refer to two-dimensional arrays, typically on the surface of a glass or polymer flat material, a filter, or a silicon wafer, upon which chemical molecular species or Biochemicals are deposited or synthesized in a predetermined spatial order enabling them to be made available as probes in a parallel manner. The present invention refers to such microarrays where the chemical, biochemical or immunological molecular species are deposited in the form of ligands above substructures, the latter being disposed within preformed open channels.

[0012] A "channel" as used herein means a three-dimensional feature in a microarray platform substrate material that, in its open form ("open channel"), provides physical external access from at least one direction and which, in its closed form ("closed channel"), is capable of conducting fluids such as liquids or gases in a directionally controlled manner. The term "closed channel" as used herein refers to channels that are closed over the entire length with the exception of openings at the entrance and exit of the channels. In addition to this, the closed channels may have any cross-sectional shape, and the cross-sections along a channel may have different types of cross-sections.

[0013] "Sub-structures". The word "substructures" in the context of "sub-structured channels" refers to cross-sectional differences of the channels along the length of open channels. The microchannel substructuring includes micro- and nano-structuring at the bottom of the open channels and / or their sidewalls, obtained by any type of material structuring process, such as wet pickling or dry etching, micromachining, electron beam processing, abrasion, hot stamping, or injection molding.

The expression "printing in the channels" relates to the process of depositing liquids containing the ligand, optionally in combination with chemical reagents that carry out the immobilization of the ligand, in open channels. This liquid deposition can be performed by any device or instrument capable of making available and placing small sample volumes in a locally addressable, accurate manner.

The term "reagent is used for any type of chemical species that participates in a chemical, biochemical or immunological interaction, with or without a molecular change of any species involved.

A "ligand" as used herein refers to chemical, biochemical or immunological species that are immobilized on the first surface of the microarray platform and participate as reagents. The immobilization of a ligand encompasses all adsorption binding methods (e.g., surface precipitation, hydrophobic interactions, ionic interactions, as well as combinations thereof) and all methods involving a covalent bonding of ligand.

[0017] "Target probe". Target probes are solute components interacting with said ligands or ligand-target complexes in reaction chambers.

The "reaction chamber" as used in the present invention is the space and physical volume where target ligands and probes undergo chemical, biochemical or immunological interactions, or participate as reagents in chemical, biochemical or immunological interactions.

Summary of the invention

The present invention introduces an apparatus and corresponding devices for use with the apparatus for performing biochemical, immunological or chemical reactions on a novel microarray platform, the assembly consisting of one or more microarray platform devices. , and the set of apparatus and platforms being suitable for performing microarray-based analyzes. Another object of the invention is to provide methods for using said device and microarray platform devices for analytical purposes.

The invention further comprises an apparatus for analytical use which allows to retain and simultaneously develop a selected amount of microarray platform devices, individually or up to six items in parallel, the microarray platform devices being placed and screwed between a structured tablet plate and a cover plate, the latter providing access and connection to an inlet port and an outlet port. Below the tablet-type plate is a heating plate allowing adjustment of the temperature in the reaction chamber to ambient temperature and beyond. In a preferred embodiment of the present invention, the second substrate of the microarray platform is in contact with the tablet plate, thereby effecting effective temperature transfer to the reaction chamber. The entire assembly of the hotplate, the shelf type plate and the cover plate, with the microarray platform devices inserted, is mounted on a base that houses the electrical wiring and the connections to the hotplate and a temperature controller unit (the latter not being part of the invention). Another external instrument is required to actuate the transport of liquid from the inlet compartment of the apparatus, feeding said liquids into and through the reaction chamber of the microarray platform device. This instrumentation does not form part of the present invention.

The invention essentially comprises a microarray platform device which may be as large as or larger than a microscope slide having first and second surfaces and one or more microstructures etched in said first surface, said microstructure in the microstructure. first substrate comprising one or more substructures and one or more biologically active sites disposed within said microstructure of the second surface or on top of said substructures, and a second microarray platform substrate forming reaction chambers small volume after assembly with the first surface of the second substrate, said first and second microarray substrates forming the microarray platform.

A preferred embodiment of the invention relates to the microarray platform device which comprises a printed network in a channel (printed in networks in an open channel) of one or more ligand-forming (bio) molecules, The ligand-forming molecules are thus deposited and preferably covalently immobilized within the channel-forming cavities prior to closure of the channels, said channel closure being effected by sealing the open channels with a second microarray platform substrate. These microarray devices can be used in combination with the single sample analysis apparatus, or for serial analysis in conjunction with, for example, robotic multiple pipetting systems.

In other preferred embodiments of the present invention, the inner surfaces of the open channel (of the second surface of the first microarray substrate) are sub-structured, so that the substructures are designed to perform a disturbance of the liquid flow. In further preferred embodiments of the present invention, said substructures are designed for confined deposition of ligand molecules at elevations, other than the bottom of the channels, thereby allowing for distinctive ligand placement and scanning. selective, vis-à-vis the elevation, of the platform by optical means, for example by confocal microscopy. Ultimately preferred substructuring elements of the present invention determine a rapid disrupted liquid flow within vertically structured micro-channels.

In other preferred embodiments of the present invention, the open channels forming a reaction chamber are etched on the second surface of the first microarray platform substrate, and the input and output connections are connections. through (holes) with autoclave structures on the first surface of the first microarray substrate, which is opposed to the reaction chamber (assembled on the second surface of the first microarray substrate).

In still other preferred embodiments, the fluid inlet ports of the microarray platforms, located at the first surface of the first microarray substrate, are connected to fluid inlets of the plate forming lid, and the overall fluid transport is actuated either by a mechanism for moving mechanical liquid, or by applying a positive or negative gas pressure (vacuum or suction). This fluid actuation is performed by a suitable instrument, said instrument not being part of the present invention.

In yet another preferred embodiment of the present invention, the apparatus shown in FIG. 1makes close-fitting adjustment of the microarray platform to the tablet-type plate by means of screws, and enables rapid and efficient temperature transport from the hotplate, via the tablet-type plate to the first surface of the second microarray substrate and the reaction chamber, which is separated by said second substrate.

Specific preferred embodiments of the present invention will become apparent from the following more detailed description of certain embodiments and claims.

Brief Description of Drawings

Fig. 1 describes the apparatus and its constituent components: the base 1, the heating plate 2, the tablet type plate 3, the microarray platform device 5 and the cover plate 6. The latter is shown here in partial section for better show the positioning of the microarray platform devices and the input 7 and output 8 connections.

Fig. Fig. 2 shows a partial overview (Fig. 2A), a longitudinal section (Fig. 2B) and a cross-section (Fig. 2C) of an assembly consisting of the tablet-like plate 3, microarray platform devices. 5, and the cover plate 6.

FIG. 3 is a drawing of the heating plate 2 and the heating pad 11.

FIG. 4 is a perspective view of the first surface of the microarray platform substrate 12.

FIG. 5 is a perspective view of the second surface of the first microarray platform substrate 13 with an engraved meander-type open channel 14. The second microarray substrate with its first surface 15 and its second surface 16 is shown above under the shape of a laminate for illustrative purposes.

FIG. 6 describes the preferred option of substructures in the reaction chamber forming open channels.

FIG. 7 is a fluorescent scanning image of fluorescent ligand molecules printed in a channel.

Detailed Description of the Invention

The design of the device according to the present invention is described in FIG. 1. The apparatus consists of a base 1, a heating plate 2 carrying a heating pad 11, a structured tablet type plate 3 for placing up to 6 microarray platform devices 6 and a plate forming lid. In a preferred embodiment, metal materials are used for the pedestal, the heating base plate and the tablet plate, while the material of the cover plate is a transparent synthetic polymer. For standard use, the base, the base heating plate, the heating pad and the overlying tablet plate are assembled and joined by screws, the heating pad being electrically connected to an electrical controller unit which does not part of the present invention.

In another aspect, the invention provides an apparatus, shown in FIG. 1, for treating a sample, in particular a chemical or biochemical sample, the apparatus further comprising computerized control means and temperature control means; the fluid activation means, the computerized control means and the temperature control means not forming part of the present invention.

The apparatus shown in FIG. 1is used in conjunction with microarray platform devices. Conveniently, the holding means comprises screw clamping means (holes 9 for positioning these screws) suitable for "sandwiching" the microarray platform devices between the tablet-type plate and the cover plate. This holding mechanism allows a close adjustment of the inlet orifice of the cover plate 17 and the inlet of the microarray platform 18, and between the exit orifice of the microarray platform 19 and the outlet orifice of the cover plate 20. A close fit between the two mentioned parts can be obtained by guiding structures which allow an autoclave sealing or any other type of liquid sealing known to the human being. job. Precise alignment of the microarray platform device and the cover plate may further be determined by receiving cavities and corresponding pins on the contact surfaces of the assembly components.

Details of the heating means are shown in FIG. 3. The metal heating plate 2, with the heating pad 11 assembled on the top, is fixed on the upper part of the base. As part of this invention, the tablet plate is individually secured by screws on the top of the heating pad. Such a configuration allows easy removal of the tablet-type plate without disconnecting the electrical connections, in the case where decontamination of the tablet-type plate is necessary.

FIG. 2A shows an assembly of the tablet plate and the cover plate with 5 microarray platform devices in place. For loading a desired number of microarray platforms 5, said microarray platforms are placed in the recesses 4 and the cover plate is placed on top, forming a "sandwich-type" assembly. Fig. 2Bet fig. 2C describe cross sections of such an assembly including microarray platform devices. It is important to note that the inlet 17 and the outlet 20 of the cover plate are spatially connected to the inlet port 18 and the outlet port 19 of the microarray platform on the first surface of the substrate. microarray platform. Liquid-tight connections are obtained by means of properly placed O-rings or equivalent fasteners of the state of the art at either connection. A tight assembly of the microarray platforms, "sandwiched" between the tablet plate and the cover plate, is reinforced by thumb screws, one on each side of any microarray platform used in the process. analytical method. This allows parallel processing of any selected number of microarray platforms, from one to six.

Details of the analytical platform according to the present invention are shown in FIG. 4and fig. 5. Microarray platforms having the conventional dimensions of a microscope slide (US standard: 1 × 3, European standard: 75 × 25 mm) are preferred because of many commercially available analytical instruments (printer microarrays, microarray reader, hybridization instruments, microarray development devices, based on the slat format) for efficient processing and microarray development of said format. Although preferred, the present invention is not limited to the slide format, and the principle of printing in a channel over a substructure can be applied to many different formats, as well as different materials.

Any type of material allowing a micro-structuring of the first microarray substrate and a substantial seal with the second microarray substrate is considered suitable for the manufacture of microarray platforms according to the invention. The preferred first substrate materials are synthetic polymers having a low fluorescence background which can be structured, for example, by injection molding or hot stamping, and which can be produced in large volumes. This first substrate may consist of homopolymers or copolymers, for example polyurethanes, polyesters, cyclic olefins, polypropylene, polycarbonate, polyethylene, polyesters, acrylates, polyamides, polyureas or other organic polymers. known to those skilled in the art. Alternatively, materials for the first microarray substrate as part of the non-plastic materials include glass, silicone materials, noble metals, quartz, composites, generally materials that can be structured to micrometric and nanometric dimensions by commonly known methods. The first microarray substrates can be microstructured into one type of material and then treated with a local or total surface coating to obtain suitable substrate properties, as required for detection (eg direct optical or fluorophore-dependent optical measurement). gold coating for the detection of molecular interactions by surface plasmon resonance, suitable waveguide coatings combined with diffraction gratings for refractometric measurements, interdigital electrodes combined with electronic connections for recording locally generated electrochemical signals) . Materials that are expensive and difficult to microstructure by themselves are the least recommended for use.

In the preferred embodiment, microstructuring of the microarray platform may be in accordance with or similar to the drawing of FIG. 4 and FIG. 5. The first substrate of the microarray platform is structured on both sides; the first surface of the first platform substrate 12 is shown in FIG. 4, and the second surface of the first substrate 13 is indicated in FIG. 5. The first surface of the first lamellar microarray substrate consists of a dedicated area for slat marking 23 and the surface of the microarray platform 12 which is in contact with the cover plate. The inlet port 18 establishes the transplateformal connection 22 to the microstructures on the second surface of the first microarray platform substrate (see Fig. 4). The area surrounding the orifice 24 includes the receiving portion of the autoclave structure on the cover plate 10. The outlet port 19 on the first surface of the first microarray platform surface makes direct contact with the outlet port of the cover plate 20, said outlet being connected to the actuating system which may be of any type of gas pump mechanism, any type of vacuum generating system, or current-driven device due to a positive pressure exerted on the conical inlet compartment on the cover plate. If the microarray platform device is used for serial analysis manually or jointly, for example, with robotic multiple pipetting systems, the area surrounding the inlet port 7 is in the form of an open container compartment. liquid.

FIG. 6 shows an exploded view of the second surface 13 of the first microarray substrate with a meander-like structure 14 showing one of many possible arrangements and designs of open channel structure. Other arrangements of open channel structures may take the form of straight lines, a spiral with, for example, an outlet at the center, or an inverted spiral arrangement. In another arrangement, individual open channel structures may be multiplexed on the second surface of the first microarray platform substrate platform. The multiplicity of said structural arrangements implies that there is a plurality of inlet ports and exit ports corresponding to the number of microstructures on said microarray platform.

Regardless of the type of the two-dimensional arrangement, the open channels in the second surface of the first microarray platform substrate 14 are converted into closed channels by overlapping the second microarray platform substrate. In a preferred arrangement, said second overlay substrate is a thin film, a flexible material generally known as a laminate, 15 & 16, capable of closing the open channels of the microarray platform after application to the first substrate substrate. Structured Solid Microarrays 13. A liquid-proof channel closure is obtained for example with laminates that reversibly or irreversibly interact with the surface 13. A liquid-proof closure may be obtained by gluing at the temperature. ambient, using commercially available laminates, or by heat sealing or welding laminates, any of the procedures mentioned corresponding to the state of the art currently used for the reversible or irreversible sealing of microplates. The sealing laminates 15 & 16 are preferably about 100 microns thick, impermeable to liquids and gases, thermally stable in the range of -40 ° C to + 100 ° C, are optically transparent to 250 nm, and are resistant to chemicals and solvents. These laminates are generally provided with a layer of pressure-sensitive adhesive on their lower surfaces. In practice, the laminates are applied in a manner similar to the adhesive tapes, and serve to permanently seal the open channels, in particular covering the entire surface 13.

The invention is not limited to lamination, but a liquid-proof channel closure can also be achieved by melting material. This applies to the particular case where hard materials are used as second microarray platform substrates (eg glass or silicone materials), or by welding of hard plastics or other procedures known to the human being. job.

FIG. 6 describes a preferred option for open channel substructures. The cross-sectional shapes of the open channels 25 may be U-shaped, rectangular, with or without rounded, V-shaped, oval, or rounded bottom features. In a preferred embodiment, the second surface of the first microarray platform substrate 13 and the first surface of the second microarray platform substrate 15 are flat, resulting in cross sections of channels having the aforementioned shapes with a flat cover. The height of the closed channels thus formed is established by the step of manufacturing the second surface of the first substrate 13. The depth of the channel is at least 30 μm and at most 400 μm, better still between 50 μm and 150 μm. The width of the open channel 25 is at least 200 μm and at most 1000 μm, more preferably between 400 μm and 800 μm. The width of the bridges 26 between the individual channels is at least 200 μm and at most about 1000 μm, more preferably between 400 μm and 600 μm. Microstructuring, complementary to the second surface of the first substrate 14, may be applied to the first surface of the second microarray platform substrate 15, particularly when hard materials are used as second substrates. This structuring can eventually result in round, oval, diamond-shaped, rectangular or square cross sections of the closed channels.

Depending on the shape and size of the closed channels, the total volume of the reaction chamber is between at least 4 .mu.l and at most 40 .mu.l, more preferably between 15 .mu.l and 25 .mu.l.

The channel printing of the invention, and the detection in a channel of chemical and biochemical reactions, may comprise sub-structured open channel topographies, said substructuring improving the molecular interactions due to local changes. fluid flow conditions. This substructuring further enhances the exposure of the immobilized ligand to reagents, rinse buffers and solvents and, in some configurations, places the capture ligands at distinctive topical elevations permitting not only confined capture ligand deposition, but also a distinctive addressable readout of generated signals, for example, by confocal microscopy or mass-sensitive optical detection, such as refractometry or surface plasmon resonance. Fig. Figure 6 shows enlarged partial views of exemplary substructures that may be integral portions of the reaction chambers. Said substructures are generated during the manufacturing of the platform by hot stamping, injection molding, ablation technologies, common in the art (for example micro-machining, laser ablation, beam ablation). electrons), by stripping procedures (wet pickling, dry pickling), or by positive printing molding. The capture ligands are printed at specific sites within the open channel using contact or non-contact microarray printing systems known to those skilled in the art.

Selected topographies of preferred substructures are further detailed in FIG. 6. The simplest version of a surface substructure in a channel consists of an arrangement of protruding disk-shaped structures 27 at the bottom of the open channels. The upper surface of said disk-shaped structures may have a minimal concavity that allows for effective local deposition and confinement of printed ligand solutions. In some other embodiments, wall-to-wall corrugated structures, with smooth transitions between alternating elevations, are introduced into the open channels, the ligands being printed on top of the elevations, or, in another embodiment, at intermittent depressions. These substructures effect efficient fluid flow distortion and promote reaction kinetics at the molecular interaction site.

The present invention requires the deposition of capture ligands in open channel structures. Ligand deposition is accomplished by contact or non-contact bio-printers, with a preference for instruments for precise two-dimensional positioning of the printing robot and accurate formation of small volumes of aliquots per deposition event. Current commercial instruments deliver liquid volumes of between 5 μl and several μl (picoliters).

The deposition of ligands can be performed with any type of instrumentation, which is generally called micro-array formation system, bio-printer, network forming system, nanotraceur or nano-printer, allowing a deposition precise locality of liquids in controlled quantities. Such microarray forming systems are commercially available, and their specifications are generally provided by the producer. Piezo-activated printing and pin deposition of liquid ligands are among the preferred printing systems, and their application refers to procedures of the state of the art. In connection with the present invention, it is essential that the fluid deposition take place from the open side of the "open channel" 25. The two-dimensional alignment of the instrument must be sufficiently adjustable and accurate to allow printing in the channels and , in the case of a substructuring of the channels, to print on or in the substructures in question. The sum of all the printed characteristics can form a two-dimensional network of deposited ligands, possibly including sample redundancies. A preferred type of printing feature is local speckling of individual ligand solutions. Aside from the speckling characteristics, microarray instrumentation can also be used to fully or partially deposit ligand solutions, either within or along a preselected structure, such as large areas within the microstrip. a channel or, as described in Example 2, a cross-sectional side-to-side of a meander-type channel arrangement, so that the result can eventually become a type of bar code by line. Similarly, the processed signal image of a printed network in a channel of a plurality of features can result in a two-dimensional bar code.

In some embodiments, a simple deposition of the ligands in the open channels may lead to physical adsorption (also called physisorption) or chemisorption of the ligands. The said sorption processes depend on the type of material used as the first microarray platform substrate, the physicochemical nature of the ligand, and the solvent. In some cases, microarray-based assays can be performed based on ligand adsorption. For example, molecular interactions based on forces such as bioaffinity and hydrophilicity or hydrophobicity may be sufficiently strong and lead to stable target-probe binding.

In some embodiments, it may be desirable to achieve a relatively strong interaction of the ligand with the channel forming material. These interactions can be achieved by means of a high affinity binding of ligand molecules, or by covalent attachment of the ligands to the material. Some of the processes leading to covalent ligand binding require preactivation of the material or attachment of reactive chemical species prior to ligand deposition. Chemical processes leading to strong binding, and in particular covalent, ligands are numerous, are well described in the literature on the subject, and are known to those skilled in the art. A covalent ligand-target binding may be performed according to the methods described in the monograph entitled Bioconjugate Techniques, Ed. G. T. Hermanson, 1996, Academy Press Inc., and in the journal "Surface immobilization of biomolecules by light," H. Sigrist; A. Collioud; J.-F. Clemence; H. Gao; R. Luginbuehl; Mr. Saenger; G. Sundarababu, Optical Engineering 1995, 34, 2339-2348, incorporated herein by reference in their entirety. The method described in the references mentioned may be modified when necessary to accommodate the material (first microarray platform substrate) and the target ligand. In view of the simplicity of the process, covalent ligand binding is preferably effected by photopolymer polymer-mediated methods, such methods involving the broad reactivity of photo-generated intermediates (particularly carbenes and nitrenes or cetyl radicals) with a wide variety of materials, including plastic polymers and elastomers.

Example 1

A polycarbonate plastic substrate (7.5 × 2.5 × 1 mm) was patterned on the second surface with a meander-type open channel, in accordance with the present invention, a receiving zone was engraved on the first surface of the plastic slide, and inlet and outlet connection holes were drilled. The second surface of the plastic substrate which exposes the meander type open channel has been laminated with an all purpose adhesive polyethylene web (eg Simport T329-1). The function of the microarray platform device was investigated by applying 10 to 50 μl of test solution to the receiving zone and subsequent application of a negative pressure at the outlet port after connecting the orifice to a peristaltic pump. Fluid samples tested for leak-tightness included, inter alia, phosphate buffered saline, water, aqueous solutions of artificial colorants (food colorants), bovine serum albumin in phosphate buffered saline , as well as samples of freshly drawn blood. The results confirm that the microarray platform device is liquid-proof, and that fluids of different compositions and densities can pass into the reaction chamber by applying a negative pressure (for example generated by means of a empty by the action of a peristaltic pump).

Example 2

A microstructured microarray platform with open channels, as detailed in Example 1, was treated with the photopolymer polymer OptoDex <®> (a product of Arrayon Biotechnology SA, Neuchâtel, Switzerland) and dried at room temperature. ambient for 2 hours under 5 × 10 <-> <2> mbar to generate a photo-activatable surface. The following ligands were dissolved in 0.5 mM sodium phosphate and 1.5 mM NaCl, pH 7.4: 0.5 mg / ml mouse immunoglobulin (mlgG); 0.5 mg / ml human immunoglobulin (hlgG). 2 μl of each sample were pipetted into separate side-by-side segments of an open channel meander-type structure. After deposition, the samples were dried at room temperature for 1 hour at 5 × 10 -3 mbar and the printed surfaces were irradiated for 4 minutes with an Oriel lamp (350 nm, 11 mW / cm 2). . After photoimmobilization, the open channels were laminated and the reaction chamber was rinsed first with 20 .mu.l of phosphate buffered saline (PBS) containing 1% bovine serum albumin, 20 .mu.l of PBS / Tween® 20, 20 μl of PBS and 20 μl of deionized water. For control purposes the microarray platform was scanned for Cy5 fluorescence with the Affymetrix 428 network scanner before further processing with a fluorescent target.

The immunostaining of the immobilized antigen was carried out by introducing 50 .mu.l of Cy5 fluorescently labeled anti-mouse antibody, the flow of fluid being entrained by applying a negative pressure with a peristaltic pump. . Incubation with the antigen was performed for 5 minutes and included reverse flow agitation of the antigen solution. After the incubation, the reaction chamber was rinsed first with 50 μl of phosphate buffered saline (PBS) containing 1% of bovine serum albumin, then 50 μl of PBS / Tween® 20, 50 μl of PBS. and 20 μl of deionized water. The laminate was removed and the microarray platform was scanned with the Affymetrix 428 network scanner. The results obtained are summarized in the following Table I.

Table I

[0057] <tb> Photo-immobilized ligand <sep> Cy5 fluorescence recovery before immunostaining (arbitrary units) <sep> Cy5 fluorescence recovery after immunostaining (arbitrary units) <tb> Channel 1 Segment: mlgG <sep> 273 ± 96 <sep> 54585 ± 2594 <tb> Channel 2 Segment: mlgG <sep> 378 ± 109 <sep> 47877 ± 3647 <tb> Channel Segment 3: hlgG <sep> 283 ± 58 <sep> 9538 ± 471

Example 3

[0058] Example 3 documents the procedures for addressable deposition in ligand molecule channels, and establishes the signal detection process by confocal microscopy. Fig. 7 shows a fluorescence scan image of a microarray platform with meander-like microstructures after deposition of fluorescently labeled ligand (Cy5-labeled OptoDex). Using the NP2 nanotracer, a product of GeSiM Ltd., Germany, which allows precise liquid sample deposition in non-contact mode, fluorescent ligand samples were injected and placed within the open channels. The instrument software allows proper positioning of the piezo pipettes, whose activation allows the deposition of single droplets having a volume of about 0.4 nl each. Fig. 7 shows that the droplets settle in the form of individual spots without coming into contact with the walls of the open channel. Stains with different ligand concentrations are distinctly revealed by fluorescence scanning with the Affymetrix 428 network scanner (product of Affymetrix Ltd., USA). In addition to this observation, it has been found that the fluorescence signal can be detected by confocal microscopy without removal of the laminate. The size of the spots and the optical resolution are comparable even if the intensity of the signal is reduced as expected due to the presence of the laminate. The results are summarized in Table II.

Table II

[0059] <tb> Detection of ligands printed in the channels <sep> Recovery of Cy5-OptoDex (arbitrary units, 2 fields x 2 columns x 6 rows: 24 spots) <tb> Without laminate <sep> 18753 ± 3013 <tb> With laminate <sep> 10446 ± 3409

Example 4

The performance of the microarray platform device and the apparatus, both of which are the subject of the present invention, was studied by analyzing the content of tetanus antibodies in human blood serum. The results of the comparative investigation, presented in Table III, were obtained by analysis of blood sera from 16 donors for the presence of anti-tetanus toxoid antibodies or by ELISA (enzyme immunoassay) procedures, which are methods of diagnosis known to those skilled in the art, either by procedures using microarrays. The microarray procedures were performed as follows. Prior to local deposition of the tetanus toxoid antigen on the microarray platform, the microarray platform was coated with a thin film layer of the OptoDex <®> photopolymer polymer, and dried. The tetanus toxoid was dissolved in phosphate buffer and the solution was printed with a robotic system in the open channels, a single droplet of this antigen solution (volume 400 μl each) having been deposited on the top of each structure protruding disk-shaped. Positive and negative characteristics, containing either human IgG immunoglobulin or mouse immunoglobulin, respectively, have also been similarly imprinted on other disk-like substrates of the microarray platform, together with standard solutions for fluorescence calibration. After printing, the microarray devices were first dried under vacuum and exposed to light (350 nm, 4 min, 10 mW / cm <2>) performing covalent (photoinduced) immobilization of the printed molecules; then the microarray platforms were stratified. After mounting the microarray printed in the apparatus described in FIG. 1, the reaction chamber was perfused first with buffer, then a sample of 2 microliters of serum, diluted in 100 microliters of dilution buffer, was introduced into the reaction chamber by actuation with a peristaltic pump, and incubated for 16 minutes by alternating cyclic movement of the sample solution. After incubation, the reaction chamber of the microarray platform was rinsed with buffer, and then perfused with fluorophore-labeled human anti-immunoglobulin. After 16 minutes, the microarray was rinsed and the microarray platform was scanned for fluorescence. Compared to the calibration standards, the fluorescence intensity was related to the immunoglobulin content of the original test sample, and the resulting anti-tetanus toxoid antibody concentration was expressed in International Units (IU / I). ).

Table III

[0061] <tb> ID of the serum <sep> Concentration of anti-tetanus toxoid antibody patient (international units / liter as in serum serum) <tb> <sep> Present microarray procedure <sep> ELISA procedure <Tb> 60052 <September> 12 <September> 9 <Tb> 71246 <September> 20 <September> 30 <Tb> 82284 <September> 51 <September> 57 <Tb> 60122 <September> 192 <September> 212 <Tb> 82106 <September> 307 <September> 401 <Tb> 82300 <September> 507 <September> 518 <Tb> 62103 <September> 628 <September> 537 <Tb> 61180 <September> 903 <September> 790 <Tb> 60394 <September> 1058 <September> 1403 <Tb> 82236 <September> 1389 <September> 1501 <Tb> 82303 <September> 1592 <September> 1657 <Tb> 60284 <September> 1626 <September> 1995 <Tb> 82316 <September> 1532 <September> 2050 <Tb> 82299 <September> 2354 <September> 2697 <Tb> 82424 <September> 4591 <September> 6106 <Tb> 81109 <September> 5761 <September> 8120

Claims (15)

Dedicated apparatus for performing multiplex analytical assays on microstructured analytical platform devices, consisting of the following parts assembled on a pedestal: a) a heating unit connected to an external electrical controller unit; b) a structured tablet type plate for placement of microarray platform devices, c) one or more microstructured analytical devices, with individually addressable reaction chambers, at least one of said devices comprising a first microstructured substrate with one or more grafted ligand (bio) molecules and a second substrate, which transforms the microstructures into the first microarray platform substrate into a channel-based reaction chamber, one or a plurality of different capture molecules being deposited and immobilized within the microstructures of the first substrate, and the second substrate being subsequently applied, forming a part of a microfluidic system, d) a cover plate providing input and output through connections to the analytical platform devices and a fluid activation system, respectively. An analytical apparatus according to claim 1, wherein a multiplex actuation system allows the simultaneous infusion of up to six microarray platform devices with reagents required for carrying out chemical, biochemical or immunological reactions within the microarray platform. reaction chambers addressed individually, the liquids being agitated by cyclic forward / backward movement of the liquids. An apparatus according to claim 1 or 2, wherein an integrated heater permits adjustment of fluid temperature in the reaction chamber of the microarray platform devices to a preselected temperature in the range of 20 ° C to 92 ° C, preferably in the range from room temperature to 40 ° C. A microarray platform device comprising a first substrate comprising a first microstructured microarray platform with one or more grafted ligand (bio) molecules and a second substrate, which transforms the microstructures in the first microarray platform substrate into a microstructured microstructure platform. channel-based reaction, wherein one or a plurality of different capture molecules are deposited and immobilized within the microstructures of the first substrate, and the second substrate is subsequently applied, forming a portion of a microfluidic system. 5. Device according to claim 4, wherein the depth of the microstructures is between 15 and 400 micrometers, preferably 100 micrometers. 6. Device according to claims 4 and 5, wherein the bridges between the microstructures are between 200 and 1000 micrometers, preferably 500 micrometers. The device according to claims 4 to 6, wherein the bottom of the microstructured surfaces has flow-distorted disc shaped substructures. 8. Device according to claims 4 to 7, wherein the elevations in the channels are disc-shaped substructures having a radius of 150 micrometers and a height of 25 to 45 micrometers. The device of claims 4 to 8, wherein the second microarray platform substrate is a transparent hard plastic material, an elastomer or a glass. 10. Device according to claims 4 to 9, wherein the capture molecules are printed and immobilized in the open channels of a first microstructured microarray platform, the total characteristic of the deposited (bio) molecules forming a network. 11. Device according to claims 4 to 10, wherein the capture molecules are immobilized at the flat bottom of the first microstructured microarray platform before application of the second microarray platform substrate. The device of claims 4 to 11, wherein the capture molecules are immobilized on the elevations of the first microstructured microarray platform prior to application of the second microarray platform substrate. The device of claims 4 to 12, wherein the binding of the reagent to the probe molecule is recorded by fluorescence detection of a assay reagent after removal of the second microarray platform substrate. The device of claims 4 to 12, wherein reagent binding to the probe molecule is recorded by fluorescence detection of a assay component without prior removal of the second microarray platform substrate. The use of the microarray platform device according to claims 4 to 14, with the apparatus according to claims 1 to 3, for microanalytical studies, in particular microarray-based assays, and for food and environmental analyzes based on microarrays.